Investigative Ophthalmology & Visual Science, Vol. 33, No. 5, April 1992
Copyright © Association for Research in Vision and Ophthalmology
Neurotronsmitters and Neuropeptides Stimulate Inositol
Phosphates and Intracellular Calcium in Cultured
Human Nonpigmented Ciliary Epithelium
Richard B. Crook and Jon R. Polansky
The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerol
pathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inositol phosphate formation by vasopressin (~3-fold), carbachol (~2-fold) and histamine (~5-fold) was
observed only after cells had been confluent for at least six days. In contrast, a response to bombesin
(~3-fold) declined with extended time in confluent culture. Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showed
a close correlation between the E C ^ of the different agonists when elevation of inositol phosphates was
compared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmacologic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order of
potencies for selective agonists and antagonists. The data suggest that the muscarinic receptor on
human NPE cells is the M 3 subtype, whereas the vasopressin receptor, as defined by its linkage
to the inositol phosphate/diacylglycerol pathway, is the V, subtype. Invest Ophthalmol Vis Sci 33:
1706-1716,1992
The role of neurotransmitters and neuropeptides in
the eye has generated considerable interest over the
last decade.12 Neuropeptides have been identified in
parasympathetic, sympathetic, and sensory nerves in
the eye,1 and several examples of neuropeptide-neurotransmitter and neuropeptide-neuropeptide colocalization have been reported.3"5 This suggests a complex pattern of peripheral innervation. Evidence suggests that neuropeptides and neurotransmitters may
play a role in the control of intraocular pressure.67
Neuropeptides and neurotransmitters have been
shown to activate second messenger pathways in the
ciliary body epithelium, the major site of aqueous humor secretion.89 Recently, the inositol phosphate/
diacylglycerol (IP/DAG) second messenger pathway
in the ciliary epithelium also has begun to be characterized.10"13 The IP/DAG pathway has been shown in
other cell types to regulate secretion, contraction, metabolism, and mitosis when activated by neuropeptides, neurotransmitters, and other factors.14 Although substantial evidence suggests that aqueous hu-
mor secretion by the ciliary epithelium is under
neural or hormonal control,615 the role of the IP/
DAG pathway in this process is unknown.
The IP/DAG pathway involves receptor-mediated
activation of phospholipase C, which catalyzes the
formation of inositol trisphosphate (InsP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. InsP3 triggers the release of Ca2+ from endoplasmic reticulum stores, and DAG activates protein kinase C. Subsequent modifications of enzyme
and other protein activities by Ca2+ binding and phosphorylation lead to the physiological response or responses of the cells to the stimulating agonist.14
We previously reported stimulation of inositol phosphate formation and intracellular calcium fluxes in
nonpigmented epithelia (NPE) by histamine.1213 In
the present study, we report receptor-mediated stimulation of inositol phosphate formation and intracellular Ca2+ by vasopressin, carbachol, and bombesin.
Materials and Methods
Chemicals
From the Cellular Pharmacology Laboratory, Department of
Ophthalmology, University of California, San Francisco, San Francisco, California.
Supported by EY07984, EY03980, and That Man May See, Inc.,
Submitted for publication: June 13, 1991; accepted October 25,
1991.
Reprint requests: Dr. Richard B. Crook, Box 0730, UCSF, San
Francisco, CA 94143.
Carbamylcholine chloride (carbachol), arginine-vasopressin, d(CH2)5Tyr(Me)2AVP, [D-Arg1, D-Trp79,
Leu1 *] substance P (spantide), and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid
(EGTA) were obtained from Sigma Chemical Co. (St.
Louis, MO). 4-Diphenylacetoxy-N-methylpiperidine
1706
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No. 5
INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polonsky
methiodide (4DAMP), pirenzepine, and methoctramine were obtained from Research Biochemicals
(Natick, MA). d(CH2)5D-IleVAVP (SKF 101398) and
d(CH2)5 Tyr(Et)VAVP (SKF101926) were gifts of Ms.
Julia Christie of Smith Kline and French Laboratories
(King of Prussia, PA). Bombesin, litorin, gastrin releasing peptide (GRP 18-27), neuromedin B, [D-Arg1,
D-Phe5, D-Trp7-9, Leu11] substance P, and [D-Arg1,
D-Pro2, D-Trp7-9, Leu11] substance P were obtained
from Bachem Bioscience (Philadelphia, PA). Deamino [Arg8] vasopressin (dAVP) and [Phe2, He3, Orn8]
vasopressin (PIOVP) were from Peninsula Laboratories (Belmont, CA). Indo-1-AM was obtained from
Molecular Probes (Eugene, OR). Genapol X-080 was
from Calbiochem (La Jolla, CA), and [3H] myoinositol (10-20 Ci/mmol) was from Amersham Corp. (Arlington Heights, IL). All other chemicals were reagent
grade and obtained from Fischer Scientific (Pittsburgh, PA).
Cell Culture
NPE were obtained and grown as described1216 on
extracellular matrix (ECM) in 24-well multiwells
(Falcon Plastics, Oxnard, CA). ECM was prepared as
described.17 The NPE used here were stocks derived
from 19-21 wk aborted human fetuses. All experiments were performed within the guidelines of the
Human Experimentation Committee at UCSF.
Briefly, cells from frozen stocks were seeded at 2-5
X 104 cells/well and grown in Growth Medium (Medium 199 plus 15% fetal calf serum (FCS), 2 mM glutamine, 20 /ig/ml gentamicin, 0.5 tig/ml fungizone
(Grand Island Biologicals, Grand Island, NY) and 0.5
mg/ml fibroblast growth factor (FGF). The cells were
grown at 37°C in a water-jacketed, CO2 incubator at
5% CO2. The medium was changed every second day.
At confluence, the cells were shifted to Maintenance
Medium (Growth Medium with 10% FCS and lacking FGF), and the medium was changed every second day.
Inositol Phosphates Measurement
At various times after they reached a confluent
state, NPE were labeled with 15 ^Ci/ml [3H] myoinositol for 20-26 hr at 37°C. The cells then were
changed to Ml99 without FCS, containing 10 mM
LiCl18 and incubated for 5 min at 37°C. Bioactive
compounds were added in XI00 concentrated solutions and the cells were incubated at 37°C for various
times. To terminate the incubation, the medium was
aspirated and the cell monolayers were lysed with
100% methanol. The cell monolayers were scraped off
the surface with a rubber policeman, and water soluble inositol phosphates were extracted by the proce-
1707
dure of Berridge et al18 modified to include an acidic
methanol phase as described by Schacht.19 Ion exchange chromatography by the method of Berridge20
was carried out as previously described.12 Total inositol phosphates (inositol monophosphate [InsP,], inositol bisphosphate [InsP2], and [InsP3]) were eluted
from 1.2 ml AG-1-X8 columns with 8 ml of 1M
HCOONH4/O.I M HCOOH. InsP,, InsP2, and InsP3
were individually eluted as described.11 Unless otherwise indicated, 1 ml of sample was added to 7.8 ml of
Scintiverse II (Fisher Scientific) and counted in a
Packard scintillation counter with 35% counting efficiency.
Intracellular Ca2+ Measurement
Cells were treated with indo-1-AM as described21
with the following modifications. Cells grown on 10
cm Petri dishes and held at confluence for various
lengths of time were removed using 0.02% trypsin and
0.05% ethylenediaminetetraacetic acid in saline solution (Cell Culture Facility, UCSF) for 5-7 min at
37°C, followed by centrifugation at 800 X g for 5 min
in a table-top centrifuge. The cell medium was decanted and the cells were resuspended at 107 cells/ml
in RPMI-1640 medium (Grand Island Biological,
Grand Island, NY) with 10% FCS and treated with 3
jiM indo-1-AM in dimethylsulfoxide (0.3%,finalconcentration) for 20 min at 37 °C, 5% CO2. The cells
were diluted to 106 cells/ml RPMI-1640 and incubated another 20 min at 37°C, 5% CO2. They were
cooled on ice, then centrifuged at 800 X g for 5 min at
4°C. The medium was decanted and the cells were
resuspended in ice-cold phosphate-buffered saline
and centrifuged as before. This was repeated three
times, with the last resuspension in Indo Buffer (25
mM HEPES, 125 mM NaCl, 5 mM KC1, 1 mM
NaHPO4, 0.5 mM MgCl2, 0.1 gm% glucose, 0.1 gm%
bovine serum albumin, pH 7.4, and 1 mM CaCl2 unless otherwise noted) at 5-10 X 106 cells/ml. For experiments without CaCl2, CaCl2 was deleted from the
medium and 1 mM EGTA was added. Cells were kept
on ice until used again.
Ca2+ measurements were carried out using a SPEX
fluorimeter with excitation and emission wavelengths
of 334 and 400 nm, respectively. Cell suspensions (2.5
ml) were placed in a quartz cuvette with a stirring
motor beneath the cuvette holder to allow mixing of
the cuvette solution. Bioactive compounds were
added as XI00 concentrated solutions to the cuvette
solution and recordings were made every 0.5 sec. At
the end of a run, intracellular calcium concentrations
were calculated with a modification of the method of
Grynkiewicz et al21—addition of 20 /x\ of the nonfluorescing nonionic detergent Genapol X-080 dis-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1992
1708
Vol. 33
which chelated Ca2+ and gave a minimum Ca2+ absorbance (Fmin). Ca2+ concentrations were calculated as
described using 250 nM as the KD for the indo-1 -Ca2+
complex.21 This method corrects for fluorescence because of unbound indo-1.
Experiments in which carbachol or vasopressin
were used to stimulate inositol phosphates formation
or intracellular Ca2+ were carried out on cells confluent 6-10 d (Fig. 1). To obtain optimal values for
bombesin stimulation of these responses, cells confluent 2-4 d were used (Fig. Id). Quantitatively similar, although smaller, responses to bombesin were observed in cells confluent 6-10 d (data not shown).
Carbachol
Data Presentation
2
4
6
8
10 12
'
2
4
6
Data in Figures 1-7 are representative experiments
carried out in duplicate. Data points are the averages
of values indicated by the error bars. Where data
spread is smaller than the size of the symbol, no error
bars are shown. These experiments were carried out at
least twice, with comparable results. Experiments
shown in Figs 9 and 10 were carried out at least five
times, with similar results. Data points in Figure 9 are
averages of duplicate or triplicate samples whose
spread is shown by the error bars. Data in "Ratio of
Observed/Predicted" column in Table 1 are means
± standard deviation from up to four experiments (n)
done in duplicate. Two typical experiments are
shown.
8 10 12
TIME IN CONFLUENT CULTURE (d)
Fig. 1. Effect of time in confluent culture on inositol phosphate
stimulation by four agonists. Cells were grown to confluency as
described in Materials and Methods. At various times thereafter
they were labeled for 24 hr with [3H] myoinositol and treated for 30
min with 10 mM LiCl and one of the following: 1 mM carbachol; 1
fj.M vasopressin; 100 nM histamine; or 10 nM bombesin. The incubations were terminated and total inositol phosphates analyzed as
described in Materials and Methods. Data are expressed as the
mean of duplicate-treated samples divided by the mean of untreated (control) samples grown for the same length of time. Control value ranged from 608 ± 82 cpm/ml (day 4) to 472 ± 18 cpm/
ml (day 10). Experimental values generally differed no more than
15% from the mean.
Results
solved cell membranes allowing maximal binding of
indo-1 by Ca2+ (Fmax). Eighty microliters of 1 M
EGTA and 280 fA 1M tris base then were added,
Monolayers confluent for 1 -2 d show elevated inositol phosphates in response to histamine,12"13 but not
in response to carbachol or vasopressin. Cells cultured
Table 1. Additivity studies
Drug
Exp. 1
Exp. 2
Ratio of
observed/
predicted
Carbachol (C)
Vasopressin (V)
Histamine (H)
Bombesin (B)
C+V
C+H
C+B
V+H
V+B
H+B
C+ V+H
C +V + B
C+H+B
V+H+B
C+V+H+B
B+N
B+L
1153 ± 10
1060 ±21
1834 ± 99
846 ± 17
1621 ± 183
1993 ± 303
1351 ±033
2263 ± 040
1478 ± 102
2169 ± 101
803 ± 028
1051 ±035
1601 ± 102
790 ± 068
1435 ± 04
2013 ±03
995 ± 43
1970 ±52
1260 ±08
1621 ±09
2610 ±072
ND*
2228 ± 201
2266 ± 087
2630 ±071
815 ±022
789 ±015
0.98 ±0.01
0.97 ±0.01
0.87 ± 0.02
0.90 ± 0.02
0.91 ±0.05
0.84 ± 0.04
1.02 ±0.05
ND
0.97 ± 0.02
0.89 ± 0.03
0.91 ±0.03
0.52 ± 0.04
0.50 ±0.01
Observed (cpm/ml)
' ND, not determined.
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N
4
4
3
4
4
4
2
2
2
2
1
2
1000
Total
IP's
/cpm\
800
Bombesin
600
Carbachol
UmlJ
400
Vasopressin
200
oo"10
9
8
7
6
5
-log [AGONIST] (M)
4
3
Fig. 2. Effect of vasopressin, carbachol, and bombesin concentration on total inositol phosphate stimulation. Cells confluent for 10
days were labeled with [3H] myoinositol for 24 hr and then treated
with 10 mM LiCl and various concentrations of vasopressin (D),
carbachol (O), or bombesin (A) for 30 min at 37 °C. The incubations were terminated and total inositol phosphates analyzed as
described in the legend to Figure 1.
for longer times after attainment of confluence developed a carbachol and a vasopressin response (Fig. 1).
At 2 d, no effect of 1 mM carbachol (Fig. la) or 1 nM
vasopressin (Fig. lb) could be seen. By 4 d, however,
3000
IPj
2000
/cpm\
\2ml/ 1000
responses to both effectors were detectable, and by 6 d
a two-fold stimulation of inositol phosphates by carbachol was achieved, while vasopressin stimulated a
three-fold rise in inositol phosphates by 10 d. For comparison, the response to histamine also was measured
(Fig. lc). Here, the response increased from 2- to 2.5fold stimulation at day 2 to afive-foldstimulation by
day 6. The neuropeptide bombesin also was found to
stimulate inositol phosphates (Fig. Id). Like the response to histamine, the response to bombesin was
present at 2 d after confluency. In contrast to other
stimulators, bombesin-specific stimulation declined
with extended time in monolayer culture, falling to
50% of maximal by day 6.
The effect of vasopressin, carbachol, and bombesin
concentration on inositol phosphate formation is
shown in Figure 2. Vasopressin achieved a maximal
increase of 2.3-fold at 0.1 jiM, with an EC50 (halfmaximal stimulatory concentration) of 200 nM. Carbachol gave about a two-fold increase at 1 mM, with
an EC50 of 40 IJM. Bombesin gave a 2.2-fold stimulation at 10 nM with an EC50 of 0.25 nM. The stimulation of InsP,, InsP2, and InsP3 in response to treatment with 1 pM vasopressin is shown in Figure 3. As
was observed with histamine,12 InsP2 and InsP3
showed early increases of 1.5- to 1.8-fold that peaked
at 1 min, while InsP, increased in a sustained fashion
for at least 30 min. Similar patterns of InsP,, InsP2,
and InsP3 formation were observed following treatment with carbachol or bombesin (data not shown).
Bombesin belongs to a family of structurally related
peptides, including GRP, litorin, and neuromedin B.
These also were tested for the ability to stimulate inositol phosphates in NPE (Fig. 4) Twenty nanomolar
IPo
/cpm\
\2ml/
1709
INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polansky
No. 5
1000
100
Total
800
IP's
/cpm\
600
l"STJ
IPs
Neuromedin B
400
/cpm\
\2ndl
200
TIME (min)
Fig. 3. Stimulation of InsP,, InsP2, and InsP3 by vasopressin.
Cells were labeled with [3H] myoinositol for 24 hr and treated with
10 mM LiCl for 30 min and 100 nM vasopressin for various lengths
of time. The incubations were terminated and inositol phosphates
were extracted as described in Materials and Methods. InsP,, InsP2,
and InsP3 were step-eluted from AG 1 X 8 columns and 2-ml aliquots counted as previously described.12
oo
10
9
8
7
6
5
-log [PEPTIDE] (M)
Fig. 4. Effect of bombesin-related peptide concentrations on total
inositol phosphate stimulation. Cells were labeled with [3H] myoinositol for 24 hr and then exposed to 10 mM LiCl and various
concentrations of peptide for 30 min at 37°C. The incubations were
terminated and inositol phosphates analyzed as in the legend to
Figure 1. Litorin (0), GRP (•), and neuromedin B (A).
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1992
litorin caused a 2.6-fold stimulation of total inositol
phosphate formation, with an EC50 of 0.8 nM. GRP
gave a maximal stimulation of 1.9-fold at 10 nM, with
an EC50 of 1.8 nM, whereas neuromedin B gave a
maximal stimulation of 2.6-fold at 10 /JM, with an
EC50 of 0.2 nM. Regarding EC50, the rank order was:
bombesin < litorin < GRP < neuromedin B. With
respect to maximal stimulation, the order was: litorin
= neuromedin B > bombesin > GRP. These peptides
may stimulate inositol phosphates via the same receptor, because treatment of cells with 10 nM bombesin
plus 10 nM neuromedin B or 10 nM litorin caused
stimulation of inositol phosphates roughly equivalent
to that caused by bombesin alone (see Table 1).
Agonist and Antagonist Studies
Acetylcholine antagonists were employed to establish rank order of effectiveness in inhibiting the carbachol-specific stimulation of inositol phosphates. Pirenzepine, an M,-specific antagonist,22 methoctramine, an M2-specific antagonist,23 and 4DAMP, an
M3-specific antagonist,24 were used. Figure 5 shows
that 4DAMP was the most potent antagonist tested,
with an IC50 (half maximal inhibitory concentration)
of 3 nM and complete inhibition of carbachol stimulation at 100 nM. Pirenzepine showed an IC50 of 3.5 /xM
with complete inhibition at 100 fxM, and methoctramine showed inhibitory effects only at concentrations
above 60 ^M. The rank order of IC5Os for the carbachol response therefore was 4DAMP < pirenzepine
< methoctramine.
Three antagonists of vasopressin then were tested
for their ability to inhibit inositol phosphates forma-
1200r
1000
Total
IP's
/cpm\
Vml )
Methoctramine
800
600
4 DAMP
400
200
10
Vol. 33
tion by vasopressin. d(CH2)5 [Tyr(Me)2]AVP, a V, selective antagonist;25 d(CH2)5 [Tyr(Et)2,Val4]AVP, a
mixed V,/V2 antagonist;26 and d(CH2)5[Ile2,Val4]AVP, a V2-selective antagonist,27 were added in increasing concentrations to cells simultaneously given
100 nM vasopressin. The results of such an experiment (Fig. 6a) show that the IC5Os of all three antagonists were roughly the same (20-40 nM). In all cases,
vasopressin-stimulated inositol phosphate formation
was completely inhibited by 10 MM antagonist.
Two agonists of vasopressin also were tested (Fig.
6b). Deamino [Arg8] dAVP, a potent V2-specific agonist in animals,28 PIOVP, a V! agonist in animals,29
and vasopressin were compared for their ability to
stimulate NPE inositol phosphates. dVAP stimulated
with an EC50 of 30 nM, about five times lower than
that of vasopressin (EC50 = 150 nM in this experiment), with a maximum attained by both peptides at
1 )LiM. PIOVP, in contrast, showed a maximal stimulation of 1.4 fold compared to a maximal stimulation
by vasopressin and dAVP of 2.3 fold. An EC50 of approximately 20 nM was observed with this partial agonist. The rank order of potency therefore was dAVP
> vasopressin, with PIOVP a submaximal stimulator.
These data present a paradox, because vasopressin receptor subtypes have been defined by second messenger linkages (V1 to cAMP formation and V2 to intracellular Ca2+ elevation 30). This is addressed in the
Discussion.
Bombesin stimulation of inositol phosphate formation was probed with three bombesin antagonists:
peptide A* ([D-Arg1, D-Pro2, D-Trp7-9, Leu11] substance P);31 peptide D* ([D-Arg1, D-Phe5, D-Trp79,
Leu11] substance P);31 and spantide ([D-Arg1, Trp7-9,
Leu11] substance P).1 In Swiss 3T3 cells, peptide D
was shown to befive-foldmore potent than peptide A
in blocking bombesin effects.31 Figure 7 shows the results of adding various concentrations of each antagonist with 1 nM bombesin to NPE cells. Peptide D
blocked stimulation of inositol phosphates by bombesin with an IC50 of 0.4-0.7 nM, with maximal inhibition achieved at 3 fiM. Peptide A inhibited with an
IC50 of 1.4 ixM, with maximal inhibition at 10 ixM.
Thus, peptide D was about 3.5 times more effective
than peptide A. Spantide inhibited with an IC50 of
0.2-0.6 /iM with total inhibition at 3 ^M. The rank
order of potencies therefore was spantide ^ peptide D
> peptide A.
-log [ANTAGONIST] (M)
Fig. 5. Effect of cholinergic antagonists on inositol phosphate
stimulation by carbachol. Cells were labeled 24 hr with [3H] myoinositol and then treated for 30 min with 10 raM LiCl and 1 mM
carbachol plus various concentrations of the following: 4DAMP
(A); pirenzipine (•); or methoctramine (O). The incubations were
terminated and inositol phosphates analyzed as described in the
legend to Figure 1. Control (LiCl alone) values were 418 ± 16
cpm/ml.
Additivity Studies
The responsiveness of NPE to several neurotransmitters and neuropeptides suggested the possibility
that in vivo NPE might be stimulated by combinations of these effectors. Numerous examples exist of
neurotransmitters and neuropeptides contained
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INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook ond Polonsky
No. 5
Fig. 6. Effect of vasopressin antagonists and agonists
on inositol phosphate stimulation by vasopressin. Cells
were labeled with [3H]
myoinositol for 24 hr and
then treated as follows, (a)
Antagonists: cells were
treated for 30 min with 10
mM LiCl and 100 nM vasopressin, plus various concentrations of d(CH2)5D-Ile
VAVP (•); d(CH2)Tyr(Me)AVP (O); or d(CH2)5Tyr(Et)
VAVP (V). The incubations
were terminated and inositol phosphates analyzed as
in the legend to Figure 1. (b)
Agonists: cells were treated
for 30 min with 10 mM LiCl
and various concentrations
of vasopressin (O), dAVP
(•), or PIOVP (V). The incubations were then terminated and inositol phosphates analyzed as in the legend to Figure 1.
b.
a.
1000r
100nM Vasopressin +
Od(CH2)Tyr(Me)AVP
ad(CH 2 )D-IleVAVP
vd(CH2)Tyr(Et)VAVP
Total
IP's
/cpm\
\ml /
1400
1000
PIOVP
800
500-
600
400-
400
Vasopressin
10
-log [ANTAGONIST] (M)
1nM Bombesin +
o Peptide A
D Peptide D
v Spantide
500
Total
IP's
/cpm\
\ml / 400
300
250oo
1600
1200
within the same neuron and presumably released together upon depolarization.32"34 Moreover, ciliary
processes are known to be supplied with both adrener-
600
1711
7
6
5
-log [ANTAGONIST] (M)
Fig. 7. Effect of bombesin antagonists on inositol phosphate stimulation by bombesin. Cells were labeled with [3H] myoinositol for
24 hr and then treated for 30 min with 10 mM LiCl and 1 nM
bombesin, plus various concentrations of the following: spantide
(V); peptide D (•); or peptide A (O). The incubations were terminated and inositol phosphates analyzed as in the legend to Figure 1.
Control (LiCl alone) values were 346 ± 17 cpm/ml.
-log [AGONIST] (M)
gic and cholinergic nerves, as well as nerves containing a variety of neuropeptides (for review, see reference 1). Accordingly, experiments were conducted in
which various combinations of carbachol, vasopressin, bombesin, and histamine were added to cells and
the inositol phosphate response ("observed") was
compared to the responses of the sum of each agonist
added individually ("predicted"). The ratio of observed to predicted will be 1 if the stimulation by
more than 1 effector is equivalent to the sum of the
responses of each effector added alone.35 Maximally
stimulating concentrations of each effector were used
in each case to simplify interpretation of the data. Table 1 shows that in all cases treatment with combinations of effectors (lines 5-15) resulted in inositol phosphate stimulations greater than observed with each
agonist alone. In most cases, the stimulations were
within 10-15% of that predicted on the basis of pure
additivity, which is consistent with the concept that
each effector stimulates inositol phosphates via a different receptor.36 No profound inhibitions or stimulations of one member of a group by the others were
observed, which suggests that the receptors for each
effector probably act independently of one another.
Stimulation by combinations of the related peptides
bombesin, neuromedin B, and litorin, which stimulate to a similar degree when added independently
(Fig. 4), were not additive (lines 16-17). Bombesin
stimulation was not further increased by neuromedin
B or litorin, which is consistent with the concept that
these peptides stimulate inositol phosphates via the
same receptor.
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1712
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992
Vol. 33
c.
Bomb
1
GP EGTA
U
O
200-
rr
<
LLJ
o<
rr
100-
100 200
300
300
400 500
0
100
200
300
TIME (sec)
2+
Fig. 8. Stimulation of intracellular Ca by vasopressin, carbachol, and bombesin. (a) Vasopressin. Cell suspensions prepared from cells
confluent for 7 d and treated with indo-1-AM as described in Materials and Methods were given 1 fiM vasopressin, and the change in
fluorescence measured and converted to molar concentrations, (b) Carbachol. Cells prepared as described in (a) were given 1 mM carbachol
and intracellular Ca2+ was measured, (c) Bombesin. Cells prepared as described were given 100 nM bombesin and intracellular Ca2+ was
measured as in (a), (d) Cells prepared in either indo buffer with 1 mM CaCl2 or no CaCl2 were treated with indo-1-AM, exposed to 1 MM
vasopressin and the intracellular Ca2+ determined as in Figure la. Upper line: with CaCl2; lower line: without CaCl2.
Intracellular Ca2+ Fluxes
Because receptor-mediated formation of inositol
phosphates in many systems has been linked to subsequent increases in intracellular Ca2+,37 we evaluated
the effect of vasopressin, carbachol, and bombesin on
intracellular Ca2+ levels in NPE cells using the fluorescent calcium probe, indo-1-AM.21 Figure 8 shows fluorescence tracings from cells treated with the three effectors. Determination of Fmax (addition of Genapol
x-080) and Fmin (addition of EGTA), which allowed
conversion of fluorescence units to Ca2+ concentrations, are also shown (Fig. 8a-c). From a resting level
of 95 nM, 1 fiM vasopressin evoked a rapid rise to 180
nM, about a two-fold increase in intracellular Ca2+
(Fig. 8a). Intracellular Ca2+ then declined to a plateau
that was maintained for at least 8 min. One millimolar carbachol (Fig. 8b) elicited a smaller response, a
1.5-fold rise to 142 nM, whereas 100 nM bombesin
(Fig. 8c) gave a two-fold elevation to 211 nM intracellular Ca2+. These responses occurred within 5-15 sec
following introduction of an agonist into the cuvette.
The biphasic Ca2+ response exhibited by stimulated
NPE is characteristic of electrically nonexcitable cells.
After an initial spike, the Ca2+ level fell to a new, sustained level.38
To determine whether extracellular Ca2+ is required for the intracellular Ca2+ flux to occur, cells
were suspended in indo buffer either lacking CaCl2 or
containing 1 mM CaCl2, and the responses to 100 nM
bombesin were compared (Fig. 8d). The initial peak,
although slightly reduced, was present in cells lacking
CaCl2 compared to cells with CaCl2. In the absence of
CaCl2, however, intracellular Ca2+ fell rapidly to baseline instead of establishing a second plateau. Thus, the
initial Ca2+ spike did not require extracellular Ca2+,
while the second phase did.
Dose-response curves for carbachol, vasopressin,
and bombesin elevations of intracellular Ca2+ are
shown in Figure 9. The EC50 for intracellular Ca2+
elevation by carbachol was 100 j*M (Fig. 9a), similar
-~
250r
Bombesin
200-
u
Vasopressin
13 150o
2
10010
8
7
6
5
-log [AGONIST] (M)
4
Fig. 9. Effect of agonist concentration on intracellular Ca2+ stimulation. Cells prepared and treated with indo-1-AM as in Figure 8a
were aliquotted, given various concentrations of vasopressin (D),
carbachol (O), or bombesin (A), and peak fluorescence was converted into molar concentrations. Each point represents an average
of three to six determinations.
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No. 5
INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polansky
1713
200Carbachol
Fig. 10. Augmentation and antagonism
of carbachol-stimulated intracellular Ca 2+
levels, (a) Effects of subsequent addition of
vasopressin. Cells prepared as in Figure 8a
were given 1 mM carbachol. When the
peak of the Ca 2+ response had been
reached, 1 /*M vasopressin was added and
intracellular Ca 2+ levels monitored as in
Figure 1 a. (b) Effect of 4DAMP. Cells stimulated with 1 mM carbachol as in (a) were
given 100 nM 4DAMP where indicated,
and intracellular Ca 2+ monitored as in (a).
4 DAMP
Carbachol
+
CO
O
f
Vasopressin
CO
<
LU
o
<
cc
100100
200
300
400
500
0
100
200
300
TIME (sec)
to that observed for inositol phosphates formation
(Fig. 2). Vasopressin gave an EC50 of 180 nM, with a
maximum at 1 nM. These values are also similar to
those for inositol phosphates stimulation (Fig. 2).
Bombesin gave an EC50 of 10 nM, or 40-fold greater
than the EC50 observed for inositol phosphate stimulation (0.25 nM). Maximum Ca2+ levels were attained
at 100 nM bombesin.
To determine whether the presence of a receptor
antagonist would cause the reversal of intracellular
Ca2+ stimulation by an agonist, we tested the effect of
the muscarinic M3 antagonist 4DAMP on carbachol
stimulation of intracellular Ca2+ (Fig. 10a). Addition
of 4DAMP after stimulation of intracellular Ca2+ with
1 mM carbachol resulted in an immediate drop of
Ca2+ to levels near baseline. This suggests that the
continued occupation of an M3 receptor by carbachol
is required for maintenance of elevated intracellular
Ca2+. We also sought to determine whether increased
inositol phosphate stimulation response to multiple
effectors would be reflected in intracellular Ca2+ levels
by testing the sequential addition of 1 mM carbachol
and 1 nM vasopressin to NPE cells. Figure 10b shows
that the maximal elevation attained by 1 mM carbachol, 160 nM, was surpassed by the subsequent addition of 1 IJM vasopressin, which gave 230 nM Ca2+. In
this case, therefore, intracellular Ca2+ levels may be
influenced by the presence of more than one agonist.
Discussion
The present data describe the responsiveness of
NPE, via the inositol phosphate arm of the IP/DAG
pathway, to vasopressin, carbachol, and bombesin.
The physiological responses of the NPE to these effec-
tors remain to be determined, but the possibility exists
that they or other agents that regulate the IP/DAG
pathway could influence aqueous humor secretion.
Vasopressin and carbachol have been reported to affect aqueous inflow and intraocular pressure,2-39 although the mechanisms underlying these effects are
unclear. Few studies have been performed on bombesin in the eye,40 but like vasopressin, it is a neuropeptide known to regulate autonomic functions.
The increase in responsiveness to histamine, carbachol, and vasopressin observed when NPE are maintained in a confluent monolayer over several days
raises the possibility that, at least in tissue culture, a
nondividing state may be a prerequisite for optimal
responsiveness. DNA synthesis in NPE cells decreases
for about 4 d in confluent culture before reaching a
minimal level (RBC, GM Lui, and JRP, manuscript
in preparation). This is approximately when significant responses to some effectors begin to appear. The
decline in responsiveness of inositol phosphate formation to bombesin after NPE reach confluency is
the exception to the pattern of observed increases.
The reason for this decline is not known, but bombesin is a potent mitogen in several cell lines29'41 and
responses to mitogens have been observed to decrease
when dividing cells cease cell division.42 Besides
changes in the extent of responsiveness, time in monolayer culture had no other effect on NPE responses to
neuropeptides and neurotransmitters described in the
present report. Whether bombesin might be a mitogen for NPE cells is unknown.
Measurement of InsPl5 InsP2, and InsP3 levels after
exposure to vasopressin or other agonists under study
showed early increases in InsP2 and InsP3 followed by
a sustained rise in InsP,. This pattern is similar to our
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992
findings for histamine12 and conforms to the generally
held view that InsP3 is the cleavage product of receptor-activated phospholipase C hydrolysis of phosphatidylinositol 4,5 bisphosphate, and therefore appears
first, with InsP2 and InsPj subsequently generated by
sequential dephosphorylations of InsP3.14 The function of InsP3 is thought to be to bind to specific sites
on the endoplasmic reticulum, causing the release of
sequestered Ca2+, which transiently elevates intracellular Ca2+ levels.1443 That vasopressin, carbachol, and
bombesin each elevated intracellular Ca2+ in NPE
cells is consistent with this view. Wax and collaborators also have reported stimulation of inositol phosphates10 and intracellular Ca 2+n by vasopressin, carbachol, and several other agents in SV40-transformed
NPE cells.
Intracellular Ca2+ Studies
The intracellular Ca2+ dose-response data show
that carbachol and vasopressin stimulated at concentrations similar to those for inositol phosphate stimulation. This is consistent with the concept that InsP3
stimulation leads to a rise in intracellular Ca2+. With
bombesin, however, significantly greater concentrations were required to achieve EC50 and maximal values relative to inositol phosphate stimulation. This
could be interpreted to mean that Ca2+ stimulation
occurs by means other than InsP3 elevation with this
agent. However, we cannot rule out the possibility
that treatment of cells with trypsin to obtain suspension cultures resulted in modified bombesin receptors, with the result that higher concentrations of this
peptide than normal were required to stimulate. Further experiments will be needed to confirm dose-dependent differences between inositol phosphate and
intracellular Ca2+ stimulations in NPE cells.
The rapid decline in intracellular Ca2+ after stimulation by agonists in the absence of extracellular Ca2+
suggests that the sustained phase of the calcium response is a result of influx of extracellular calcium
that replenishes depleted intracellular stores, as described in other systems.4445 The calcium responses
reported in the study generally occurred after 5-10 sec
and peaked at 10-15 sec after the addition of agonist
(Fig. 8). InsP3 in these and prior studies12 could be
seen at 30-45 sec, but the experimental protocol in
the present studies did not allow for sufficiently short
incubation times (1-5 sec) that would permit the
order of appearance of changes in InsP3 and Ca2+ to
be determined. Although current belief is that an elevation of InsP3 precedes an elevation of cytosolic
Ca2+, evidence that contradicts this sequence has been
reported.46
Many cellular responses to neuronal stimuli occur
over millisecond time periods rather than seconds or
Vol. 33
minutes. Methods such as patch clamping and integration of membrane fragments into lipid bilayers will
be needed to accurately measure such responses.
Agonist and Antagonist Studies
Pharmacological studies of receptors for vasopressin, carbachol, and bombesin using agonists and antagonists allowed preliminary characterization of
these receptors. In animals, two types of peripheral
receptors are known for vasopressin. V, receptors
modulate pressor and glycogenolytic responses
through a Ca2+-dependent pathway, whereas V2 receptors modulate antidiuretic responses via a cAMPdependent pathway in renal tubules.30 With this criterion, the vasopressin receptor on NPE cells is V,. However, our finding that the V2-specific agonist dAVP
was 4-5 times more effective than vasopressin in stimulating inositol phosphates, whereas the V,-specific
agonist PIOVP only stimulated half as well, is consistent with a V2 subtype. This apparent contradiction
may be resolved by reports that vasopressin antagonist and agonist responses vary among species.47"49
Thus, agonist and antagonist data derived from animal studies may not apply to human vasopressin receptors. It is noteworthy that SKF 101926, a strong
antidiuretic antagonist in rats, behaves in man as a
V,/V2 mixed antagonist,47 which is consistent with
our antagonist data. Except for platelet receptors,48>50>51 vasopressin receptors have not been well
characterized in human tissue. The present study,
therefore, defines the vasopressin receptor on NPE
cells as V, based on linkage to intracellular Ca2+. However, the present study also provides evidence that the
pharmacological characteristics of this receptor may
be different from V\ receptors in other species.
Cholinergic receptors are either of the nicotinic or
muscarinic subtypes. Nicotinic receptors are located
in autonomic ganglionic synapses and striated muscles. Muscarinic receptors are found in smooth muscle and glands.38 Muscarinic receptors are subdivided
pharmacologically into Ml5 M2, and M3 subtypes.24
The antagonist rank order determined for the NPE
cholinergic receptor was: 4DAMP > pirenzepine
> methoctramine. Tubocurarine, a nicotinic receptor
agonist, had no effect on NPE cells at concentrations
of up to 1 mM (data not shown). The greater efficiency (by three logs) of 4DAMP compared to pirenzepine or methoctramine is consistent with the NPE
receptor being of the M3 subtype. This also is consistent with results using SV40-transformed human ciliary epithelial cells.10 Recombinant DNA methods recently have been used to detect four genes for muscarinic receptor proteins,52 so pharmacological analysis
may not be distinguishing all subtypes. Further work
will be needed to determine whether more than one
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No. 5
INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook ond Polansky
class of muscarinic receptor is present in human NPE
cells.
Bombesin receptor antagonist studies gave a rank
order of potencies of spantide > peptide D, with peptide A appearing to be a partial agonist. The potencies
of peptides D and A are similar to those observed in
Swiss 3T3 cells and human small cell lung cancer
SCLC cells.30 However, spantide was considerably
less potent in those studies than in the present experiments, suggesting that differences exist between the
NPE receptor and those on Swiss 3T3 or small cell
lung cancer cells. For example, the substance P derivatives used in the present study, which possess the highest specificity reported to date,30 also bind to vasopressin receptors.30'53 The bombesin effect on NPE
cells is probably not achieved through binding to vasopressin receptors, however, because additivity experiments showed that inositol phosphate stimulation
by maximal concentrations of bombesin plus vasopressin given together caused a response nearly equal
to the sum of the responses caused by either effector
alone. Such a result suggests separate sets of receptors
for vasopressin and bombesin.35 If bombesin acted
through vasopressin receptors, the combination of
bombesin plus vasopressin would have been expected
to give maximal stimulation between that of vasopressin or bombesin alone. The same argument applies to carbachol and histamine and suggests that
each effector acts via receptor population distinct
from the others.
The cells used in the present study were of fetal
origin. Thus, there could be differences between the
responses reported here and those of adult cells. Furthermore, responses observed in cells maintained in
tissue culture may differ from responses of cells in
vivo, because of alterations of neuronal and tonic environments. Based on these cautions, the responsiveness of cultured NPE cells to vasopressin, carbachol,
and bombesin, in addition to histamine, raises the possibility that these bioactive compounds might influence aspects of ciliary epithelium physiology in vivo.
We recently have found that stimulation of the DAGprotein kinase C arm of the IP/DAG pathway causes
alterations in K+ transport in NPE (RBC, DK von
Brauchitsch, and JRP, in preparation). Further study
of this signalling pathway in the ciliary epithelium
may provide information about physiological mechanisms that play a regulatory role in ion transport. This
could be relevant to the study of aqueous humor inflow.
Key words: neuropeptides, carbachol, ciliary epithelium,
inositol phosphates, calcium
Acknowledgments
The authors wish to thank Drs. Nejat Duzgunes, Demetrios Papahadjopoulos, and Arthur Weiss for the use of their
1715
fluorimeters; Drs. Mark Goldsmith and Dennis Alperts for
instruction in calcium measurements; Ms. Tracy Hydorn
for ECM preparation; and Richard Dye for manuscript
word processing.
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