AMER. ZOOL., 35:483-489 (1995)
Transcaltachia (the Rapid Hormonal Stimulation of Intestinal Calcium
Transport): A Component of Adaptation to Calcium Needs and
Calcium Availability1
ANTHONY W. NORMAN
Department of Biochemistry, and
Division of Biomedical Sciences, University of California, Riverside, California 92521
SYNOPSIS. Calcium is one of the key elements required for maintenance
and survival of life in animals with both endo- and exoskeletons. Because
there is a wide variation in dietary calcium (dependent upon the local
habitat) and dietary practices, and because there is a changing physiological need throughout life {e.g., for mammals, during growth, puberty,
pregnancy, lactation, and menopause), it is essential that the process of
intestinal calcium absorption be adaptable and responsive to both the
dietary and physiological circumstances. This article reviews the evidence
that transcaltachia, or the rapid stimulation of intestinal Ca2+ transport
by the steroid hormone, la,25(OH)2-vitamin D 3 , as studied in the chicken,
meets many of the objectives of an adaptive intestinal calcium transport
process. Transcaltachia is studied in a perfused chick duodenum, where
45
Ca2+ is placed in the lumen and potential agonists are perfused into the
celiac artery; the transcaltachic reponse represents the stimulation within
4-8 min of the transfer of 45Ca2+ from the lumen to the vascular perfusate.
la,25(OH)2-vitamin D 3 stimulation of transcaltachia occurs via nongenomic mechanisms which involve a plasma membrane receptor for the
secosteroid and the coupled opening of voltage-gated Ca2+ channels on
the basal lateral membrane of the intestinal epithelial cell and the activation of the second messengers, protein kinase C and cAMP.
INTRODUCTION
Calcium is one of the key elements
required for maintenance and survival of
all animals with either an exoskeleton or
endoskeleton. The bodily calcium content
of a 70 kg adult human will range from 1,200
to 1,500 g and proportionate amounts of
calcium are present in other animals with
an endoskeleton (Hegsted et al, 1963). The
vast majority (99%) of the calcium is associated with the endoskeleton, but there are
significant quantities associated with the
intracellular and extracellular fluid compartments (Norman, 1979). The ability of
a calcium-requiring organism to survive may
depend upon its ability to devise an efficient
' From the Symposium on Comparative Gastroin-
^ ^ ^ X S ^ t ^ ^ o S
adaptive mechanism for Ca2+ absorption
(Malm, 1958). In most animals, the path of
Ca2+
entrance is through the intestine but
in
saltwater fish a significant proportion of
Ca2+ ma b e
y absorbed across the gills (Sund e l 1 et al
- * 992 >- Y e t m t h e circumstances
of excess dietary Ca 2+ , it is not appropriate
t 0 carr
y out an unrestrained absorption of
t h e Ca2+
> a s t h l s m a y l e a d t o hypercalcemia
a n d lts
associated adverse physiological
problems, including soft tissue Ca2+ depoSltion a n d
formation of kidney stones,
Accordingly, most animals have devised a
Process of calcium absorption which can be
either
up-regulated or down-regulated to suit
their
immediate physiological and environmental circumstances (Nordin, 1988; Sundell et al, 1991).
The prime determinant of the changing
Ca2+ re q U i re ment, is the process of bodily
?*d skeletal ^owth. As discussed by Garn
of Zoologists, 27-30 December 1992, at Vancouver, (Gam, 1970),formanfrombirth to the onset
British Columbia, Canada.
of puberty there must be an average net
483
484
ANTHONY W. NORMAN
retention of approximately 100 mg of calcium/day; during puberty the daily net
retention increases to 200 mg/day for
females and 280 mg/day for males. Finally,
for adults the average net retention decreases
to 10-30 mg/day. To further illustrate the
importance of adaptation of intestinal Ca2+
absorption, Figure 1 reports nutritional balance studies of the relationship between
dietary intake of calcium and the extent of
intestinal Ca 2+ absorption in a population
of normal humans (Nordin, 1988). It is
apparent that the process of intestinal Ca2+
absorption is saturable and that there is an
upper limit to the amount of calcium that
may be absorbed even in the face of very
high dietary calcium intakes. It can be
extrapolated that an adult individual will
likely require daily dietary access to 400500 mg calcium to be able to effect a net
absorption of 100-150 mg Ca 2+ /day. In
normal adults it is essential that the daily
net absorption be this finite value even
though there is no net change in the skeletal
content of calcium; the 100-150 mg of net
calcium absorbed will all be excreted under
normal physiological circumstances in the
urine or sweat. Also reviewed in Figure 1 is
an indication of how the net calcium
absorption amounts must change during
puberty, pregnancy (where there is a dramatic increase), lactation and menopause
(where there is a modest increase). These
periods of increased and decreased calcium
needs imply the existence of an adaptive
intestinal Ca 2+ absorption mechanism that
can be modulated to provide the calcium
required by the organism to meet its current
physiological processes.
It is the thesis of the author that the prime
regulation of the process of intestinal calcium absorption is based on the vitamin D
endocrine system (see Fig. 2) and the
sequential metabolism by the liver and the
kidney of the parent vitamin D 3 into its hormonally active forms, 1 a, 25 -dihydroxyvitamin D 3 [la,25(OH)2D3] and 24R,25-dihydroxyvitamin D 3 [24R,25(OH) 2 D 3 ]
(Norman, 1987;Cancela#a/., 1988; Nemere and Norman, 1991). The kidney is the
central endocrine gland that produces in
regulated quantities the two key dihydroxylated metabolites, la,25(OH) 2 D 3 and
MT»KE (ms/<U>)
FIG. 1. Relationship of dietary calcium intake to net
calcium absorption as determined by calcium balance
studies in normal adults. The changes in the required
net calcium absorbed is indicated for a variety of physiological states. (Modified from Nordin, 1988.)
24R,25(OH)2D3. Both the 25(OH)D3-1hydroxylase and 25(OH)D3-24-hydroxylase are separate cytochrome P-450 enzymes
found in the mitochondria of the proximal
tubule of the kidney (Henry, 1992). It is
essential to appreciate that all vertebrate
classes have been shown to have a functional 25(OH)D3-1 -hydroxylase (Henry and
Norman, 1975), which is consistent with the
author's contention that la,25(OH)2D3 is
an essential regulator of the process of adaptive intestinal Ca2+ absorption.
Another key component of the vitamin
D endocrine system is the presence of recepENDOCRINE
S Y S T E M OF V I T A M I N
! INTESTINE")
1
'
D ACTION
*• Absorption of
CotP
.Mobilization of
Co + P
,. Reobsorption of
Ca+P
FIG. 2. Summary of the vitamin D endocrine system
as pertains to calcium homeostasis and the regulation
of intestinal Ca2+ absorption. For a more detailed discussion of the complete vitamin D endocrine system
see the references by Minghetti and Norman (1988)
and Reichel et al., (1989).
la,25(OH)2D3 MEDIATED TRANSCALTACHIA
tors for la,25(OH)2D3 in target tissues
(Minghetti and Norman, 1988). Receptors
for la,25(OH)2D3 have been localized in the
intestine, kidney, and bone, the three classical target organs of vitamin D action. In
the past decade, the la,25(OH) 2 D 3 receptor
has also been found in many other tissues
in vertebrate organisms, including numerous cancer cell lines, pituitary, skin, hematopoietic cells, pancreas, and brain (Minghetti and Norman, 1988; Reichel et al,
1989). Both the avian and human
la,25(OH)2D3 receptors have been cloned
and shown to be members of the nuclear
transacting receptor family that includes
estrogen, progesterone, glucocorticoid, thyroxine, aldosterone, and retinoic acid receptors (Lowe et al, 1992).
In the intestine there are two fundamental
mechanisms by which la,25(OH) 2 D 3 regulates the process of intestinal Ca2+ absorption. First la,25(OH)2D3 through its nuclear
receptor in the intestinal epithelial cells
induces the biosynthesis of a calcium binding protein, calbindin-D28K, which has been
implicated in the process of intestinal Ca2+
absorption (Nemere et al, 1986; Norman,
1984). Calbindin-D28K belongs to the superfamily of Ca2+ binding proteins which
includes calmodulin, parvalbumin, and troponin C (Minghetti et al, 1988); these proteins bind their ligand Ca2+ through the use .
of an "EF-hand" motif which represents a
particular sequence of amino acids organized in a helix-loop-helix fashion so that
the Ca2+ is chelated with a Kd of 1-10 x
10- 8 M.
More recently a second involvement of
la,25(OH)2D3 in intestinal Ca2+ absorption
has been identified. This process involves
the biological response of transcaltachia.
Transcaltachia is defined as "the rapid hormonal stimulation of intestinal Ca2+ transport" and was discovered in 1984 in the
laboratory of the author (Nemere et al,
1984). The study of transcaltachia involves
the vascular perfusion of the duodenum of
vitamin D-replete chicks (Nemere et al,
1984; Yoshimoto et al, 1986). It should be
recalled that the intestinal epithelial cell
across which Ca2+ is translocated is asymmetric; thus the highly invaginated brush
border membrane faces the lumen of the
485
intestine, where the dietary Ca2+ is presented. In this model, the Ca2+ moves
through the brush border membrane and
becomes localized in lysosomal vesicles by
a process that may involve endocytosis.
After uptake into the cell and movement of
the Ca2+ across the cell in the lysosomal
vesicles (Nemere et al, 1986), the transported Ca2+ exits across the basal lateral
membrane by an exocytosis process. This
perfused intestine system has the advantage
that potential agonists can be presented
directly to the vasculature bathing the basal
lateral membrane of the intestinal epithelial
cell which mimics the delivery of the hormone under in vivo circumstances.
By using this system, it was possible to
demonstrate that the addition of
la,25(OH)2D3 to the vascular perfusate
could stimulate the transfer of 45Ca2+ from
the lumen to the perfusate within 4-8 minutes (see Figs. 3, 4). Figure 3 presents the
results of a dose-response study with
la,25(OH)2D3. Panel 3A illustrates the
rapidity of the onset of stimulation of transcaltachia by the introduction of la,25(OH)2D3. Within 2-4 mins, there is a statistically significant increase in the rate of
appearance of 45Ca2+ in the vascular perfusate. Panel 3B summarizes the response
of the transcaltachic response of the duodenum to varying concentrations of
la,25(OH)2D3. There is an apparent maximal stimulation of transcaltachia at the
physiological concentration of 650 pM,
while there is a down regulation in the presence of pharmacological concentrations of
the la,25(OH) 2 D 3 . Possibly reduction in
transcaltachia in the presence of unphysiologic la,25(OH) 2 D 3 concentrations represents an adaptive response to avoid the
adverse consequences of hypercalcemia.
Figure 4 presents the results of an experiment to determine the location in the intestinal epithelial cell of the cellular components participating in the signal transduction
process to generate the transcaltachic
response. When la,25(OH) 2 D 3 was perfused through the celiac artery, there was a
stimulation of transcaltachia; however,
when the la,25(OH) 2 D 3 was placed in the
lumen of the duodenum (adjacent to the
brush border membranes), no stimulation
486
ANTHONY W. NORMAN
D CONTROL
O l3O(pM)
• 650 "
A l300(pM)
• 6500 "
12
I
O
o
16 20 24
TIME (mm)
28
32
36
6 8 10 12 14 16 18 2O 22 24 26 28 30 32 34 36 38 40
TIME (mm)
FIG. 4. Transcaltachia is stimulated by vascular but
not lumenal la,25(OH)2D3 application in the perfused
chick intestine. In each of three experiments, the lumen
of a vitamin D-replete chick duodenum wasfilledwith
«Ca 2+ m buffered saline containing 130 pmol of
la,25(OH)2D3 per ml (•-•); two additional duodena
were filled with 45Ca2+ in buffered saline, one of which
was perfused with (A-A) and one without (O-O) 130
pmol of la,25(OH)2D3. (Modified from Zhou and Norman, 1992.)
4 -
with the basal lateral membrane of the
intestinal cell.
One of our early eiforts in assessing the
o
physiological importance of transcaltachia
in the process of intestinal Ca 2+ absorption
ft
was to learn what consequences hypercalo
O
cemia might have on transcaltachia. We
IT)
found that perfusion of the duodena with
elevated concentrations of Ca2+ (concentrations equivalent to a plasma Ca2+ of 12 mg/
!
!
100 ml) effectively blocked the response to
0
130 650 1300 6500
a physiological and pharmacological conl,25(0H) 2 D 3 (pM)
centration of la,25(OH) 2 D 3 (Yoshimoto et
FIG. 3. Stimulation of transcaltachia by la,25(OH)2D3 ah, 1986). This is an important result in that
in the perfused duodenum of the chicken. (Panel A) it provides evidence for the adaptability of
Each duodenum, filled with «Ca 2+ (5 /iCi/ml) in GBSS the process of transcaltachia in accordance
was vascularly perfused at 25°C for the first 20 min with the ongoing physiological reality. In
with control medium (GBSS) containing 0.125% BSA
and 0.05% y\ ethanol per ml) and then with the indi- circumstances of hypercalcemia, it would be
cated concentration of la,25(OH)2D3. Values are the disadvantageous for la,25(OH) 2 D 3 to stimmean ± SEM for 3 duodena per group. (Panel B) The ulate the further absorption of Ca 2+ . This
treated/control data for 43Ca2+ from panel A at 40 min
result is in accordance with the J. C. Waterare replotted to yield a dose response curve for
low definition of adaptation, "the good fit
la,25(OH)2D3 stimulation of transcaltachia.
of the organism to the environment"
(Waterlow, 1985).
Table 1 summarizes our efforts to bioof transcaltachia could be observed over the chemically define the process of transcal40 min. time interval studied (Nemere et tachia through application of various agoai, 1984). Thus, we have concluded that nists and inhibitors. Since transcaltachia is
the cellular response elements for transcal- not inhibited by actinomycin D, an inhibtachia are located close to or are associated itor of DNA directed RNA synthesis, and
3 -
Si
la,25(OH)2D3 MEDIATED TRANSCALTACHIA
TABLE 1. Stimulators and inhibitors oftranscaltachia.
Part A: Stimulators
Compound
1,25(OH)2D3
1,25(OH)2D,
Forskolin
BAY K 8644
BAY K 8644
TPA
Cell location
Basal lateral
Brush border
Basal lateral
Basal lateral
Brush border
Basal lateral
Stimulates
transcaltachia
Yes
No
Yes
Yes
No
Yes
Part B: Inhibitors
Compound
Actinomycin D
Leupeptin
Pepstatin
Monensin
Colchicine
Cytochalasin B
Verapamil
Nifedipin
Staurosporine
Process
Nuclear transcription
Cathepsin B
Cathepsin D, pepsin
Golgi
Microtubules
Microfilaments
Ca2+ channel
antagonist
Ca2+ channel
antagonist
Protein kinase C
Inhibits
transcaltachia
No
Yes
No
No
Yes
No
Yes
Yes
Yes
The results in this table were abstracted from the
following publications from the laboratory of the author (Nemere et al, 1984; Nemere and Norman, 1987;
de Boland and Norman, 19906).
because the onset of the transport of 45Ca2+
(Fig. 1) occurs within minutes of application
of agonists to the basal lateral surface of the
intestinal cell, we have concluded that this
biological process is nongenomic in character (Nemere et al., 1984; Nemere and
Norman, 1987). Clear evidence that
la,25(OH) 2 D 3 stimulated transcaltachia
involves the opening of Ca2+ channels was
achieved using the dihydropyridine agonist,
BAY K-8644 (de Boland et al., 1990) as well
as through use of the inhibitors nifedipine
and verapamil (Nemere and Norman, 1987).
Further, the BAY K-8644 only stimulated
transcaltachia when it was applied to the
basal lateral surface but not the brush border membrane of the intestinal cell. These
results collectively suggest that la,25(OH)2D3 stimulated transcaltachia obligatorily involves the opening of Ca2+ channels
which results in an increase in intracellular
Ca2+ concentration. Consistent with this
proposal is our observation that perfusion
of the duodena with 150 mM K + , effectively
depolarizes the basal lateral membrane of
the epithelial cell and stimulates transcal-
487
tachia (de Boland and Norman, 1990a).
Quite independently it has been reported
that la,25(OH)2D3 can mediate opening of
voltage gated Ca2+ channels in rat osteoblast cells (Caffrey and Farach-Carson,
1989).
As related above, our model of intestinal
Ca2+ transport involves the movement of
the Ca2+ through the interior of the intestinal cell in lysosomal-like vesicles containing calbindin-D28K (Nemere et al., 1986;
Nemere and Norman, 1988,1989), possibly
in association with microtubules (Nemere
et al., 1987). Thus inhibition of transcaltachia by colchicine, an inhibitor of microtubules, and by leupeptin, an inhibitor of
lysosomal cathepsin B is consistent with the
proposed model.
We have also found that both forskolin,
an agonist for protein kinase A and TPA
(12-O-tetradecanoylphorbol-13-acetate), an
agonist for protein kinase C, stimulate transcaltachia (de Boland and Norman, 19906).
Consistent with the proposed involvement
of protein kinase C was the ability of staurosporine to inhibit transcaltachia. Collectively these results suggest that phosphorylation events, mediated by protein kinases
A and C are involved in the very early cellular events which occur in the intestinal
cell in response to the application of
la,25(OH)2D3.
Figure 5 presents a schematic summary
of the potential signal transduction mechanisms for transcaltachia. As reviewed
above there is evidence of the involvement
of Ca2+ channels, intracellular Ca 2+ , phosphoproteins, and possibly diacylglycerol
(DAG) and IP3. These results suggest the
involvement in the basal lateral membrane
of the so called G-proteins which are known
regulators of adenyl cyclase, protein kinase
C and Ca2+ channels. In this model the
increase in intracellular Ca2+ that occurs as
a consequence of the l,25(OH)2D3-mediated opening of Ca2+ channels is postulated
to function as one of several second messengers which mediated the exocytosis of
the Ca2+-bearing lysosomal vesicles that
constitute the transepithelial transfer of the
dietary Ca 2+ . An as yet unresolved point is,
what cellular moiety interacts with the
secosteroid agonist of transcaltachia, namely
488
ANTHONY W. NORMAN
cium availability as well as to changing
physiological calcium requirements.
ACKNOWLEDGMENTS
This work was supported in part by
USPHS grant DK-09012-28.
REFERENCES
FIG. 5. Schematic model illustrating transcaltachia
signal transduction pathways in the chick intestine.
DBP = the plasma vitamin D binding protein which
transports vitamin D seco steroids in the blood compartment; calbindin-D28K = the vitamin D-induced calcium binding protein, of 28 kD. PKC = protein kinase
C; IP3 = inositol triphosphate; PIP2 = phosphoinostitol-diphosphate; DAG = diacylglycerol; P-protein =
an unknown phosphorylated protein which may function as a second messenger.
la,25(OH) 2 D 3 ? Current evidence suggests
that there is a membrane response element
located in the basal lateral membrane of the
epithelial cell, i.e., putative receptor, which
has a separate and distinct ligand binding
domain from that of the well-studied nuclear
la,25(OH) 2 D 3 receptor (Zhou and Norman,
1992). Thus we were able to identify vitamin D secosteroids which could bind well
to the nuclear receptor and stimulate gene
expression of calbindin-D2gK mRNA but
which were ineffective at stimulating transcaltachia; conversely we also identified other
analogs of la,25(OH) 2 D 3 which could not
bind effectively to the nuclear la,25(OH)2D3
receptor, but which were potent agonists of
transcaltachia.
An important challenge for the immediate future is to isolate and biochemically
characterize this membrane receptor for
la,25(OH) 2 D 3 and to describe how it mediates the opening of Ca2+ channels so as to
stimulate the exocytosis of the membrane
vesicle bearing 45 Ca 2+ . Parallel to these
studies there should be an evaluation of the
species distribution (in animals with both
exo- and endoskeletons) distribution of
transcaltachia and an assessment of whether
it can contribute to the organism's adaptive
responses to changing environmental cal-
Caffrey, J. M. and M. C. Farach-Carson. 1989. Vitamin D 3 metabolites modulate dihydropyridinesensitive calcium currents in clonal rat osteosarcoma cells. J. Biol. Chem. 264:20265-20274.
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Adenosine 3', 5' Monophosphate-dependent protein kinase in the 1,25-dihydroxyvitamin D3mediated rapid stimulation of intestinal calcium
transport (transcaltachia). Endocrinology 127:39—
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