ABSTRACT
The sodium-dicarboxylate co-transporter NaDC1/SLC13A2, located at the apical membrane of
the proximal tubule, reabsorbs Krebs cycle intermediates from the glomerular filtrate and is
thought to be responsible for the bulk of citrate transport in the nephron. Previous studies in our
lab have shown that NaDC1 is not calcium-sensitive and that transport of citrate and succinate in
opossum kidney (OK) proximal tubule cells is calcium-sensitive. When apical extracellular
calcium is lowered apical citrate transport increases significantly in OK cells. In this study we
demonstrate that stimulating the Calcium-Sensing Receptor (CaSR) by addition of spermine in
normal and low extracellular calcium results in inhibition of succinate transport indicating that the
CaSR plays a role in calcium-sensitive dicarboxylate transport regulation. Thapsigargin,
commonly used to increase intracellular calcium, also inhibited succinate transport in normal and
low extracellular calcium. This indicates that the CaSR signals through the guanine nucleotide
binding protein Gq. In addition to increased intracellular calcium, Gq signaling also activates
Protein Kinase C (PKC). The PKC activator, Phorbol 12-Myristate 13-Acetate (PMA), inhibited
dicarboxylate transport in low extracellular calcium only indicating that PKC regulates calciumsensitive transport specifically. Also investigated were the potential roles of the Gi and Gs
pathways. Gi and Gs inhibit and activate adenylate cyclase respectively. Inhibiting G i with
Pertussis Toxin and treating cells with 8-Br-cAMP had no effect on transport indicating that
neither Gi nor Gs regulate dicarboxylate transport. Inhibiting adenylate cyclase with MDL 12,330A
decreased dicarboxylate transport in normal and low extracellular calcium. However, 8-Br-cAMP
did not reverse these effects indicating that MDL 12,330A is inhibiting transport in some way
other than Gi signaling. To determine that in OK cells if ligand biased signaling of the CaSR
favors Gq at the expense of Gi and Gs, ELISA assays were used to measure intracellular cAMP
concentration. We found that calcium and spermine do not change intracellular levels of cAMP.
Thus, our studies on Gq, Gi and Gs signaling reveal that there is ligand biased signaling of the
CaSR in favor of the Gq pathway and that dicarboxylate transport is regulated by the CaSR → Gq
→ PKC pathway.
SIGNALING PATHWAYS OF DICARBOXYLATE TRANSPORT IN A PROXIMAL TUBULE
CELL LINE
A DISSERTATION
SUBMITTED ON THE 21 of APRIL 2017
TO THE GRADUATE PROGRAM OF BIOMEDICAL SCIENCE
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
OF THE SCHOOL OF MEDICINE
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
BY
Ryan Walker
APPROVED:
Kathleen S. Hering-Smith, M.S., Ph.D.
Advisor
L. Lee Hamm, M.D.
L. Gabriel Navar, Ph.D.
Kenneth D. Mitchell, Ph.D.
Solange Nakhoul Abdulnour-, Ph.D.
Ryosuke Sato, Ph.D.
AKNOWLEDGEMENTS
This has been the greatest accomplishment of my life. I am the first in my family to earn
a PhD. It was a wonderful and challenging experience that lead me in a direction that I did not
previously expect. I started as a molecular biologist working in the Tulane Center for Gene
Therapy (now referred to as the Tulane Center for Stem Cell Research and Regenerative
Medicine). My first mentor, Radhika Pochampally, encouraged me to join the PhD. program in
Biomedical Science at Tulane University and continued to encourage me after she moved her lab
to Jackson State University. I chose to stay at Tulane and joined the physiology department after
taking the systems biology course that widened my focus beyond signal transduction pathways.
There was a great deal of trial and error before the project that I am currently investigating
became my primary interest. I have many people to thank for this. Dr. Radhika Pochampally was
ever encouraging and selfless in her devotion to nourishing young minds and advising them on
how to shape their future in a highly competitive field. I would also like to thank Patrice Penfornis.
He was the lab manager in Dr. Radhika Pochampally's lab who taught me much of my bench
work and had a comedic stress free personality.
I would also like to thank my current mentor who guided me through the last several
years in the physiology department. Dr. Kathleen Hering-Smith was a hands on principle
investigator who personally trained me in radioisotope bench work in which I had no previous
experience. She also provided much needed advice on scientific writing. Her door is always
open and her passion for what she does is infectious. She had the patience to allow me to try
several different projects before I finally settled for the one that I am currently investigating. I also
learned that having at least one project fail is an important lesson for graduate students in this
field because failure is an important part of the scientific process.
I would like to thank my committee member Dr. Lee Hamm. As the Dean of Tulane
Medical School he has many responsibilities and a very busy schedule, but somehow manages
to make time for the students and our lab in particular. As a leading researcher in the field of
i
renal physiology he has worked closely with Dr. Kathleen Hering-Smith for decades. His calm
demeanor and integrity makes him approachable, dependable and his input has been invaluable.
The Chair of physiology Dr. Gabriel Navar gave me the push I needed to improve my
presentation skills. The criticism I received from him during my first seminar was quite scathing.
However, after the following seminar he shook my hand and said my presentation skills have
improved dramatically. His critique is challenging and productive and he encourages feedback
from his graduate students as well to insure that they get the most out of their learning experience
in the physiology department.
The CaSR is one of the main focuses of this study and because of this, Dr. Solange
Nakhoul Abdulnour was added to the committee. Her expertise in CaSR research has been of
great importance. Her advice has improved both my research and my writing. She has brought
to my attention important information that was crucial to this study and I am very thankful for her
contributions.
I would also like to thank the austere but friendly Dr. Kenneth Mitchell who has a no
nonsense demeanor and gives suggestions and advice that are both helpful and strait to the point.
As a committee member he has been encouraging and has always made himself available for
any questions I may have had.
My other committee member Dr. Ryosuke Sato has provided me with valuable advice
concerning how a scientific thesis should be written. More specifically, ways in which I can
reword phrases that were grammatically acceptable, but not quite suited for a scientific paper.
Also, as a scientist in physiology who focuses on signal transduction pathways he provided
valuable advise that related to my specific area of research in which most of my focus is on signal
transduction pathways.
I would also like to thank the journal club members who shared their research, ideas,
papers of common interest, and in general broadened the scope of information beyond that of my
own specific topic. Some I have already mentioned, Dr. Kathleen Hering-Smith, Dr. Lee Hamm,
ii
Dr. Solange Nakhoul Abdulnour. Others include the members of our lab, Dr. Weitao Huang, Dr.
Shijia Zhang, Dr. Santosh Yadav, Dr. Weibo Mao, Dr. Miao Yu and Joycelynn Coleman-Barnett, a
research specialist who assisted in much of my bench work. Other members of the journal club
include collaborators Dr. Fred Teran, Dr. Altaf Khan, Dr. Tolga Caner who was always
encouraging and willing to give advice as he graduated before I did and Dr. Nazi Nakhoul who
also provided valuable advice and did an excellent job of teaching the renal section of systems
biology.
iii
TABLE OF CONTENTS
Acknowledgments
i
List of Figures
vi
List of Tables
vi
Introduction
1. Citrate Transport in the Proximal Tubule
1
2. Indications of Novel apical Calcium-Sensitive Dicarboxylate Transport Process
1
3. The Calcium-Sensing Receptor
4
a. Discovery
4
b. Structure
4
c. Ligands
5
d. Ligand Biased Signaling
5
I. Functional Selectivity of G-Protein Coupled Receptors
5
II. G-protein Pathways
8
4. Calcium-Sensing Receptor in the Kidney
9
a. Location
9
b. Tubule Specific Function
9
Hypothesis, Specific Aims and Rationale
11
Specific Aims
1. Role of the Calcium-Sensing Receptor in Calcium-Sensitive Dicarboxylate Transport 11
2. Variations in Intracellular Calcium and Dicarboxylate Transport Regulation
12
3. Protein Kinase C Activation and Calcium-Sensitive Dicarboxylate Transport
Regulation
13
4. Role of G-Protein Signaling in Dicarboxylate Transport Regulation
16
iv
a. Gi Signaling and Dicarboxylate Transport
16
b. Gi or Gs Regulation of Dicarboxylate Transport
16
Methods
1. Cell Culture of Opossum Kidney Proximal Tubule Cell Line
19
2. Measuring Dicarboxylate Transport
19
3. Pharmacologicals
20
4. Cyclic Adenosine Monophosphate Competitive Binding Assays
23
Results
1. Calcium-Sensing Receptor Activation in Normal and Low Extracellular Calcium
25
2. Effects of Thapsigargin on Dicarboxylate Transport Regulation
28
3. Activation of Protein Kinase C in Normal and Low Extracellular Calcium
31
4. Dicarboxylate Transport Inhibition, the Calcium-Sensing Receptor, and Gq Signaling
34
a. Inhibition of Gi in Normal and Low Extracellular Calcium
34
b. Investigating the Effects of Spermine and Calcium on Intracellular cAMP Levels
44
Discussion
47
1. Calcium-Sensing Receptor Activation and Calcium-Sensitive Dicarboxylate Transport 48
2. Increased Intracellular Calcium and Dicarboxylate Transport
49
3. Protein Kinase C Activation and Calcium-Sensitive Dicarboxylate Transport
51
4. Ligand Biased Signaling, the Gi and Gs Pathways, and Dicarboxylate Transport
Regulation
52
Conclusion
55
List of References
57
Biography
65
v
List of Figures
1. Ligand Biased Signaling of the Calcium-Sensing Receptor
7
2. Calcium-Sensing Receptor → Gq → Protein Kinase C Signaling
15
3. Calcium-Sensing Receptor, Gi and Gs Pathways
18
4. Calcium-Sensing Receptor, → Gq → Protein Kinase C Signaling and CaSR Activation 26
5. Inhibition of Dicarboxylate Transport by Spermine
27
6. Diagram of Calcium-Sensing Receptor → Gq → Signaling and Thapsigargin
29
7. Dicarboxylate Transport Inhibition by Thapsigargin
30
8. Phorbol 12-Myristate 13-Acetate Activation of Protein Kinase C
32
9. Protein Kinase C Activation and Dicarboxylate Transport
33
10. Gi Signaling Cascade
35
11. Pertussis Toxin and Dicarboxylate Transport
36
12. Application of MDL 12,330A and 8-Br-cAMP to Mimic Gi and Gs Signaling
40
13. Adenylate Cyclase Inhibition and Dicarboxylate Transport
41
14. Effects of 8-Br-cAMP on Dicarboxylate Transport
42
15. Dicarboxylate Transport and MDL 12,330A + 8-Br-cAMP Application
43
16. Intracellular Cyclic Adenosine Monophosphate Levels
46
17. The Calcium-Sensing Receptor, Gq, Gi and Gs Signaling Cascades Investigated
56
List of Tables
1. Novel Calcium-Sensitive Citrate Transport Process in OK Proximal Tubule Cells
3
2. Pharmacological Agents, Dose and Duration
22
vi
1
Introduction
1. Citrate Transport in the Proximal Tubule
Citrate is freely filtered at the glomerulus and reabsorbed at the proximal tubule
1-3.
Citrate in urine is a potent inhibitor of nephrolithiasis 4 and by complexing calcium it prevents the
formation of insoluble complexes with oxalate and other anions. Because the reabsorption of
filtered citrate occurs only in the proximal tubule, urinary citrate concentration is determined by
proximal tubule reabsorption 1,3. Citrate is transported at the apical membrane of the proximal
tubule in its divalent form even though citrate exists primarily as a trivalent anion species at
physiological pH. The sodium-dicarboxylate co-transporter (NaDC1), also known as solute
carrier family 13 A member 2 (SLC13A2), is a multi-membrane spanning transporter responsible
for reabsorbing dicarboxylic acid Krebs cycle intermediates from the glomerular filtrate 5,6; NaDC1
is thought to be responsible for the bulk of citrate transport at the apical membrane of the
proximal tubule 7.
Proximal tubule apical citrate transport is sodium-dependent and pH-sensitive 8,9. Citrate
is also transported from peritubular circulation on the basolateral membrane of proximal tubule
cells 8,10,11. The sodium-dicarboxylate co-transporter (NaDC3) also known as solute carrier family
13 A member 3 (SLC13A3) is a high affinity dicarboxylate transporter located on the basolateral
membrane of the proximal tubule 12,13. Competitive inhibition of citrate transport with other
tricarboxylates also suggests the presence of a tricarboxylate transporter in OK cells
11,
although
this transporter has not yet been identified.
2. Indications of Novel Apical Calcium-Sensitive Dicarboxylate Transport Process
NaDC1 is a low affinity citrate transporter located in the apical membrane of the proximal
tubule and small intestine. It is thought to be responsible for nearly all citrate reabsorption in the
nephron and thus plays an essential role in the regulation of urinary citrate. Citrate is the most
2
important endogenous chelator of urinary calcium. Hypocitraturia has been linked to
nephrolithiasis, thus renal handling of citrate is of great physiological importance
4,6,14.
As was mentioned earlier NaDC1 is thought to be responsible for the majority of citrate
transport at the apical membrane of the proximal tubule even though it is predominantly trivalent
at normal physiological pH
1,3.
Previous studies have demonstrated that citrate and succinate
transport in the opossum kidney (OK) proximal tubule cell line is increased in response to
reduced extracellular calcium, however NaDC1 has not been found to be calcium-sensitive 15-17.
Also, in OK cells studied in low extracellular calcium (<60 µM), citrate is transported in the
divalent form. Lowering extracellular pH from 7.4 to 7.1 increases citrate transport since the
concentration of the divalent form of citrate increases as pH is decreased. This does not occur
with succinate, which is a dicarboxylic acid 15-17.
In low extracellular calcium (<60 µM) there is competitive inhibition between citrate and
the dicarboxylate succinate, whereas in normal extracellular calcium (1.2 mM) there is no pH
sensitivity or competitive inhibition with succinate and tricarboxylate transport predominates
16.
However, the tricarboxylate transport process is located on the basolateral membrane whereas
our studies focus on the apical membrane 11,15-17. This evidence indicates that there is a novel
citrate transport process at the apical membrane that has not yet been identified.
3
Table 1. Novel Calcium-Sensitive Citrate Transport Process in OK Proximal Tubule Cells
4
3. The Calcium-Sensing Receptor
a. Discovery
Sydney Ringer discovered that calcium was essential for the contraction of isolated
hearts in the 19th century 18. The levels of calcium released or absorbed by certain tissues
regulates calcium homeostasis in response to hormones released via calcium-sensing processes
19.
An increase or decrease of calcium in serum results in decreased or increased parathyroid
hormone (PTH) levels, respectively 20. It was later discovered that PTH regulation is contingent
upon extracellular calcium levels which may be associated with intracellular calcium mobilization.
This is likely the result of inositol triphosphate (IP3) and diacylglycerol (DAG) production, both of
which are associated with receptor-mediated increases in intracellular calcium and protein kinase
C (PKC) activation. Further studies revealed that these processes were still present when
parathyroid cells were stimulated by extracellular calcium in the absence of calcium influx from
the extracellular environment. Taken together, these studies revealed that there was likely a
receptor on the surface of parathyroid cells that regulates PTH secretion through intracellular
calcium mobilization 21-24. In 1993 the calcium-sensing receptor (CaSR) was cloned from bovine
parathyroid by Brown et al 25.
b. Structure
The CaSR is a class C G-protein coupled receptor (GPCR). Class C GPCRs are
characterized as having a very large extracellular domain, seven membrane spanning-helices
and an intracellular domain. In 1995 Aida et al cloned a human kidney CaSR. This was found to
have a long hydrophilic 612 amino acid N-terminal domain, 250 amino acid seven membranespanning helices and a small hydrophilic 216 amino acid C-terminal domain 26. Bai et al later
found that the CaSR on the surface of transfected human embryonic kidney cells 293 (HEK293)
form dimers through intermolecular disulfide bonds 27. Calcium binding occurs at the extracellular
domain of the CaSR which consists of a bi-lobed Venus-Flytrap structure with cysteine residues
critical for dimerization 28,29.
5
The CaSR normally resides in the cell surface membranes as homodimers or
heterodimers activating various G-proteins based on conformational changes elicited by
orthosteric ligands (Type I) and enhanced by ligands that bind allosterically (Type II)
19,25,30.
Orthosteric ligands such as spermine bind directly to the site where a primary ligand would bind,
i.e. the same binding site where calcium binds the CaSR. Allosteric ligands such as cinacalcet
bind peripherally to the primary binding site, enhancing the effects of the primary ligands rather
than activating the receptor directly. Because they bind orthosterically Type I agonists activate
the CaSR whether calcium is present or not, whereas Type II agonists require the presence of
calcium as stated earlier. Because we are studying dicarboxylate transport in both normal (1.2
mM) and low (<60 µM) extracellular calcium, we use spermine rather than cinacalcet as is shown
in Figure 1.
c. Ligands
Based on potency three groups of CaSR agonists include strong agonists (Gd 3+, La3+,
and Yt3+), medium-strength agonists (Ca2+, Ba2+, Sr2+, Cd2+, and Pb2+) and weak to ineffective
agonists (Mg2+, Ga3+, Fe3+, and Na+) 31. The CaSR is generally inactive at calcium levels below
0.2 mM with activation thresholds that can vary from 0.5 to 2 mM depending on cell type and
levels of expression 32. In 2004 Chang and Shoback proposed a model for the interaction
between the CaSR and its agonists that involves varying activation states
31.
In its inactive state
the Venus-Flytrap domain is open with electron donors on the CaSR shielded by H2O via
hydrogen bonding. Increasing calcium results in the displacement of H 2O around the electron
donors stabilizing a closed activated state.
d. Ligand Biased Signaling
I. Functional Selectivity of GPCR
Ligand biased signaling, also known as functional selectivity, is a relatively new concept
in GPCR signaling. The traditional model developed over two decades ago was a two-state
model where GPCRs isomerize between an inactive (R) state and an active (R*) state
33,34.
It is
6
now believed that various ligands can result in a multitude of conformations resulting in the
activation of different G-protein pathways as illustrated in Figure 1. Many studies have
demonstrated this behavior. For example, a study by Cordeaux et al suggests that the adenine
A1 receptor can adopt agonist-specific conformations arising from small changes in ligand
structure which result in differential activation of G i, Gs and Gq 35. Davey et al showed that an
allosteric modulator of the CaSR that has been used in clinical trials exhibits stimulus bias,
favoring intracellular calcium mobilization relative to extracellular signaling regulated kinase 1 and
2 (ERK1/2) phosphorylation 36 and Thomsen et al showed that strontium biases CaSR signaling
toward ERK1/2 signaling 37. The CaSR can couple to Gq, Gi and Gs signaling proteins 38,39, all of
which have been found in the proximal tubule 40,41.
7
Figure 1. Ligand Biased Signaling of the CaSR
Ligand biased signaling of the CaSR results in activation of a particular type of G-protein (Gx is
used in all examples because the G-protein that the receptor activates varies depending on cell
context) and depends on either the concentration of extracellular calcium present (A, Normal Ca 2+
(1.2 mM) or B, Low Ca2+ (<60 µM), the allosteric enhancement of CaSR activity by the binding of
calcimimetics (C), or the orthosteric binding of polyamines such as spermine (D). Adapted from
SC Brennan, 2013.
8
II. G-protein pathways
The binding of extracellular ligands can induce intracellular signaling through multiple Gproteins. A specific G-protein is predominantly activated depending on the ligand that binds
42.
One example of this is the activation of the metabotropic glutamate receptor 1 alpha (mGluR1α)
receptor. Glutamate recruits Gq and Gs, but in the presence of gadolinium the bias shifts to Gq 43.
Another example involves positive and negative modulators of the CaSR. In the absence
of allosteric ligands extracellular calcium binding resulted in an increase in intracellular calcium
and pERK1/2 in the HEK293-TREx c-myc-CaSR cell line. The presence of allosteric ligands
resulted in a significant preference for intracellular calcium modulation compared to pERK1/2
36.
Thomson et al showed that barium has a bias toward Gi compared to Gq and polyamines have a
bias toward ERK1/2 phosphorylation compared to Gq signaling 44.
Activation of Gq has been shown to cause the activation of phospholipase C (PLC)
45.
PLC then cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and
diacylglycerol (DAG). The soluble IP3 binds its receptor on the endoplasmic reticulum leading to
the release of intracellular calcium stores. The increased intracellular calcium along with the
insoluble DAG activates PKC as shown in Figure 2. Gi also may or may not be involved with the
activation of PKC in the proximal tubule. In human embryonic kidney cells (HEK293) transfected
with human CaSR, activation of PLC and subsequent activation of PKC was a G q-dependent and
Gi-independent process 46. However in medullary thick ascending limb cells Gq and Gi were both
involved in PKC activation 47. In this study we will investigate the potential roles of Gq, Gi or Gs in
dicarboxylate transport in opossum kidney (OK) proximal tubule cells which is a well established
proximal tubule cell culture model 11,15-17.
9
4. CaSR in the Kidney
a. Location
The CaSR is present throughout various segments of the nephron and with alternating
polarities. This indicates varying responses to circulating divalent ions at both the lumen and
basolateral aspects of the nephron.
In the proximal tubule of the rat kidney, the CaSR was shown to be located apically at the
base of the brush boarder. CaSR concentration is greatest in the early segment of the proximal
tubule (S1) and decreases through the middle (S2) and last (S3) segments of the proximal tubule
48.
The CaSR is expressed in the basolateral membrane throughout the thick ascending limb and
the macula densa. In the distal convoluted tubule the CaSR is expressed on the apical
membrane in humans 49 and on the basolateral membrane in rats 48 with concentration
diminishing toward the end of the distal convoluted tubule. The CaSR was found apically in some,
but not all Type A intercalated cells in the cortical collecting duct
48.
It is also expressed apically
in the inner medullary collecting duct and in the apical membrane of principle cells
50.
b. Tubule Specific Functions
In the proximal tubule CaSR activity has been reported to dampen the effect of 1,25dihydroxyvitamin D (D3) resulting in decreased reabsorption of renal calcium while increasing
secretion independent of
51.
Perfusion studies in the thick ascending limb have shown that in
hypercalcemic rats, calcium and magnesium reabsorption is inhibited by increases in calcium
concentration at the basolateral membrane where the CaSR is located 52. The increased
extracellular calcium also inhibits renal outer medullary potassium channels
53.
In the distal
convoluted and connecting tubules the CaSR and the Calcium-Selective Transient Potential
Vanilloid-subtype 5 channel (TRPV5) co-localize at the luminal membrane and activation of the
CaSR results in TRPV5-mediated calcium influx 49. In the collecting duct, CaSR activation
prevents nephrolithiasis by promoting H+-ATPase-mediated H+ excretion and downregulation of
Aquaporin 2, thus resulting in urinary acidification and polyuria
54.
The various functions of the
10
CaSR throughout the nephron show how this GPCR can have vastly different behaviors
depending on the polarity and the cell type 55. In our current study, we are investigating what may
be yet another function in which the CaSR prevents stone formation, and is located the proximal
tubule.
11
Hypothesis, Specific Aims and Rationale
Citrate in urine is a potent inhibitor of nephrolithiasis, freely filtered at the glomerulus and
reabsorbed in the proximal tubule 1,2,4,8. Calcium is solubilized by citrate and thus prevents the
formation of insoluble complexes with oxalate and other anions. Urinary citrate concentration is
determined by the proximal tubule where it is reabsorbed 3.
NaDC1 is thought to be responsible for the majority of citrate transport at the apical
membrane of the proximal tubule. Previous studies in our lab have demonstrated that citrate and
succinate transport in OK cells is increased in response to reduced extracellular calcium and that
NaDC1 has not been found to be calcium-sensitive 15-17. This would indicate that there is a novel
apical calcium-sensitive dicarboxylate transport process that has not yet been identified.
The overall hypothesis is that normal levels of extracellular calcium inhibit dicarboxylate
transport via CaSR activation of one of three G-proteins either by stimulating PKC or by inhibiting
or activating adenylate cyclase respectively. It is also important to determine if ligand biased
signaling has an effect on dicarboxylate transport regulation as the CaSR has many agonists.
Specific Aim 1
Role of the CaSR in calcium-sensitive dicarboxylate transport.
Hypothesis Specific Aim 1
CaSR activation inhibits dicarboxylate transport in normal extracellular calcium in OK
proximal tubule cells. Spermine, the orthosteric agonist of the CaSR should mimic the effect that
normal extracellular calcium has on dicarboxylate transport inhibition.
Rationale
If signaling via CaSR regulates apical dicarboxylate transport in normal extracellular
calcium at the proximal tubule, this effect should be demonstrable using agonists of the CaSR.
Calcimimetics are small, phenylalkylamine derivatives that activate the CaSR. Some bind
12
allosterically to the CaSR as Type II agonists that modify the EC50 of the receptor for ionized
calcium. Cinacalcet, a thoroughly studied calcimimetic is commonly used due to its optimal
pharmacokinetics and bioavailability 56. For our purposes, calcimimetics like the Type I agonist
spermine are most useful since in low extracellular calcium spermine can bind orthosterically,
activating the CaSR even in the absence of calcium 57; unlike Type II agonists such as cinacalcet
that require the presence of calcium for CaSR activation.
If CaSR activation is responsible for the differences in dicarboxylate transport under
varying extracellular calcium levels in the lumen of the proximal tubule, then spermine in low (<60
µM) extracellular calcium should mimic the effects of normal (1.2 mM) extracellular calcium by
inhibiting dicarboxylate transport through increased CaSR activity. These experiments will
provide evidence that in normal extracellular calcium dicarboxylate transport inhibition may be
due to CaSR activation. The CaSR is generally inactive at calcium levels below 0.2 mM with
activation thresholds that can vary from 0.5 to 2 mM depending on cell type and levels of
expression 32. Since the CaSR may not be fully activated at 1.2 mM it is also possible be that
spermine increases CaSR activity even further in normal extracellular calcium, thus having a
compounding effect on dicarboxylate transport inhibition.
Specific Aim 2
Variations in intracellular calcium and dicarboxylate transport regulation.
Hypothesis Specific Aim 2
Addition of thapsigargin commonly used to increase intracellular calcium inhibits
dicarboxylate transport in low extracellular calcium, thus simulating the effects of normal
extracellular calcium and spermine.
Rationale
Reducing extracellular calcium from 1.2 mM to <60 µM results in a significant increase in
citrate and succinate transport in OK cells
15-17.
It is known that increases in intracellular calcium
13
can be triggered by increases in extracellular calcium 58-61. There is also evidence that lower
extracellular calcium can lead to lower intracellular calcium 60. As shown in Figure 2, a rise in
intracellular calcium can be maintained by treatment with thapsigargin resulting in PKC activation
which regulates many plasma membrane transporters found in the proximal tubule 58,59,62-65.
Thapsigargin inhibits the Ca2+-ATPase responsible for pumping released intracellular calcium
back into the endoplasmic reticulum. After treatment with thapsigargin there is a spike in
intracellular calcium caused by depletion of intracellular stores. As was stated earlier, reducing
extracellular calcium leads to increased citrate and succinate transport in OK cells. It is not yet
known if elevated intracellular calcium is involved with calcium-sensitive dicarboxylate transport
regulation. If increased intracellular calcium inhibits dicarboxylate transport in normal
extracellular calcium, then reducing intracellular calcium by lowering extracellular calcium to <60
µM could lead to increased dicarboxylate transport. The question being investigated here is,
does thapsigargin treatment prior to succinate transport measurement lead to inhibition of
dicarboxylate transport in the proximal tubule?
Specific Aim 3
PKC activation and dicarboxylate transport regulation.
Hypothesis Specific Aim 3
The PKC activator Phorbol 12-Myristate 13-Acetate (PMA) inhibits dicarboxylate
transport in OK proximal tubule cell monolayers.
Rationale
In addition to providing evidence that dicarboxylate transport is regulated by CaSR
activation, it is also important to determine what signal transduction pathway this may be
occurring through. The CaSR is well known for activating PKC. There is a good possibility that
the CaSR may inhibit dicarboxylate transport via PKC activation based on previous studies
demonstrating that PKC is often activated by the CaSR via G q signaling 59.
14
So far we have been focusing on the Gq pathway. We have provided evidence that the
CaSR activator spermine results in transport inhibition in both normal and low extracellular
calcium. Using thapsigargin, a pharmacological used to increase intracellular calcium, yielded
results very similar to that of spermine in that both pharmacologicals inhibited succinate transport
in normal and low extracellular calcium. This would indicate that dicarboxylate transport inhibition
is caused by CaSR activation followed by Gq activation and subsequent rise in intracellular
calcium. As is shown in Figure 2, a rise in intracellular calcium along with the formation of DAG
may activate PKC.
PKC has been found to regulate many plasma membrane transporters found in the
proximal tubule, such as Type II sodium-phosphate transporters, organic anion transporter 1,
renal betaine transporter, NaDC1 and NaDC3 58,62-65. PKC has been shown to inhibit
dicarboxylate transport, but in more than one way. When NaDC1 was over expressed in
Xenopus Oocytes, dicarboxylate transport was inhibited by activation of PKC 66. Only part of this
inhibition was due to internalization of NaDC1, a non-calcium-sensitive dicarboxylate transporter
16.
There is another mode of dicarboxylate transport inhibition caused by PKC activation that
does not involve internalization and mutational studies done on the consensus phosphorylation
sites on NaDC1 indicate that it is not likely NaDC1 is phosphorylated at those sites 66. Based on
the study in Oocytes it is clear that PKC activation may inhibit dicarboxylate transport via NaDC1
but it is not yet known if PKC activation has an effect on calcium-sensitive dicarboxylate transport.
Therefore using PMA addition to activate PKC in OK cells we investigated its potential changes
on calcium-sensitive dicarboxylate transport.
Kempson et al demonstrated that the betaine/GABA transporter (BGT1) is calciumsensitive and this likely occurs through PKC activation. Using thapsigargin they showed that PKC
activation resulted in internalization of the BGT1 and thus inhibition of transport 58. It may be that
the apical calcium-sensitive dicarboxylate transporter is being internalized by PKC activation and
this can be investigated by direct activation of PKC with PMA.
15
Figure 2. CaSR → Gq → PKC Signaling
Flowchart outlining the CaSR → Gq → PKC signal transduction pathway that is being investigated
with the use of the CaSR activator spermine, the PKC activator PMA and thapsigargin to increase
intracellular calcium.
16
Specific Aim 4
Role of G-protein signaling in dicarboxylate transport regulation.
Hypothesis Specific Aim 4
Dicarboxylate transport regulation via the CaSR in OK proximal tubule cells occurs
through the Gq pathway and not Gi or Gs.
Aim 4a
Gi signaling and dicarboxylate transport.
Rationale
Aside from activating the Gq protein and the PKC pathway, the CaSR also signals via the
Gi protein which is known to inhibit adenylate cyclase and cAMP production
38.
It is not yet known
if Gi is involved in the activation of PKC in the proximal tubule or if dicarboxylate transport
inhibition occurs through inhibition of adenylate cyclase and cAMP production via the G i protein.
If the CaSR were inhibiting dicarboxylate transport via Gi activation then inhibiting Gi with
pertussis toxin (PTX) would result in increased dicarboxylate transport in normal extracellular
calcium.
If CaSR activation inhibits dicarboxylate transport via Gi which in turn inhibits adenylate
cyclase, then inhibiting adenylate cyclase with N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2imine-hydrochloride (MDL 12,330A) would also inhibit dicarboxylate transport in low extracellular
calcium. If MDL 12,330A does inhibit dicarboxylate transport via adenylate cyclase inhibition, the
addition of 8-Br-cAMP should reverse the effects of MDL 12,330A.
Aim 4b
Gi or Gs regulation of dicarboxylate transport.
17
Rationale
As was discussed earlier in addition to Gq, Gi and Gs proteins are also associated with
the CaSR 38,39 and have also been found in the proximal tubule 40,41. To further investigate if
CaSR activation via extracellular calcium or spermine results in G i or Gs activation in OK proximal
tubule cells, intracellular cAMP levels can be measured using ELISA assays. G i activation has
been shown to reduce intracellular cAMP concentrations by inhibiting adenylate cyclase
67.
Gs
activation increases cAMP by stimulating adenylate cyclase 67. Results from the ELISA assays
should show if the two ligands (calcium and spermine) have a bias toward G i or Gs and if the two
ligands have the same bias favoring one G-protein over another. If there is no significant change
in cAMP these results should provide further evidence that the bias is toward Gq.
18
Figure 3. CaSR, Gi and Gs Pathways
Flowchart outlining the CaSR, Gi, and Gs adenylate cyclase signal transduction pathways.
These pathways are being investigated with the use of the Gi inhibitor PTX, the adenylate cyclase
inhibitor MDL 12,330A (used to mimic Gi or reverse the effects of Gs) and 8-Br-cAMP to mimic Gs
or reverse the effects of Gi or MDL 12,330A.
19
Methods
1. Cell Culture of Opossum Kidney Proximal Tubule Cell Line
OK cells were cultured and uptake assays performed as previously described
11,15-17.
Briefly OK cells are maintained in MEM containing 26 mM HCO 3- supplemented with 10 % FCS
(Invitrogen) 25 mM HEPES, 11 mM L-glutamine and 100 IU/ml penicillin in a humidified
atmosphere of 5 % CO2 at 37ºC. Cell monolayers were grown on 24-well plates (Corning-Costar),
wells = 1.9 cm2, with media changes every 2 days. After reaching confluence, cell monolayers
were changed to serum-free media for a minimum of 24 hours before experiments were
performed.
2. Measuring Dicarboxylate Transport
Succinate transport was measured by uptake of radiolabeled ( 14C)-succinate on confluent
OK cell monolayers. Just before uptake measurements cells were rinsed free of media and
incubated for 2 min at 37ºC in a buffer containing either normal (1.2 mM) or low (<60 µM) calcium.
The remaining components of the buffer were in mM: 109 NaCl, 3 KCl, 2 KH2PO4, 1 MgSO4, 5
alanine, 8.3 glucose, 1 sodium acetate, 25 HEPES; osmolality of 290 mM H2O and pH 7.4. For
each uptake assay 200 ml of normal and low calcium solutions were aliquoted into their
respective flasks and were incubated in a water bath at 37º C. The solutions were then
equilibrated by bubbling with O2 for 1 hour 1,3,11,15-17.
Uptake was performed at 37º C using the 12 central wells of 24 well plates. Then 6 wells
are incubated with normal calcium (1.2 mM) and 6 wells are incubated with low calcium (<60 µM).
Next, 3 wells of each, normal and low calcium, would then be treated with pharmacologicals
shown in table 2 (also explained in the following section) and the other half of the plate would be
treated with the appropriate vehicle controls. Just before uptake culture media was aspirated and
the cell monolayers would be rinsed 2 times with the appropriate buffer (i.e. 1.2 mM or <60 µM
calcium) without isotope followed by 2 minute incubation using the same buffer at 37º C. Next
20
the incubation buffers were aspirated and 0.4 ml of the appropriate uptake buffer with 0.626
mCi/ml [1,4-14C] succinate (Moravek Biochemicals) was added to individual wells. The final
concentration of succinate was ~0.004 mM. The uptake solution also contained [ 3H]-mannitol
(Perkin Elmer) to determine the residual extracellular volume. After 3 minutes, the uptake buffer
was removed and the wells were rinsed 3 times with ice cold 0.1 M MgCl2. The monolayers were
then lysed with 1 ml of 0.1N NaOH for 5 minutes. The wells were then scraped and the lysate
from each well was then transferred to individual 7 ml glass scintillation vials (Fisher Scientific)
and 5 ml of scintillation cocktail (Perkin Elmer) was added to each vial for liquid scintillation
counting 11,15-17.
Uptake was calculated from measured 14C radioactivity per well using the Beckman
Coulter Liquid Scintillation Counter model LS 6500. Appropriate windows and crossover
calculations were used to distinguish between 3H and 14C. Uptake was further factored for
residual extracellular volume that was not removed by the triplicate rinsing. Because variability
can occur in transport rates, each experiment was performed in a paired fashion, such that both
low (<60 µM) and normal (1.2 mM) calcium solutions were used. There were a minimum of 4
experiments for each 4 plate experiment. Data are expressed as means ± SE. All statistical
analysis was performed using one way ANOVA. Statistical significance was defined as P<0.05.
3. Pharmacologicals
Unless otherwise stated all pharmacologicals were purchased through Sigma Aldrich. All
uptakes were performed in which half of the wells (both normal and low calcium) were treated
with various pharmacologicals. Table 2 shows the pharmacological agents, vehicle, dose,
duration and references used in these studies. All pharmacologicals used for uptake experiments
were used to determine their effects on succinate transport. Spermine is a known CaSR activator
57,68.
Thapsigargin is commonly used to increase intracellular calcium
69.
PMA is a PKC
activator 63,64. MDL 12,330A is an adenylate cyclase inhibitor; however, it can also have
secondary effects that likely do not involve adenylate cyclase 70. MDL 12,330A has been shown
to block certain calcium channels in endocrine cells and also has nonspecific effects on glycine
21
transport in certain retinal cells independent of adenylate cyclase possibly due to toxic effects
resulting in decreased cell viability 71,72. MDL 12,330A can also inhibit phosphodiesterase which
could mask its effect on adenylate cyclase 73. 8-Br-cAMP is cell membrane permeable and can
be used to simulate an increase in endogenous intracellular cAMP
74.
PTX is a Gi inhibitor 75.
Forskolin is an adenylate cyclase activator used in the cAMP ELISA experiments 70. Spermine is
a CaSR activator and was used in both uptake and ELISA experiments 57,68.
22
Table 2. Pharmacological Agents, Dose and Duration
Pharmacological
Dose
Duration
Vehicle
Ref
Spermine
Thapsigargin
PMA
MDL-12,330A
1 mM
10 μM
50 nM
50 μM
3' During Uptake
30' Prior
30' Prior
30' Prior
H2O
DMSO
DMSO
DMSO
57, 68
69
63, 64
70
8Br-cAMP
100 μM
30' Prior
H2O
74
MDL + 8Br
50 μM + 100 μM
30' Prior
DMSO + H2O
70, 74
PTX
100 ng/ml
Overnight
H2O
75
Forskolin
10 μM
30' Prior
H2O
70
23
4. cAMP Competitive Binding Assays
A series of colorimetric cAMP ELISA competitive binding assays (Cayman Chemical,
581001) were performed on 96 well plates, standard curves were performed in duplicate and
samples in triplicate. As was done with uptake experiments OK cells were grown on 24 well
plates. Buffers containing either normal (1.2 mM) or low (<60 µM) calcium were incubated at 37º
C. The remaining components of the buffer were in mM: 109 NaCl, 3 KCl, 2 KH 2PO4, 1 MgSO4,
5 alanine, 8.3 glucose, 1 sodium acetate, 25 HEPES; osmolality of 290 mM H2O and pH 7.4.
MgSO4 was not included in low calcium solutions as Mg2+ is a CaSR activator and in low calcium
buffer Mg2+ may be more likely to interfere. For each assay normal and low calcium solutions
were aliquoted into their respective flasks and were incubated in a water bath at 37º C. The
solutions were then equilibrated by bubbling with O 2 for 1 hour. Cell monolayers were rinsed
twice in normal calcium buffer or low calcium buffer. Unlike the uptake experiments these plates
were then returned to the incubator in their respective buffers for 25 minutes. Since buffered in
HEPES this should not change the pH. One of these buffers included normal calcium with 10 µM
forskolin because a total treatment time of 30 minutes is required with forskolin. Solutions were
aspirated and replaced by the same buffers with the exception of one containing normal calcium
with 1 mM spermine as treatment time for spermine is much shorter than that of forskolin. All
wells were then incubated in a water bath at 37º C for five minutes. This was necessary so that
the wells treated with forskolin had a total treatment time of 30 minutes. Solutions were then
aspirated and 54 µl of 0.1 M HCl was added to each 1.9 cm2 well and incubated for 20 minutes at
room temperature. Cells were scraped off and dissociated by pipetting up and down. After
transferring to centrifuge tubes lysates were centrifuged at 1,000 x g for 10 minutes and
supernatants were transferred to their respective tubes.
Standard curve preparation goes as follows: The cAMP EIA standard is reconstituted
with 1 ml of EIA buffer (Standard A). 80 µl of standard A is aliquoted into 2.92 ml EIA Buffer
(Standard B) for a concentration of 200 pmol/ml. A serial dilution is then prepared in tubes from 0
(EIA buffer only with no cAMP) to 8 (lowest concentration of cAMP). Tubes 1-8 in pmol/ml
24
contain 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 respectively. Standards and samples
(with EIA buffer added for 2 dilutions) are then acetylated by adding 100 µl of 4 M KOH and 25 µl
acetic anhydride, vortexed for 15 seconds followed by the addition of 25 µl KOH and briefly
vortexed again. These concentrations are based on the 500 µl standards and must be adjusted
when acetylating samples and is based on sample size.
The ELISA cAMP EIA kit (Cayman Chemical 581001) includes 96 well mouse anti-rabbit
IgG coated 96 well plates. Plate set up requires two blank wells (nothing added to the wells at
this stage), two nonspecific binding wells (50 µl of EIA buffer + 50 µl of tube 0 of standard curve),
3 maximum binding wells (50 µl of tube 0), standard curve wells in duplicate (50 µl from tubes 18), sample wells in triplicate (50 µl of test samples). To all wells except the blank wells and the
total activity wells 50 µl of cAMP acetylcholinesterase (AChE) tracer is added. To all wells
(except total activity, nonspecific binding, and blank wells) 50 µl of cAMP ELISA antiserum is
added. Plates are then covered and incubated for 18 hours at 4º C on a rocking platform.
Plates are then developed by emptying the wells and rinsing 5 times with wash buffer
then adding 200 µl of Ellman's Reagent to each well and 5 µl of tracer to the total activity well.
The plate is then covered and placed on an orbital shaker protected from light for 100 minutes at
room temperature. The plates were read at 412 nm. Absorbance readings for all wells in
duplicate or triplicate are averaged. The total activity well is only used as a diagnostic tool and is
not included in the calculations. Nonspecific binding is subtracted from maximum binding which
yields the corrected maximum binding B0. Nonspecific binding is subtracted from all standard
and sample averages and divided by B0 (B/B0) which is binding divided by maximum binding).
The equation used to linearize the standard curve is logit (B/B0) = ln [B/B0/(1-B/B0)]. Sample
concentrations can then be calculated using the standard curve.
Data are expressed as means ± SE. Each mean is derived from 4 individual experiments
studied on 4 separate days. One way ANOVA was used for statistical comparison in all ELISA
assays. Statistical significance was defined as P<0.05.
25
Results
1. CaSR Activation in Normal and Low Extracellular Calcium
Extracellular calcium inhibits dicarboxylate transport at the apical membrane of OK
proximal tubule cells 15-17 and we suspect that this is the result of CaSR activity. If the CaSR
regulates dicarboxylate transport then apical addition of CaSR agonists may be used to test this
hypothesis as shown in Figure 4. Because normal and low extracellular calcium is used in our
dicarboxylate transport assays, spermine (as shown in Figure 4) was our agonist of choice due to
its ability to activate the CaSR in low extracellular calcium unlike the more commonly used Type 2
agonist cinacalcet, which requires the presence of calcium.
Figure 5 shows inhibitory effects of the Type1 agonist spermine on succinate transport in
both normal and low extracellular calcium. Bar 1 on the far left side is the control representing
succinate transport at 100% (% of control) in normal extracellular calcium. Bar 2 represents
succinate transport in normal extracellular calcium with 1mM spermine. The addition of spermine
reduced transport in normal extracellular calcium (100 ± 0.00 to 71.6 ± 1.7 % of control, p<0.05).
Bar 3 represents succinate transport in low extracellular calcium vehicle control in which transport
was increased compared to control Bar 1. Bar 4 represents succinate transport in low
extracellular calcium with 1 mM spermine. Addition of spermine decreased transport in low
extracellular calcium from (174 ± 29.8 to 123 ± 25.3 % of control, p<0.01). The decrease in
transport resulting from the CaSR activator spermine indicates luminal calcium regulation of
dicarboxylate transport via the CaSR.
26
Figure 4. CaSR → Gq → PKC Signaling and CaSR Activation
Flowchart depicting the CaSR → Gq → PKC pathway investigated by use of the CaSR activator
spermine pertaining to the succinate transport experiments shown in Figure 5.
27
Figure 5. Inhibition of Dicarboxylate Transport by Spermine
Succinate transport was increased in low (<60 µM) compared to normal (1.2 mM) extracellular
Ca2+ in vehicle controls (VC) shown in Bar 3 vs. Bar 1. The addition of 1 mM spermine (Sp) for 3
minutes inhibited transport in both normal and low calcium. In normal extracellular calcium
spermine decreased transport (100 ± 0.0 to 71.6 ± 1.7 % of control) shown in Bars 1 and 2. In
low extracellular calcium spermine decreased transport (174 ± 29.8 to 123 ± 25.3 % of control)
shown in Bars 3 and 4, thus CaSR activation by spermine decreases dicarboxylate transport in
proximal tubule cell monolayers. # Indicates p<0.05, * indicates p<0.01.
28
2. Effects of Thapsigargin on Dicarboxylate Transport Regulation
We have demonstrated that CaSR activation by spermine results in the inhibition of
dicarboxylate transport in OK proximal tubule cells in normal and low extracellular calcium. The
next step in our study was to determine which G-protein pathway is being activated as the CaSR
generally couples to one G-protein at the expense of another depending on cell type and polarity.
Gq is often associated with CaSR signaling and as is shown in Figure 6, Gq activation results in
increased intracellular calcium via PLC activation, which then helps to activate PKC.
To investigate whether CaSR activation is regulating dicarboxylate transport through G q
activation, we began by examining the effects that raising intracellular calcium would have on
transport by pretreating OK cells with thapsigargin (commonly used to increase intracellular
calcium). This was followed by 14C-succinate transport assays in both normal and low
extracellular calcium.
Figure 7 shows the effects of thapsigargin on succinate transport in both normal and low
extracellular calcium. Lowering extracellular calcium significantly increased succinate transport
demonstrating the inhibitory effect that normal levels of extracellular calcium have on
dicarboxylate transport. Bar 1 on the far left side of the graph is the vehicle control representing
succinate transport at 100% in normal extracellular calcium. Bar 2 represents succinate transport
in normal extracellular calcium with thapsigargin which results in decreased transport (100 ± 0.00
to 73.5 ± 2.9 % of control, p<0.01). Bar 3 represents succinate transport in low extracellular
calcium vehicle control in which transport was increased compared to Bar 1 (normal extracellular
calcium, vehicle control. Bar 4 represents succinate transport in low extracellular calcium with
thapsigargin. Comparing Bars 3 and 4, apical addition of thapsigargin decreased transport in low
extracellular calcium as well (134 ± 7.2 to 100 ± 8.2 % of control, p<0.01). This indicates that
increases in intracellular calcium via Gq signaling results in decreased dicarboxylate transport due
to activation of the CaSR in normal extracellular calcium.
29
Figure 6. Diagram of CaSR → Gq → PKC Signaling and Thapsigargin
Flowchart depicting the CaSR → Gq → PKC pathway investigated by use of thapsigargin to
increase intracellular calcium; this pertains to the succinate transport experiments shown in
Figure 7.
30
Figure 7. Dicarboxylate Transport Inhibition by Thapsigargin
Succinate transport was increased in low (<60 µM) compared to normal (1.2 mM) extracellular
calcium in vehicle controls (VC) shown in Bars 1 and 3. The addition of 10 µM thapsigargin
(Thaps) inhibited transport in both normal and low extracellular calcium (Bars 2 and 4). In normal
extracellular calcium thapsigargin decreased transport (100 ± 0.00 to 73.5 ± 2.9 % of control)
shown in Bars 1 and 2. In low calcium thapsigargin decreased transport (134 ± 7.2 to 100 ± 8.2 %
of control) shown in Bars 3 and 4. This provides evidence that dicarboxylate transport inhibition
is facilitated through Gq signaling. * Indicates p<0.01.
31
3. Activation of PKC via PMA in Normal and Low Extracellular Calcium
So far we have demonstrated that dicarboxylate transport is inhibited via CaSR activation
and provided evidence that this is accomplished through Gq signaling.
As is shown in Figure 8, Gq activation leads to an increase in intracellular calcium
followed by PKC activation; PKC is known to regulate many cell membrane transporters
58,62-65.
To further investigate whether the CaSR regulation of dicarboxylate transport in OK proximal
tubule cells occurs through Gq signaling, OK cells were pretreated with the PKC activator PMA.
Figure 9 shows the effects of PMA on succinate transport in both normal and low extracellular
calcium.
Bar 1 on the far left side is the vehicle control representing succinate transport at 100% in
normal extracellular calcium. Bar 2 represents succinate transport in normal extracellular calcium
with 50 nM PMA in which transport was unchanged (100 ± 0.00 to 100 ± 5.9 % of control, p=NS).
Bar 3 represents succinate transport in low extracellular calcium in which transport was increased
compared to vehicle control in normal extracellular calcium shown in Bar 1. Bar 4 represents
succinate transport in low extracellular calcium with PMA. Comparing Bars 3 and 4, apical
addition of PMA decreased transport in low extracellular calcium (143 ± 7.8 to 118 ± 6.6 % of
control, p<0.01). This indicates that PKC activation may regulate transport in low extracellular
calcium specifically.
Taken together the thapsigargin and PMA results indicate that CaSR activation by
extracellular calcium in OK cells results in Gq signaling and is therefore the likely pathway of
apical dicarboxylate transport regulation.
32
Figure 8. PMA Activation of PKC
Flowchart depicting the CaSR → Gq → PKC pathway investigated by use of PMA to activate PKC
pertaining to the succinate transport experiments shown in Figure 9.
33
Figure 9. PKC Activation and Dicarboxylate Transport
As shown previously, succinate transport increases in low (<60 µM) compared to that in normal
(1.2 mM) extracellular calcium in vehicle controls (VC) shown in Bars 1 vs. 3. The addition of 50
nM PMA resulted in a decrease in succinate transport only in low extracellular calcium (143 ± 7.8
to 118 ± 6.6 % of control) shown in Bars 3 and 4. PMA did not affect transport in normal
extracellular calcium (100 ± 0.00 to 100 ± 5.9 % of control) shown in Bars 1 vs. 2. This indicates
that PKC activation may restore the inhibitory effects of calcium on dicarboxylate transport. *
Indicates p<0.01.
34
4. Dicarboxylate Transport Inhibition, the CaSR and Gq Signaling Bias
4a. Inhibition of Gi in Normal and Low Extracellular Calcium
Our studies using spermine to activate the CaSR, thapsigargin to increase intracellular
calcium and the PKC activator PMA, have demonstrated that dicarboxylate transport regulation
likely occurs through CaSR → Gq → PKC signaling. It is important to take into account that the
CaSR is a unique and dynamic GPCR, exhibiting behaviors that vary dramatically depending on
polarity and tissue specificity. It is also used as a prime example in studies delineating the
nuances of ligand biased signaling where one pathway is favored at the expense of another. Two
other G-proteins that the CaSR is known to signal through and have a bias for depending on
polarity and cell type are Gi and Gs. Though our evidence indicates that the CaSR in OK
proximal tubule cell monolayers regulates dicarboxylate transport with a bias for Gq it is still
possible that other pathways are involved. Gi is an endogenous inhibitor of adenylate cyclase. If
CaSR activation inhibits dicarboxylate transport via Gi, it would be expected that inhibiting Gi
would increase transport by increasing intracellular cAMP concentrations as is shown in Figure
10.
In Figure 11 Bar 1 on the far left side is the vehicle control representing succinate
transport at 100% in normal extracellular calcium. Bar 2 represents succinate transport in normal
extracellular calcium with the 100 ng/ml of the Gi inhibitor PTX in which transport was unaffected
(100 ± 0.00 to 107 ± 1.9 % of control, p=NS). Bar 3 represents succinate transport in low
extracellular calcium vehicle control in which transport was increased compared to Bar 1 (normal
extracellular calcium vehicle control). Bar 4 represents succinate transport in low extracellular
calcium with PTX. Bars 3 and 4 show that addition of PTX in low extracellular calcium also had
no effect on transport (174 ± 24 to 178 ± 15 % of control, p=NS).
Therefore Since addition of PTX did not result in increases in transport, Gi is likely not
involved in the regulation of dicarboxylate transport in OK proximal tubule cells.
35
Figure 10. Gi Signaling Cascade
Flowchart depicting the CaSR → Gi → adenylate cyclase pathway investigated by use of PTX to
inhibit Gi pertaining to the succinate transport experiments shown in Figure 11.
36
Figure 11. PTX and Dicarboxylate Transport
Succinate transport was increased in low (<60 µM) Bar 3 compared to normal (1.2 mM)
extracellular calcium in vehicle controls (VC) shown in Bar 1. Overnight pretreatment of 100
ng/ml PTX had no effect on transport in normal extracellular calcium (100 ± 0.00 to 107 ± 1.9 %
of control) shown in Bars 1 vs. 2. PTX also had no effect in low extracellular calcium (174 ± 24 to
178 ± 15 % of control) shown in Bars 3 vs. 4. This indicates that Gi signaling is not involved with
dicarboxylate transport regulation. * Indicates p<0.01.
37
Figure 11 shows that inhibition of Gi with PTX had no effect on dicarboxylate transport in
either normal or low extracellular calcium conditions. This indicates that Gi is not involved in
dicarboxylate transport regulation. However to determine if direct inhibition of adenylate cyclase
results in dicarboxylate transport inhibition we tested a known exogenous inhibitor of adenylate
cyclase, MDL 12,330A, to mimic Gi activation. If Gi signaling decreases dicarboxylate transport
via adenylate cyclase inhibition, it would be expected that MDL 12,330A would also inhibit
dicarboxylate transport.
Figure 13 shows that apical addition of MDL 12,330A inhibited succinate transport in both
normal and low extracellular calcium. Bar 1 on the far left side is the vehicle control representing
succinate transport at 100% in normal extracellular calcium. Bar 2 represents succinate transport
in normal extracellular calcium with MDL 12,220A, where transport was decreased (100 ± 0.00 to
53.7 ± 7.4 % of control, p<0.01). Bar 3 represents succinate transport in low extracellular calcium
vehicle control in which transport was increased compared to Bar 1. Bar 4 represents succinate
transport in low extracellular calcium with MDL 12,330A. Bars 3 and 4 show that apical addition
of MDL 12,330A decreased transport in low extracellular calcium as well (146 ± 6.4 to 88.7 ± 9.0 %
of control, p<0.01) thus suggesting that directly blocking adenylate cyclase may result in
dicarboxylate transport inhibition. This is in direct contrast to the evidence provided by the PTX
studies in which Gi inhibition had no effect on dicarboxylate transport.
As shown in Figure 12 Gi has been shown to inhibit adenylate cyclase and this results in
a decrease in cAMP production 38. Gs is an adenylate cyclase activator that increases cAMP 76.
Because of the opposing effects of Gi and Gs on adenylate cyclase, the membrane permeable 8Br-cAMP can be used to study both pathways simultaneously. In Figure 11 we show that
inhibiting Gi with PTX had no effect on dicarboxylate transport yet, directly inhibiting adenylate
cyclase with MDL 12,330A decreased dicarboxylate transport to an extent similar to that of
spermine via activation of the CaSR as is shown in Figures 5 and 13. Because of these varying
results, further studies on Gi signaling were needed to resolve the contrasting evidence from the
PTX and MDL 12,330A studies. If CaSR activation results in decreased dicarboxylate transport
38
in normal extracellular calcium via Gi inhibition of adenylate cyclase, then the addition of 8-BrcAMP would be expected to either reverse or counter this inhibition due to increased cAMP.
However, if CaSR activation decreases dicarboxylate transport in normal extracellular calcium via
Gs signaling and thus adenylate cyclase activation, this would indicate that increased cAMP
results in the decrease in dicarboxylate transport in normal extracellular calcium.
The results in Figure 14 show that 8-Br-cAMP had no effect on succinate transport in
either normal or low extracellular calcium. Bar 1 on the far left side is the vehicle control
representing succinate transport at 100% in normal extracellular calcium. Bar 2 represents
succinate transport in normal extracellular calcium plus 8-Br-cAMP in which transport was
unchanged in normal extracellular calcium (100 ± 0.00 to 94.7 ± 1.7 % of control, p=NS). Bar 3
represents succinate transport in low extracellular calcium in which transport was increased
compared to Bar1. Bar 4 represents succinate transport in low extracellular calcium plus cAMP.
Bars 3 and 4 show that apical addition of 8-Br-cAMP also had no effect in low extracellular
calcium (154 ± 9.9 to 131 ± 9.6 % of control, p=NS). These results indicate that Gi and Gs are not
involved in dicarboxylate transport regulation. Also of great importance, these results align with
those of PTX (Figure 11), an inhibitor of Gi which also had no effect on dicarboxylate transport,
and adds weight to the idea that the conflicting evidence provided by the MDL 12,330A
experiments are not the result of inhibiting adenylate cyclase, but some other mechanism not
involved with Gi signaling. There are many examples of secondary mechanisms and non specific
effects of MDL 12,330A. MDL 12,330A has been shown to block certain calcium channels in
endocrine cells 72 and also has nonspecific effects on glycine transport in certain retinal cells
independent of adenylate cyclase inhibition possibly due to toxic effects resulting in decreased
cell viability 71. The toxic effects mentioned could be the result of high levels of DMSO. MDL
12,330A can also inhibit phosphodiesterase which could mask its effect on adenylate cyclase
negating the specific effects intended by its use 73.
To investigate whether inhibition of dicarboxylate transport via MDL 12,330A is reversed
by 8-Br-cAMP as is shown in the flowchart in Figure 12, a different series of experiments was
39
performed where the control wells were treated with MDL 12,330A in both normal and low
extracellular calcium and experimental wells were treated with a combination of MDL 12,330A +
8-Br-cAMP as shown in Table 2.
As shown in Figure 15, all wells were treated with MDL 12,330A. Bar 1 on the far left
side is the vehicle control representing succinate transport at 100% in normal extracellular
calcium with MDL 12,330A. Bar 2 represents succinate transport in normal extracellular calcium
with MDL 12,330A + 8-Br-cAMP in which the addition of 8-Br-cAMP had no effect on transport
(100 ± 0.00 to 100 ± 5.1 % of control, p=NS). Bar 3 represents succinate transport in low
extracellular calcium vehicle control with MDL 12,330A. Bars 1 vs. 3 show that lowering
extracellular calcium had no significant effect on transport (100 ± 0.00 to 116 ± 9.2 % of control,
p=NS). Bar 4 represents succinate transport in low extracellular calcium with MDL 12,330A + 8Br-cAMP. Bars 3 vs. 4 show that 8-Br-cAMP did not reverse the effect of MDL 12,330A in low
extracellular calcium (116 ± 9.2 to 133 ± 8.7 % of control, p=NS). Bar 5 represents succinate
transport in low extracellular calcium with MDL 12,330A + 200 µM 8-Br-cAMP which is twice the
amount of 8-Br-cAMP used earlier. Bars 3 vs. 5 show that in low extracellular calcium 8-Br-cAMP
did not reverse the effects of MDL 12,330A even when the concentration of 8-Br-cAMP is doubled
(116 ± 9.2 to 130 ± 11.3 % of control, p=NS). This along with the previous 8-Br-cAMP studies
provides further evidence that MDL 12,330A is not inhibiting dicarboxylate transport by inhibiting
adenylate cyclase, but possibly through some other mechanism that does not involve Gi signaling.
So far, most evidence indicates that dicarboxylate transport in normal extracellular
calcium is regulated by CaSR activity with a bias toward Gq and not Gi or Gs. The only conflicting
evidence is that from the MDL 12,330A experiments shown in Figure 13. This has been
countered by evidence provided by PTX and 8-Br-cAMP shown in Figures 11,14 and 15; however
to be thorough and to further investigate Gs signaling, a series of experiments was performed to
measure cAMP levels in response to calcium and spermine activation of the CaSR.
40
Figure 12. Application of MDL 12,330A and 8-Br-cAMP to Mimic Gi and Gs
Flowchart depicting the CaSR, Gi, Gs and adenylate cyclase pathways investigated. Cells were
treated with MDL 12,330A to mimic Gi inhibition of adenylate cyclase in order to determine if Gi is
involved in extracellular calcium mediated dicarboxylate transport inhibition via the CaSR as
shown in Figure 13
As shown in Figure 14, cells were treated with 8-Br-cAMP to mimic Gs signaling or
counter Gi signaling in order to determine if Gi inhibits dicarboxylate transport via adenylate
cyclase inhibition or if Gs inhibits dicarboxylate transport via adenylate cyclase stimulation. Cells
were treated with MDL 12,330A + 8-Br-cAMP as shown in Figure 15. This was done to
determine if the effects of MDL 12,330A would be reversed by 8-Br-cAMP.
Intracellular cAMP ELISA concentration was measured with a series of cAMP ELISA
assays as shown in Figure 16. This was done to determine if Gi or Gs signaling occurs in
response to CaSR activation via extracellular calcium or spermine.
41
Figure 13. Adenylate Cyclase inhibition and Dicarboxylate Transport
Succinate transport was increased in low (<60 µM) compared to normal (1.2 mM) extracellular
calcium in vehicle controls (VC) shown in bars 1 and 3. 30 minute pretreatment of 50 µM MDL
12,330A resulted in reduced dicarboxylate transport in normal extracellular calcium (100 ± 0.00 to
53.7 ± 7.4 % of control) shown in bars 1 and 2. MDL 12,330A also inhibited transport in low
extracellular calcium (146 ± 6.4 to 88.7 ± 9.0 % of control) shown in bars 3 and 4. This evidence
is contrary to results of PTX shown in Figure 11 where Gi inhibitor had no effect on transport,
whereas mimicking Gi with MDL 12,330A as shown in this figure did affect transport. This may be
the result of secondary effects of MDL 12,330A unrelated to adenylate cyclase inhibition as was
described earlier. * Indicates p<0.01.
42
Figure 14. Effects of 8-Br-cAMP on Dicarboxylate Transport
Succinate transport was increased in low (<60 µM) compared to normal (1.2 mM) extracellular
calcium in vehicle controls (VC) shown in bars 1 and 3. 30 minute pretreatment of 100 µM 8-BrcAMP did not have an effect on succinate transport in normal extracellular calcium (100 ± 0.00 to
94.7 ± 1.7 % of control) shown in bars 1 and 2. 8-Br-cAMP also had no effect in low extracellular
calcium (154 ± 9.9 to 131 ± 9.6 % of control) shown in bars 3 and 4. This suggests that
dicarboxylate transport is not regulated by Gi or Gs. * Indicates p<0.01.
43
Figure 15. Dicarboxylate Transport and MDL 12,330A + 8-Br-cAMP Application
In this series of experiments MDL 12,330A is the control and is used to block adenylate cyclase
from increasing cAMP. When comparing these controls in normal and low extracellular calcium,
the calcium-sensitivity is not detected, i.e. there is no significant difference between succinate
transport in normal and low extracellular calcium (100 ± 0.00 to 116 ± 9.2 % of control) shown in
Bars 1 vs. 3. However, the MDL 12,330A blocking adenylate cyclase is not reversed by addition
of 8-Br-cAMP in normal extracellular calcium (100 ± 0.00 to 100 ± 5.1 % of control) shown in Bars
1 vs. 2. The effects of MDL 12,330A (blocking adenylate cyclase) were not reversed by the
addition of 8-Br-cAMP in low extracellular calcium (116 ± 9.2 to 133 ± 8.7 % of control) shown in
Bars 3 vs. 4, or in low extracellular calcium where 8-Br-cAMP concentrations were doubled to 200
µM (116 ± 9.2 to 130 ± 11.3 % of control) shown in Bars 3 vs. 5. This provides further evidence
that MDL 12,330A did not inhibit dicarboxylate transport via adenylate cyclase inhibition.
44
4b. Investigating the Effects of Spermine and Calcium on Intracellular cAMP Levels
Thus far the evidence provided herein by the CaSR-Gq signaling studies indicates that
the CaSR regulates dicarboxylate transport in normal extracellular calcium in OK proximal tubule
cell monolayers via Gq-PKC signaling. The results of Gi and Gs signaling studies indicate that the
CaSR does not regulate dicarboxylate transport via Gi or Gs signaling. To further verify this
conclusion a series of experiments was performed using ELISA assays to measure cAMP levels
in response to spermine (a CaSR activator) and extracellular calcium (the primary ligand of the
CaSR). Endogenously Gi is an inhibitor of adenylate cyclase and Gs is an activator. If the CaSR
in OK proximal tubule cells had a bias for Gi, CaSR activation would reduce cAMP levels due to
adenylate cyclase inhibition. If the CaSR had a bias for Gs, CaSR activation would increase
cAMP levels due to adenylate cyclase activation. Because the CaSR is known for ligand biased
signaling, the property of a receptor in which different ligands can activate different pathways, it is
important to test the effects of extracellular calcium and spermine on endogenous cAMP levels to
determine if they have a similar bias.
The results of these studies shown in Figure 16 demonstrate that normal extracellular
calcium had little effect on cAMP levels when compared to low extracellular calcium (0.3347 ±
0.1033 to 0.2716 ± 0.0949 pmol/ml, p=NS) as is shown in Bars 1 vs. 2 on the far left of the figure.
This indicates that activation of the CaSR in OK proximal tubule cells does not signal via the Gi or
Gs. The CaSR activator spermine also had little effect on cAMP levels in normal extracellular
calcium compared to normal extracellular calcium alone (0.3347 ± 0.1033 to 0.3541 ± 0.1364
pmol/ml, p=NS) as shown in Bars 1 vs. 3. This indicates that both spermine and extracellular
calcium likely activate the same G-protein signaling pathway of the CaSR. Forskolin is an
adenylate cyclase activator and is used as a control to increase intracellular cAMP. As was
expected, forskolin in normal extracellular calcium resulted in a large increase in cAMP levels
compared to normal extracellular calcium alone (0.3347 ± 0.1033 to 1.0081 ± 0.2279 pmol/ml,
p<0.05) as shown in Bars 1 vs. 4. These results indicate that CaSR activation does not result in
Gi or Gs signaling in OK proximal tubule cells.
45
Taken together, studies on Gq, Gi and Gs signaling pathways indicate that both normal
extracellular calcium and spermine activate the CaSR in OK proximal tubule cells. The results
shown in Figures 5,7,9,11,14,15 and 16 suggest that the bias is toward Gq signaling and that
dicarboxylate transport regulation in normal extracellular calcium in OK proximal tubule cells
occurs via the CaSR → Gq → PKC pathway.
46
Figure 16. Intracellular cAMP Levels
The adenylate cyclase activator forskolin (10 µM for 30 minutes) in normal extracellular calcium
(1.2 mM) increased endogenous intracellular cAMP compared to normal extracellular calcium
(0.3347 ± 0.1033 to 1.0081 ± 0.2279 pmol/ml) shown in Bars 4 vs. 1. However, there was no
significant change in intracellular cAMP levels between normal and low extracellular calcium (<60
µM) (0.3347 ± 0.1033 to 0.2716 ± 0.0949 pmol/ml) shown in Bars 1 vs. 2. The CaSR activator
spermine (1 mM for 5 minutes) in normal extracellular calcium had no significant effect on
intracellular cAMP levels compared to normal extracellular calcium control (0.3347 ± 0.1033 to
0.3541 ± 0.1364 pmol/ml) shown in Bars 3 vs. 1. This indicates that CaSR activation in OK cells
does not result in Gi or Gs signaling in a way that would affect adenylate cyclase activity. #
Indicates p<0.05.
47
Discussion
Citrate in urine is a potent inhibitor of nephrolithiasis and urinary citrate concentration is
determined by proximal tubule reabsorption 3,4. It makes sense that the lowering of extracellular
calcium results in increases in citrate transport at the apical membrane of the proximal tubule
would result because with less calcium in the lumen one would require less citrate to complex the
calcium and thus keep calcium soluble in the urine. It could be that if extracellular calcium did not
decrease reabsorption of dicarboxylates to the extent needed to solubilize calcium the result
would be increased stone formation.
Recently, there is some evidence that the CaSR is involved. For example, in many stone
forming patients single nucleotide polymorphisms (SNP) were found in the CaSR gene that result
in lower expression levels the CaSR 77. If the CaSR inhibits dicarboxylate transport, it could be
that SNPs that reduce CaSR expression would lead to increased citrate transport and stone
formation. To understand the cause and perhaps develop new treatments for stone formers with
low urinary citrate it is important that we have a firm understanding of how the dicarboxylate
transport processes in the proximal tubule function and what factors may interfere with how they
function. The non-calcium-sensitive NaDC1 cotransporter was thought to be responsible for all of
the citrate reabsorption on the apical side of the proximal tubule. However, previous studies in
our lab have detected a calcium-sensitive transport process that has not as yet been identified
and may play an important role in preventing stone formation by preventing citrate reabsorption in
response to increased luminal calcium concentration in the proximal tubule
15-17.
In this study, we
are attempting to elucidate the signal transduction pathways via the CaSR (which in the proximal
tubule is located on the apical membrane) that regulate dicarboxylate transport in OK proximal
tubule cells.
48
1. CaSR Activation and Calcium-Sensitive Dicarboxylate Transport
Hering-Smith et al have previously shown that when apical extracellular calcium is
reduced dicarboxylate transport is increased, thus extracellular calcium is an inhibitor of
dicarboxylate reabsorption in the proximal tubule 15-17. It is logical for a process such as this to
occur since when less calcium is present in the lumen less citrate would be required to solubilize
the calcium; thus increased reabsorption of dicarboxylate could be used inside proximal tubule
cells for metabolic purposes 8,9.
To determine the mechanism by which dicarboxylate transport inhibition takes place in
normal extracellular calcium the CaSR was investigated since it is also present in the apical
membrane of the proximal tubule and it regulates many different processes throughout the
nephron 48,55,78. The fact that the CaSR is expressed on the apical membrane in some regions
and the basolateral membrane in others suggests that systems are being regulated through
detection of calcium in both the lumen and the basolateral aspects of the nephron. The many
segment specific functions of the CaSR throughout the nephron have been widely studied
49,51-54.
One example is in the proximal tubule where the CaSR expressed on the apical membrane
regulates phosphate excretion 79. In the thick ascending limb, where much of the luminal calcium
is reabsorbed, the CaSR is expressed on the basolateral membrane. In this segment of the
nephron it is the increase in plasma calcium concentration that inhibits luminal reabsorption
52.
There is also evidence that the CaSR in juxtaglomerular cells inhibits renin secretion 80. Other
studies have suggested that the CaSR may inhibit stone formation through multiple functions in
different parts of the nephron. For example, Topala et al demonstrated that when luminal
calcium is increased in the distal convoluted tubule and connecting tubule the CaSR co-localizes
with TPRV5 49. CaSR mediated activation of this channel resulted in increased calcium
reabsorption. Another study by Renkema et al, demonstrated that even if the CaSR-TRPV5
system fails there are other compensatory mechanisms further downstream in the nephron 54. In
this study which investigated these compensatory mechanisms, TPRV5-/- mice did not form
stones. In these mice activation of the CaSR in the collecting duct resulted in H +-ATPase-
49
mediated H+ excretion acidifying the urine. Aquaporin 2 was also downregulated in the collecting
duct leading to polyuria. Both polyuria and acidification of the urine help prevent kidney stones.
In the same study, to further verify that the CaSR was preventing stone formation, a subunit of
the H+-ATPase was mutated to express inactive H+-ATPase in TPRV5-/- mice resulting in calcium
phosphate precipitation 54.
In this study we are investigating potential mechanisms in the proximal tubule that may
help prevent calcium stone formation via CaSR activation and subsequent inhibition of citrate
reabsorption from the lumen. As shown in Figure 5 the addition of the CaSR activator spermine
in low extracellular calcium resulted in inhibition of dicarboxylate transport similar to that which is
seen in normal extracellular calcium. This is an important observation because the activation of
the CaSR is restoring the dicarboxylate transport inhibition that is seen at normal extracellular
calcium. Since the addition of spermine in low extracellular calcium results in approximately the
same amount of transport inhibition in normal extracellular calcium alone demonstrates that the
CaSR is indeed responsible for dicarboxylate transport regulation. The CaSR is generally
inactive at calcium levels below 0.2 mM with activation thresholds that can vary from 0.5 to 2 mM
depending on cell type and levels of expression 32. As shown in Figure 5 (second bar from the
left) the addition of spermine in normal extracellular calcium resulted in further inhibition of
dicarboxylate transport. This may be a compounding effect in which spermine increases CaSR
activity beyond that which is observed in normal extracellular calcium (1.2 mM).
2. Increased Intracellular Calcium and Dicarboxylate Transport
The CaSR in the proximal tubule can be associated with the Gq, Gi or Gs proteins. The
G-protein through which it signals depends upon CaSR conformation 31,68,81. Perhaps this
conformation changes in response to changes in extracellular calcium concentration. As shown
in Figure 5 we have demonstrated using spermine that normal extracellular calcium may inhibit
dicarboxylate transport via CaSR activation. To further understand this process it is important to
examine the possible signal transduction pathways that are involved. Activation of G q causes the
50
activation of PLC resulting in increased intracellular calcium and PKC activation as is outlined in
the flowchart in Figure 2 59. In HEK293 cells transfected with human CaSR, activation of PLC
and subsequent activation of PKC is a Gq-dependent and Gi-independent process 46.
Thapsigargin is often used to increase intracellular calcium via inhibition of the Ca 2+-ATPase
responsible for pumping released intracellular calcium back into the endoplasmic reticulum.
Similar to the dicarboxylate transport processes we are currently studying, the Sodium-GABA
cotransporter is also inhibited by extracellular calcium
58.
This transporter was inhibited by
thapsigargin induced increase in intracellular calcium and possibly subsequent PKC activation 58.
Figure 7 shows that thapsigargin treatment results in dicarboxylate transport inhibition in both
normal and low extracellular calcium. These results, being similar to the spermine results in
Figure 5, provide evidence that dicarboxylate transport could be regulated by the CaSR via the
Gq pathway. As was described earlier it is Gq activation that results in increased intracellular
calcium followed by PKC activation and the CaSR has been thoroughly investigated due to its
propensity for ligand biased signaling 36,37,44-46,59. Though many GPCRs including the CaSR are
known to activate more than one G protein at a time, ligand biased signaling involves a strong
bias toward one pathway at the expense of other pathways that would be activated under
alternative circumstances such as exposure to other ligands, polarization (apical or basolateral),
renal segment such as proximal tubule, thick ascending limb, connecting tubule, collecting duct
and intracellular and extracellular conditions such as calcium concentration and pH. The CaSR
activates various G protein pathways such as Gq and Gi preferentially throughout different
segments of the nephron. The importance of this is evident when considering the multitude of
functions described earlier. It is imperative that calcium concentrations are detected and
regulated in the lumen of the nephron along with interstitial regions and plasma. It was initially
presumed that the sole purpose of the CaSR is to maintain plasma calcium homeostasis via PTH
regulation. Bone tissue is used as a storage system in which calcium can be withdrawn to
maintain adequate plasma concentration. Increased plasma calcium concentration increases
CaSR activity resulting in PTH downregulation. Since the discovery of the CaSR many additional
functions have been discovered, many of which are independent of PTH activity and do not
51
necessarily involve plasma calcium homeostasis. As was described above the CaSR prevents
calcium stone formation via segment specific functions throughout the nephron, possibly due to
the utilization of various G protein pathways in which the ligand biased signaling of the CaSR
changes as a result of nephron segment location and polarization (apical or basolateral)
78.
It is
clear that mechanisms in other segments of the nephron prevent stone formation via CaSR
activation 49,54. However, in the proximal tubule such mechanisms have not been discovered. In
the current study we provide evidence that there is an additional mechanism in the proximal
tubule in which the CaSR could prevent calcium stone formation via dicarboxylate transport
inhibition. The effects of thapsigargin in normal extracellular calcium shown in Figure 7 indicate
that this occurs via Gq signaling. The PKC activator PMA was used to further investigate the Gq
pathway and dicarboxylate transport regulation. We found that PKC activation resulted in
decreased succinate transport in low extracellular calcium only, indicating that this signaling
pathway regulates the dicarboxylate transport process specifically.
3. PKC Activation and Calcium-Sensitive Dicarboxylate Transport
In 1999 Pajor inhibited succinate transport by 95 % in Xenopus oocytes expressing rabbit
NaDC1 in response to PKC activators such as PMA 66. PKA activation had no effect on transport.
Mutating two consensus phosphorylation sites in NaDC1 did not prevent inhibition caused by
PMA. Cytochalasin D prevented some transport inhibition likely due to disruption of
microfilaments and endocytosis. Although succinate transport was inhibited up to 95 % only 30 %
of NaDC1 was internalized 66. This indicated that succinate transport was being inhibited by
endocytosis and reduced transporter activity in rabbit NaDC1. Succinate transport in oocytes
expressing opossum NaDC1, studied in low extracellular calcium, was significantly reduced 16. In
OK cells, low extracellular calcium increases succinate transport indicating a transport process
that is not likely mediated by NaDC1. We have been investigating the potential pathways
involved in dicarboxylate transport inhibition in response to CaSR activation. Specifically, we
have been focusing on the Gq pathway (Figure 2). As was explained earlier, activation of the Gq
52
pathway results in a rise in intracellular calcium and subsequent activation of PKC
59.
Using
spermine and thapsigargin as is shown in Figures 5 and 7 we provided evidence that both
(spermine used to activate the CaSR) as well as thapsigargin (commonly used to increase
intracellular calcium) resulted in dicarboxylate transport inhibition. In the results shown in Figure
9 OK cells treated with the PKC activator PMA resulted in a significant decrease in dicarboxylate
transport in low extracellular calcium only. This indicates that PKC activation restores the
inhibitory effect normal extracellular calcium (1.2 mM) has on dicarboxylate transport. Taken
together, we can conclude from the results of spermine, thapsigargin and PMA experiments
(Figures 5, 7, and 9) that dicarboxylate transport inhibition normal extracellular calcium occurs via
the CaSR → Gq → PKC pathway outlined in Figure 2.
4. Ligand Biased Signaling, the Gi and Gs Pathways, and Dicarboxylate Transport
Regulation
Ligand biased signaling in GPCRs is a relatively recent model in which different ligands
stabilize specific conformations that lead to a preference toward the activation of one pathway in
favor of another 42. We have demonstrated with the transport experiments shown in Figures 5, 7
and 9 that dicarboxylate transport inhibition in OK proximal tubule cells occurs via the CaSR →
Gq → PKC pathway as is outlined in Figure 2. Aside from Gq, CaSR activation is also known to
activate the Gi or Gs pathways which could result in changes in cAMP levels via adenylate
cyclase inhibition or activation, respectively 38,39,76.
To investigate whether the Gi or Gs pathways are involved in dicarboxylate transport
regulation, we began by using the Gi inhibitor PTX. If dicarboxylate transport inhibition via CaSR
activation were the result of Gi signaling it would be expected that Gi inhibition would increase
transport in normal extracellular calcium. Figure 11 shows that the addition of PTX had no effect
on dicarboxylate transport in normal and low calcium conditions. This indicates that G i is not
likely involved in dicarboxylate transport regulation. These results are somewhat contradicted by
the results shown in Figure 13. Using MDL 12,330A to inhibit adenylate cyclase as a way of
53
mimicking Gi signaling reduced transport in both normal and low extracellular calcium. This is
what would be expected if dicarboxylate transport was being inhibited by the CaSR via G i
activation. It is possible that MDL 12,330A may likely be inhibiting transport in some other way
that does not involve adenylate cyclase inhibition. Previous studies by Gadea et al indicate that
MDL 12,330A has secondary effects unrelated to adenylate cyclase inhibition
71.
It has been
shown to block certain calcium channels in endocrine cells and also has nonspecific effects on
glycine transport in certain retinal cells independent of adenylate cyclase possibly due to toxic
effects resulting in decreased cell viability 71,72. MDL 12,330A can also inhibit phosphodiesterase
which could mask its effect on adenylate cyclase by preventing the degradation of intracellular
cAMP 73.
If MDL 12,330A used to mimic Gi is inhibiting transport by decreasing cAMP levels via
adenylate cyclase inhibition, then the addition of 8-Br-cAMP should reverse these effects.
However, as is shown in Figure 15 this did not happen. Also If Gi inhibits transport by reducing
cAMP levels then addition of 8-Br-cAMP would be expected to increase transport; however,
Figure 14 shows that 8-Br-cAMP by itself had no effect on dicarboxylate transport. Since 8-BrcAMP used alone had little effect on dicarboxylate transport this indicates that there is no
involvement of either Gi or Gs as both G-proteins are inhibitors and activators of adenylate
cyclase respectively. The MDL 12,330A results in Figure 13 indicate that dicarboxylate transport
may be regulated by Gi. However the secondary effects of MDL 12,330A found in other studies
as well as the PTX (Figure 11) and 8-Br-cAMP (Figures 14 and 15) results in the current study
provide evidence to the contrary. Due to this conflicting evidence further investigation was
necessary.
To resolve the conflicting evidence presented by the MDL 12,330A studies and to
demonstrate that the CaSR is not activating Gi or Gs in OK proximal tubule cells, a final set of
experiments were performed. cAMP ELISA assays were used to measure cAMP levels in
response to various pharmacological treatments in OK proximal tubule cells. If CaSR activation
resulted in Gi activation, there would be a reduction in intracellular cAMP levels and if G s is
54
activated, there would be an increase in intracellular cAMP since they inhibit and activate
adenylate cyclase respectively. As shown in Figure 16 there was little change in cAMP levels
when comparing normal and low extracellular calcium indicating that extracellular calcium does
not activate Gi or Gs via the CaSR. The CaSR activator spermine also had no effect on cAMP
levels. This along with the PTX and 8-Br-cAMP studies is sufficient evidence to conclude that
CaSR activation in OK proximal tubule cells does not result in G i or Gs signaling. In the previous
sections it was concluded that CaSR activation inhibits dicarboxylate transport via Gq signaling.
All of the evidence together suggests that both normal extracellular calcium and spermine bias
the CaSR toward Gq at the expense of Gi and Gs in OK proximal tubule cells. Previous studies in
this lab have demonstrated that there is a calcium-sensitive dicarboxylate transport process in OK
proximal tubule cells that is likely not occurring via NaDC1. The current study has elucidated the
CaSR → Gq → PKC signal transduction pathway that regulates apical proximal tubule
dicarboxylate transport.
55
Conclusion
Citrate in urine is a potent endogenous inhibitor of nephrolithiasis and urinary citrate
concentration is determined by proximal tubule reabsorption
3,4.
The non-calcium-sensitive
NaDC1 transporter was originally thought to be responsible for all of the citrate transport on the
apical side of the proximal tubule. However, previous studies in this lab have detected a calciumsensitive transport process that has not been identified and may play an important role in stone
formation in patients with low urinary citrate 15-17. Other studies have demonstrated that calcium
stone formation may be prevented through multiple mechanisms in separate segments of the
nephron regulated by the CaSR 49,54. Thus herein we are investigating a calcium-sensitive
transport process in the proximal tubule in which normal levels of luminal calcium decreases
dicarboxylate reabsorption and may be yet another mechanism in which the CaSR helps to
prevent kidney stones. The data are clear that normal extracellular calcium (1.2 mM) and
spermine (both of which result in CaSR activation), G q signaling, thapsigargin (commonly used to
increase intracellular calcium), PMA (PKC activator) and reduced apical dicarboxylate transport
are linked. The cAMP assays, 8-Br-cAMP and PTX data all suggest that normal extracellular
calcium and the subsequent activation of the CaSR does not result in either Gi or Gs signaling in
OK proximal tubule cells. However, the MDL 12,330A data suggests that lowering cAMP may
alter dicarboxylate transport. Since 8-Br-cAMP did not significantly reverse the effects of MDL
12,330A, this suggests that MDL 12,330A may be altering dicarboxylate transport through some
other mechanism entirely. This is quite possible based on evidence of secondary effects of MDL
12,330A. Whether dicarboxylate transport is being regulated through phosphorylation,
internalization or some other mechanism cannot now be known. We have however provided
evidence that dicarboxylate transport inhibition in normal extracellular calcium is occurring via the
CaSR → Gq → PKC pathway and is independent of Gi or Gs signaling.
56
Figure 17. The CaSR, Gq, Gi and Gs Signaling Cascades Investigated
An overview of G-protein pathways studied and pharmacologicals used in our investigation to
determine the role extracellular calcium plays apical dicarboxylate transport regulation in the
proximal tubule. Time of treatment, concentration and vehicle controls are outlined in Table 2
57
List of References
1.
Brennan, T.S., Klahr, S. & Hamm, L.L. Citrate transport in rabbit nephron. Am J Physiol
251, F683-689 (1986).
2.
Grollman, A.P., Walker, W.G., Harrison, H.C. & Harrison, H.E. Site of Reabsorption of
Citrate and Calcium in the Renal Tubule of the Dog. Am J Physiol 205, 697-701 (1963).
3.
Brennan, S., Hering-Smith, K. & Hamm, L.L. Effect of pH on citrate reabsorption in the
proximal convoluted tubule. Am J Physiol 255, F301-306 (1988).
4.
Hamm, L.L. & Hering-Smith, K.S. Pathophysiology of hypocitraturic nephrolithiasis.
Endocrinol Metab Clin North Am 31, 885-893, viii (2002).
5.
Pajor, A.M. Sodium-coupled transporters for Krebs cycle intermediates. Annu Rev
Physiol 61, 663-682 (1999).
6.
Bai, X.Y., et al. Membrane topology structure of human high-affinity, sodium-dependent
dicarboxylate transporter. FASEB J 21, 2409-2417 (2007).
7.
Pajor, A.M. Citrate transport by the kidney and intestine. Semin Nephrol 19, 195-200
(1999).
8.
Simpson, D.P. Citrate excretion: a window on renal metabolism. Am J Physiol 244, F223234 (1983).
9.
Hamm, L.L. Renal handling of citrate. Kidney Int 38, 728-735 (1990).
10.
Baruch, S.B., Burich, R.L., Eun, C.K. & King, V.F. Renal metabolism of citrate. Med Clin
North Am 59, 569-582 (1975).
11.
Law, D., Hering-Smith, K.S. & Hamm, L.L. Citrate transport in proximal cell line. Am J
Physiol 263, C220-225 (1992).
12.
Chen, X.Z., Shayakul, C., Berger, U.V., Tian, W. & Hediger, M.A. Characterization of a
rat Na+-dicarboxylate cotransporter. J Biol Chem 273, 20972-20981 (1998).
13.
Sekine, T., et al. Cloning, functional characterization, and localization of a rat renal Na+dicarboxylate transporter. Am J Physiol 275, F298-305 (1998).
14.
Bai, X., et al. Identification of basolateral membrane targeting signal of human sodiumdependent dicarboxylate transporter 3. J Cell Physiol 206, 821-830 (2006).
58
15.
Hering-Smith, K.S., Gambala, C.T. & Hamm, L.L. Citrate and succinate transport in
proximal tubule cells. Am J Physiol Renal Physiol 278, F492-498 (2000).
16.
Hering-Smith, K.S., Schiro, F.R., Pajor, A.M. & Hamm, L.L. Calcium sensitivity of
dicarboxylate transport in cultured proximal tubule cells. Am J Physiol Renal Physiol 300,
F425-432 (2011).
17.
Hering-Smith, K.S., et al. Localization of the calcium-regulated citrate transport process in
proximal tubule cells. Urolithiasis 42, 209-219 (2014).
18.
Ringer, S. A further Contribution regarding the influence of the different Constituents of
the Blood on the Contraction of the Heart. J Physiol 4, 29-42.23 (1883).
19.
Brown, E.M. & MacLeod, R.J. Extracellular calcium sensing and extracellular calcium
signaling. Physiol Rev 81, 239-297 (2001).
20.
Sherwood, L.M., Potts, J.T., Care, A.D., Mayer, G.P. & Aurbach, G.D. Evaluation by
radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature
209, 52-55 (1966).
21.
Kifor, O. & Brown, E.M. Relationship between diacylglycerol levels and extracellular
Ca2+ in dispersed bovine parathyroid cells. Endocrinology 123, 2723-2729 (1988).
22.
Shoback, D.M., Membreno, L.A. & McGhee, J.G. High calcium and other divalent cations
increase inositol trisphosphate in bovine parathyroid cells. Endocrinology 123, 382-389
(1988).
23.
Nemeth, E.F. & Scarpa, A. Rapid mobilization of cellular Ca2+ in bovine parathyroid cells
evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor. J
Biol Chem 262, 5188-5196 (1987).
24.
Nemeth, E.F. & Carafoli, E. The role of extracellular calcium in the regulation of
intracellular calcium and cell function. Cell Calcium 11, 319-321 (1990).
25.
Brown, E.M., et al. Cloning and characterization of an extracellular Ca(2+)-sensing
receptor from bovine parathyroid. Nature 366, 575-580 (1993).
59
26.
Aida, K., Koishi, S., Tawata, M. & Onaya, T. Molecular cloning of a putative Ca(2+)sensing receptor cDNA from human kidney. Biochem Biophys Res Commun 214, 524529 (1995).
27.
Bai, M., Trivedi, S. & Brown, E.M. Dimerization of the extracellular calcium-sensing
receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem 273,
23605-23610 (1998).
28.
Ray, K., et al. Identification of the cysteine residues in the amino-terminal extracellular
domain of the human Ca(2+) receptor critical for dimerization. Implications for function of
monomeric Ca(2+) receptor. J Biol Chem 274, 27642-27650 (1999).
29.
Silve, C., et al. Delineating a Ca2+ binding pocket within the venus flytrap module of the
human calcium-sensing receptor. J Biol Chem 280, 37917-37923 (2005).
30.
Hu, J. & Spiegel, A.M. Structure and function of the human calcium-sensing receptor:
insights from natural and engineered mutations and allosteric modulators. J Cell Mol Med
11, 908-922 (2007).
31.
Chang, W. & Shoback, D. Extracellular Ca2+-sensing receptors--an overview. Cell
Calcium 35, 183-196 (2004).
32.
Conigrave, A.D. & Ward, D.T. Calcium-sensing receptor (CaSR): pharmacological
properties and signaling pathways. Best Pract Res Clin Endocrinol Metab 27, 315-331
(2013).
33.
Samama, P., Cotecchia, S., Costa, T. & Lefkowitz, R.J. A mutation-induced activated
state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol
Chem 268, 4625-4636 (1993).
34.
Gether, U., Lin, S. & Kobilka, B.K. Fluorescent labeling of purified beta 2 adrenergic
receptor. Evidence for ligand-specific conformational changes. J Biol Chem 270, 2826828275 (1995).
35.
Cordeaux, Y., Ijzerman, A.P. & Hill, S.J. Coupling of the human A1 adenosine receptor to
different heterotrimeric G proteins: evidence for agonist-specific G protein activation. Br J
Pharmacol 143, 705-714 (2004).
60
36.
Davey, A.E., et al. Positive and negative allosteric modulators promote biased signaling
at the calcium-sensing receptor. Endocrinology 153, 1232-1241 (2012).
37.
Thomsen, A.R., et al. Strontium is a biased agonist of the calcium-sensing receptor in rat
medullary thyroid carcinoma 6-23 cells. J Pharmacol Exp Ther 343, 638-649 (2012).
38.
Hofer, A.M. & Brown, E.M. Extracellular calcium sensing and signalling. Nat Rev Mol Cell
Biol 4, 530-538 (2003).
39.
Hernández-Bedolla, M.A., et al. Calcium-sensing-receptor (CaSR) controls IL-6 secretion
in metastatic breast cancer MDA-MB-231 cells by a dual mechanism revealed by agonist
and inverse-agonist modulators. Mol Cell Endocrinol 436, 159-168 (2016).
40.
Zhu, Y., et al. Ablation of the Stimulatory G Protein α-Subunit in Renal Proximal Tubules
Leads to Parathyroid Hormone-Resistance With Increased Renal Cyp24a1 mRNA
Abundance and Reduced Serum 1,25-Dihydroxyvitamin D. Endocrinology 157, 497-507
(2016).
41.
Rangel, L.B., et al. PI-PLCbeta is involved in the modulation of the proximal tubule Na+ATPase by angiotensin II. Regul Pept 127, 177-182 (2005).
42.
Jensen, A.A. & Bräuner-Osborne, H. Allosteric modulation of the calcium-sensing
receptor. Curr Neuropharmacol 5, 180-186 (2007).
43.
Tateyama, M. & Kubo, Y. Dual signaling is differentially activated by different active
states of the metabotropic glutamate receptor 1alpha. Proc Natl Acad Sci U S A 103,
1124-1128 (2006).
44.
Thomsen, A.R., Hvidtfeldt, M. & Bräuner-Osborne, H. Biased agonism of the calciumsensing receptor. Cell Calcium 51, 107-116 (2012).
45.
Wu, D.Q., Lee, C.H., Rhee, S.G. & Simon, M.I. Activation of phospholipase C by the
alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells. J Biol Chem 267,
1811-1817 (1992).
46.
Kifor, O., et al. Regulation of MAP kinase by calcium-sensing receptor in bovine
parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 280, F291302 (2001).
61
47.
Abdullah, H.I., Pedraza, P.L., McGiff, J.C. & Ferreri, N.R. CaR activation increases TNF
production by mTAL cells via a Gi-dependent mechanism. Am J Physiol Renal Physiol
294, F345-354 (2008).
48.
Riccardi, D., et al. Localization of the extracellular Ca2+/polyvalent cation-sensing protein
in rat kidney. Am J Physiol 274, F611-622 (1998).
49.
Topala, C.N., et al. Activation of the Ca2+-sensing receptor stimulates the activity of the
epithelial Ca2+ channel TRPV5. Cell Calcium 45, 331-339 (2009).
50.
Sands, J.M., et al. Apical extracellular calcium/polyvalent cation-sensing receptor
regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting
duct. J Clin Invest 99, 1399-1405 (1997).
51.
Egbuna, O., et al. The full-length calcium-sensing receptor dampens the calcemic
response to 1alpha,25(OH)2 vitamin D3 in vivo independently of parathyroid hormone.
Am J Physiol Renal Physiol 297, F720-728 (2009).
52.
Quamme, G.A. Effect of hypercalcemia on renal tubular handling of calcium and
magnesium. Can J Physiol Pharmacol 60, 1275-1280 (1982).
53.
Wang, W.H., Lu, M. & Hebert, S.C. Cytochrome P-450 metabolites mediate extracellular
Ca(2+)-induced inhibition of apical K+ channels in the TAL. Am J Physiol 271, C103-111
(1996).
54.
Renkema, K.Y., et al. The calcium-sensing receptor promotes urinary acidification to
prevent nephrolithiasis. J Am Soc Nephrol 20, 1705-1713 (2009).
55.
Riccardi, D. & Brown, E.M. Physiology and pathophysiology of the calcium-sensing
receptor in the kidney. Am J Physiol Renal Physiol 298, F485-499 (2010).
56.
Hebert, S.C. Therapeutic use of calcimimetics. Annu Rev Med 57, 349-364 (2006).
57.
Petrel, C., et al. Positive and negative allosteric modulators of the Ca2+-sensing receptor
interact within overlapping but not identical binding sites in the transmembrane domain. J
Biol Chem 279, 18990-18997 (2004).
58.
Kempson, S.A., Edwards, J.M. & Sturek, M. Inhibition of the renal betaine transporter by
calcium ions. Am J Physiol Renal Physiol 291, F305-313 (2006).
62
59.
Wang, D., McGiff, J.C. & Ferreri, N.R. Regulation of cyclooxygenase isoforms in the renal
thick ascending limb: effects of extracellular calcium. J Physiol Pharmacol 51, 587-595
(2000).
60.
Ohkura, K., Hori, H. & Terada, H. Effect of extracellular calcium on the intracellular
calcium level of newborn rat skin basal cells. Biol Pharm Bull 21, 1-4 (1998).
61.
Chen, C.J., Anast, C.S., Posillico, J.T. & Brown, E.M. Effects of extracellular calcium and
magnesium on cytosolic calcium concentration in Fura-2-loaded bovine parathyroid cells.
J Bone Miner Res 2, 319-327 (1987).
62.
Beckman, M.L., Bernstein, E.M. & Quick, M.W. Multiple G protein-coupled receptors
initiate protein kinase C redistribution of GABA transporters in hippocampal neurons. J
Neurosci 19, RC9 (1999).
63.
Forster, I.C., et al. Protein kinase C activators induce membrane retrieval of type II Na+phosphate cotransporters expressed in Xenopus oocytes. J Physiol 517 ( Pt 2), 327-340
(1999).
64.
Hagos, Y., et al. Regulation of sodium-dicarboxylate cotransporter-3 from winter flounder
kidney by protein kinase C. Am J Physiol Renal Physiol 286, F86-93 (2004).
65.
Wolff, N.A., et al. Protein kinase C activation downregulates human organic anion
transporter 1-mediated transport through carrier internalization. J Am Soc Nephrol 14,
1959-1968 (2003).
66.
Pajor, A.M. & Sun, N. Protein kinase C-mediated regulation of the renal
Na(+)/dicarboxylate cotransporter, NaDC-1. Biochim Biophys Acta 1420, 223-230 (1999).
67.
Sunahara, R.K. & Taussig, R. Isoforms of mammalian adenylyl cyclase: multiplicities of
signaling. Mol Interv 2, 168-184 (2002).
68.
Brennan, S.C., et al. Calcium sensing receptor signalling in physiology and cancer.
Biochim Biophys Acta 1833, 1732-1744 (2013).
69.
Harriman, J.F., Liu, X.L., Aleo, M.D., Machaca, K. & Schnellmann, R.G. Endoplasmic
reticulum Ca(2+) signaling and calpains mediate renal cell death. Cell Death Differ 9,
734-741 (2002).
63
70.
Emery, A.C., Eiden, M.V. & Eiden, L.E. A new site and mechanism of action for the
widely used adenylate cyclase inhibitor SQ22,536. Mol Pharmacol 83, 95-105 (2013).
71.
Gadea, A., López, E. & López-Colomé, A.M. The adenylate cyclase inhibitor MDL12330A has a non-specific effect on glycine transport in Müller cells from the retina. Brain
Res 838, 200-204 (1999).
72.
Rampe, D., Triggle, D.J. & Brown, A.M. Electrophysiologic and biochemical studies on
the putative Ca++ channel blocker MDL 12,330A in an endocrine cell. J Pharmacol Exp
Ther 243, 402-407 (1987).
73.
Lippe, C. & Ardizzone, C. Actions of vasopressin and isoprenaline on the ionic transport
across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors
MDL12330A and SQ22536. Comp Biochem Physiol C 99, 209-211 (1991).
74.
Yamaguchi, T., et al. Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from
autosomal-dominant polycystic kidneys. Kidney Int 63, 1983-1994 (2003).
75.
Siebler, T., et al. Pertussis toxin sensitive G-proteins are not involved in the mitogenic
signaling pathway of insulin-like growth factor-I in normal rat kidney epithelial (NRKE)
cells. Regul Pept 62, 65-71 (1996).
76.
Nasman, J., Kukkonen, J.P., Ammoun, S. & Akerman, K.E. Role of G-protein availability
in differential signaling by alpha 2-adrenoceptors. Biochem Pharmacol 62, 913-922
(2001).
77.
Vezzoli, G., Terranegra, A. & Soldati, L. Calcium-sensing receptor gene polymorphisms
in patients with calcium nephrolithiasis. Curr Opin Nephrol Hypertens 21, 355-361 (2012).
78.
Magno, A.L., Ward, B.K. & Ratajczak, T. The calcium-sensing receptor: a molecular
perspective. Endocr Rev 32, 3-30 (2011).
79.
Ba, J., Brown, D. & Friedman, P.A. Calcium-sensing receptor regulation of PTHinhibitable proximal tubule phosphate transport. Am J Physiol Renal Physiol 285, F12331243 (2003).
64
80.
Ortiz-Capisano, M.C., Liao, T.D., Ortiz, P.A. & Beierwaltes, W.H. Calcium-dependent
phosphodiesterase 1C inhibits renin release from isolated juxtaglomerular cells. Am J
Physiol Regul Integr Comp Physiol 297, R1469-1476 (2009).
81.
Kenakin, T. Functional selectivity and biased receptor signaling. J Pharmacol Exp Ther
336, 296-302 (2011).
65
Biography
Ryan Walker was born in Forest Grove Oregon 1979. He put himself through his first years of
school while working as a construction worker in Texas. After earning his associates degree in
science at San Antonio College he earned a fully paid scholarship from Tulane University based
on academic achievement. While working on his bachelor degree in cell and molecular biology
he began working for Dr. Radhika Pochampally at the Tulane Center for Gene Therapy. While
there he had two publications. One was the characterization of several different osteosarcoma
cell lines harvested from amputations provided by Ochsner hospital and the other involved the
encapsulation of curcumin within cyclodextrins within liposomes to increase bioavailability of a
known anti inflammatory substance with the potential of being used as an adjuvant in cancer
treatment. After a year working in gene therapy he joined the PhD. program in Biomedical
Science at Tulane medical school. He left his lab in gene therapy and began work in the
Physiology Department at Tulane University School of Medicine where he studied calciumsensitive dicarboxylate transport and the CaSR under the guidance of his mentor Dr. Kathleen
Hering-Smith.