neurotransmitters and neuropeptides stimulate inositol...

Investigative Ophthalmology & Visual Science, Vol. 33, No. 5, April 1992Copyright © Association for Research in Vision and Ophthalmology

Neurotronsmitters and Neuropeptides Stimulate InositolPhosphates and Intracellular Calcium in Cultured

Human Nonpigmented Ciliary EpitheliumRichard B. Crook and Jon R. Polansky

The effects of several neurotransmitters and neuropeptides on the inositol phosphate/diacylglycerolpathway were examined in human nonpigmented ciliary epithelial cells. Maximal stimulation of inosi-tol phosphate formation by vasopressin (~3-fold), carbachol (~2-fold) and histamine (~5-fold) wasobserved 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 bisphos-phate, and inositol trisphosphate all were stimulated by these agonists. Dose-response studies showeda close correlation between the E C ^ of the different agonists when elevation of inositol phosphates wascompared to stimulation of intracellular Ca2+, with the exception of bombesin. Preliminary pharmaco-logic characterization of the receptors for vasopressin, carbachol, and bombesin provided rank order ofpotencies for selective agonists and antagonists. The data suggest that the muscarinic receptor onhuman NPE cells is the M3 subtype, whereas the vasopressin receptor, as defined by its linkageto the inositol phosphate/diacylglycerol pathway, is the V, subtype. Invest Ophthalmol Vis Sci 33:1706-1716,1992

The role of neurotransmitters and neuropeptides inthe eye has generated considerable interest over thelast decade.12 Neuropeptides have been identified inparasympathetic, sympathetic, and sensory nerves inthe eye,1 and several examples of neuropeptide-neuro-transmitter and neuropeptide-neuropeptide colocal-ization have been reported.3"5 This suggests a com-plex pattern of peripheral innervation. Evidence sug-gests that neuropeptides and neurotransmitters mayplay a role in the control of intraocular pressure.67

Neuropeptides and neurotransmitters have beenshown to activate second messenger pathways in theciliary body epithelium, the major site of aqueous hu-mor secretion.89 Recently, the inositol phosphate/diacylglycerol (IP/DAG) second messenger pathwayin the ciliary epithelium also has begun to be charac-terized.10"13 The IP/DAG pathway has been shown inother cell types to regulate secretion, contraction, me-tabolism, and mitosis when activated by neuropep-tides, neurotransmitters, and other factors.14 Al-though substantial evidence suggests that aqueous hu-

From the Cellular Pharmacology Laboratory, Department ofOphthalmology, University of California, San Francisco, San Fran-cisco, 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.

mor secretion by the ciliary epithelium is underneural or hormonal control,615 the role of the IP/DAG pathway in this process is unknown.

The IP/DAG pathway involves receptor-mediatedactivation of phospholipase C, which catalyzes theformation of inositol trisphosphate (InsP3) and diac-ylglycerol (DAG) from phosphatidylinositol 4,5-bis-phosphate. InsP3 triggers the release of Ca2+ from en-doplasmic reticulum stores, and DAG activates pro-tein kinase C. Subsequent modifications of enzymeand other protein activities by Ca2+ binding and phos-phorylation lead to the physiological response or re-sponses of the cells to the stimulating agonist.14

We previously reported stimulation of inositol phos-phate formation and intracellular calcium fluxes innonpigmented epithelia (NPE) by histamine.1213 Inthe present study, we report receptor-mediated stimu-lation of inositol phosphate formation and intracellu-lar Ca2+ by vasopressin, carbachol, and bombesin.

Materials and Methods

Chemicals

Carbamylcholine chloride (carbachol), arginine-va-sopressin, 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

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No. 5 INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polonsky 1707

methiodide (4DAMP), pirenzepine, and methoctra-mine were obtained from Research Biochemicals(Natick, MA). d(CH2)5D-IleVAVP (SKF 101398) andd(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 re-leasing 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 obtainedfrom Bachem Bioscience (Philadelphia, PA). Dea-mino [Arg8] vasopressin (dAVP) and [Phe2, He3, Orn8]vasopressin (PIOVP) were from Peninsula Laborato-ries (Belmont, CA). Indo-1-AM was obtained fromMolecular Probes (Eugene, OR). Genapol X-080 wasfrom Calbiochem (La Jolla, CA), and [3H] myoinosi-tol (10-20 Ci/mmol) was from Amersham Corp. (Ar-lington Heights, IL). All other chemicals were reagentgrade and obtained from Fischer Scientific (Pitts-burgh, PA).

Cell Culture

NPE were obtained and grown as described1216 onextracellular matrix (ECM) in 24-well multiwells(Falcon Plastics, Oxnard, CA). ECM was prepared asdescribed.17 The NPE used here were stocks derivedfrom 19-21 wk aborted human fetuses. All experi-ments were performed within the guidelines of theHuman Experimentation Committee at UCSF.Briefly, cells from frozen stocks were seeded at 2-5X 104 cells/well and grown in Growth Medium (Me-dium 199 plus 15% fetal calf serum (FCS), 2 mM glu-tamine, 20 /ig/ml gentamicin, 0.5 tig/ml fungizone(Grand Island Biologicals, Grand Island, NY) and 0.5mg/ml fibroblast growth factor (FGF). The cells weregrown at 37°C in a water-jacketed, CO2 incubator at5% CO2. The medium was changed every second day.At confluence, the cells were shifted to MaintenanceMedium (Growth Medium with 10% FCS and lack-ing FGF), and the medium was changed every sec-ond day.

Inositol Phosphates Measurement

At various times after they reached a confluentstate, NPE were labeled with 15 ^Ci/ml [3H] myoino-sitol for 20-26 hr at 37°C. The cells then werechanged to Ml99 without FCS, containing 10 mMLiCl18 and incubated for 5 min at 37°C. Bioactivecompounds were added in XI00 concentrated solu-tions and the cells were incubated at 37°C for varioustimes. To terminate the incubation, the medium wasaspirated and the cell monolayers were lysed with100% methanol. The cell monolayers were scraped offthe surface with a rubber policeman, and water solu-ble inositol phosphates were extracted by the proce-

dure of Berridge et al18 modified to include an acidicmethanol phase as described by Schacht.19 Ion ex-change chromatography by the method of Berridge20

was carried out as previously described.12 Total inosi-tol phosphates (inositol monophosphate [InsP,], inosi-tol bisphosphate [InsP2], and [InsP3]) were elutedfrom 1.2 ml AG-1-X8 columns with 8 ml of 1MHCOONH4/O.I M HCOOH. InsP,, InsP2, and InsP3

were individually eluted as described.11 Unless other-wise indicated, 1 ml of sample was added to 7.8 ml ofScintiverse II (Fisher Scientific) and counted in aPackard scintillation counter with 35% counting effi-ciency.

Intracellular Ca2+ Measurement

Cells were treated with indo-1-AM as described21

with the following modifications. Cells grown on 10cm Petri dishes and held at confluence for variouslengths of time were removed using 0.02% trypsin and0.05% ethylenediaminetetraacetic acid in saline solu-tion (Cell Culture Facility, UCSF) for 5-7 min at37°C, followed by centrifugation at 800 X g for 5 minin a table-top centrifuge. The cell medium was de-canted and the cells were resuspended at 107 cells/mlin RPMI-1640 medium (Grand Island Biological,Grand Island, NY) with 10% FCS and treated with 3jiM indo-1-AM in dimethylsulfoxide (0.3%, final con-centration) for 20 min at 37 °C, 5% CO2. The cellswere diluted to 106 cells/ml RPMI-1640 and incu-bated another 20 min at 37°C, 5% CO2. They werecooled on ice, then centrifuged at 800 X g for 5 min at4°C. The medium was decanted and the cells wereresuspended in ice-cold phosphate-buffered salineand centrifuged as before. This was repeated threetimes, with the last resuspension in Indo Buffer (25mM HEPES, 125 mM NaCl, 5 mM KC1, 1 mMNaHPO4, 0.5 mM MgCl2, 0.1 gm% glucose, 0.1 gm%bovine serum albumin, pH 7.4, and 1 mM CaCl2 un-less otherwise noted) at 5-10 X 106 cells/ml. For ex-periments without CaCl2, CaCl2 was deleted from themedium and 1 mM EGTA was added. Cells were kepton ice until used again.

Ca2+ measurements were carried out using a SPEXfluorimeter with excitation and emission wavelengthsof 334 and 400 nm, respectively. Cell suspensions (2.5ml) were placed in a quartz cuvette with a stirringmotor beneath the cuvette holder to allow mixing ofthe cuvette solution. Bioactive compounds wereadded as XI00 concentrated solutions to the cuvettesolution and recordings were made every 0.5 sec. Atthe end of a run, intracellular calcium concentrationswere calculated with a modification of the method ofGrynkiewicz et al21—addition of 20 /x\ of the non-fluorescing nonionic detergent Genapol X-080 dis-


1708 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1992 Vol. 33

Carbachol

2 4 6 8 10 12 ' 2 4 6 8 10 12

TIME IN CONFLUENT CULTURE (d)

Fig. 1. Effect of time in confluent culture on inositol phosphatestimulation by four agonists. Cells were grown to confluency asdescribed in Materials and Methods. At various times thereafterthey were labeled for 24 hr with [3H] myoinositol and treated for 30min with 10 mM LiCl and one of the following: 1 mM carbachol; 1fj.M vasopressin; 100 nM histamine; or 10 nM bombesin. The incu-bations were terminated and total inositol phosphates analyzed asdescribed in Materials and Methods. Data are expressed as themean of duplicate-treated samples divided by the mean of un-treated (control) samples grown for the same length of time. Con-trol value ranged from 608 ± 82 cpm/ml (day 4) to 472 ± 18 cpm/ml (day 10). Experimental values generally differed no more than15% from the mean.

solved cell membranes allowing maximal binding ofindo-1 by Ca2+ (Fmax). Eighty microliters of 1 MEGTA and 280 fA 1M tris base then were added,

Table 1. Additivity studies

which chelated Ca2+ and gave a minimum Ca2+ absor-bance (Fmin). Ca2+ concentrations were calculated asdescribed using 250 nM as the KD for the indo-1 -Ca2+

complex.21 This method corrects for fluorescence be-cause of unbound indo-1.

Experiments in which carbachol or vasopressinwere used to stimulate inositol phosphates formationor intracellular Ca2+ were carried out on cells con-fluent 6-10 d (Fig. 1). To obtain optimal values forbombesin stimulation of these responses, cells con-fluent 2-4 d were used (Fig. Id). Quantitatively simi-lar, although smaller, responses to bombesin were ob-served in cells confluent 6-10 d (data not shown).

Data Presentation

Data in Figures 1-7 are representative experimentscarried out in duplicate. Data points are the averagesof values indicated by the error bars. Where dataspread is smaller than the size of the symbol, no errorbars are shown. These experiments were carried out atleast twice, with comparable results. Experimentsshown in Figs 9 and 10 were carried out at least fivetimes, with similar results. Data points in Figure 9 areaverages of duplicate or triplicate samples whosespread is shown by the error bars. Data in "Ratio ofObserved/Predicted" column in Table 1 are means± standard deviation from up to four experiments (n)done in duplicate. Two typical experiments areshown.

Results

Monolayers confluent for 1 -2 d show elevated inosi-tol phosphates in response to histamine,12"13 but notin response to carbachol or vasopressin. Cells cultured

Drug

Carbachol (C)Vasopressin (V)Histamine (H)Bombesin (B)C + VC + HC + BV + HV + BH + BC + V + HC +V + BC + H + BV + H + BC + V + H + BB + NB + L

Observed (cpm/ml)

Exp. 1

1153 ± 101060 ±211834 ± 99846 ± 17

1621 ± 1831993 ± 3031351 ±0332263 ± 0401478 ± 1022169 ± 101

Exp. 2

803 ± 0281051 ±0351601 ± 102790 ± 068

1435 ± 042013 ±03995 ± 43

1970 ±521260 ±081621 ±092610 ±072

ND*2228 ± 2012266 ± 0872630 ±071815 ±022789 ±015

Ratio of

observed/predicted

0.98 ±0.010.97 ±0.010.87 ± 0.020.90 ± 0.020.91 ±0.050.84 ± 0.041.02 ±0.05

ND0.97 ± 0.020.89 ± 0.030.91 ±0.030.52 ± 0.040.50 ±0.01

N

4434442

22212

' ND, not determined.


No. 5 INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polansky 1709

1000

Total 8 0 0

IP's/cpm\ 6 0 0

UmlJ400

200

Bombesin

Carbachol

Vasopressin

o o " 1 0 9 8 7 6 5 4 3

-log [AGONIST] (M)

Fig. 2. Effect of vasopressin, carbachol, and bombesin concentra-tion on total inositol phosphate stimulation. Cells confluent for 10days were labeled with [3H] myoinositol for 24 hr and then treatedwith 10 mM LiCl and various concentrations of vasopressin (D),carbachol (O), or bombesin (A) for 30 min at 37 °C. The incuba-tions were terminated and total inositol phosphates analyzed asdescribed in the legend to Figure 1.

for longer times after attainment of confluence devel-oped a carbachol and a vasopressin response (Fig. 1).At 2 d, no effect of 1 mM carbachol (Fig. la) or 1 nMvasopressin (Fig. lb) could be seen. By 4 d, however,

3000

IPj 2000

/cpm\\2ml/ 1000

IPo/cpm\\2ml/ 100

IPs/cpm\\2ndl

TIME (min)

Fig. 3. Stimulation of InsP,, InsP2, and InsP3 by vasopressin.Cells were labeled with [3H] myoinositol for 24 hr and treated with10 mM LiCl for 30 min and 100 nM vasopressin for various lengthsof time. The incubations were terminated and inositol phosphateswere extracted as described in Materials and Methods. InsP,, InsP2,and InsP3 were step-eluted from AG 1 X 8 columns and 2-ml ali-quots counted as previously described.12

responses to both effectors were detectable, and by 6 da two-fold stimulation of inositol phosphates by car-bachol was achieved, while vasopressin stimulated athree-fold rise in inositol phosphates by 10 d. For com-parison, the response to histamine also was measured(Fig. lc). Here, the response increased from 2- to 2.5-fold stimulation at day 2 to a five-fold stimulation byday 6. The neuropeptide bombesin also was found tostimulate inositol phosphates (Fig. Id). Like the re-sponse to histamine, the response to bombesin waspresent at 2 d after confluency. In contrast to otherstimulators, bombesin-specific stimulation declinedwith extended time in monolayer culture, falling to50% of maximal by day 6.

The effect of vasopressin, carbachol, and bombesinconcentration on inositol phosphate formation isshown in Figure 2. Vasopressin achieved a maximalincrease of 2.3-fold at 0.1 jiM, with an EC50 (half-maximal stimulatory concentration) of 200 nM. Car-bachol gave about a two-fold increase at 1 mM, withan EC50 of 40 IJM. Bombesin gave a 2.2-fold stimula-tion at 10 nM with an EC50 of 0.25 nM. The stimula-tion of InsP,, InsP2, and InsP3 in response to treat-ment with 1 pM vasopressin is shown in Figure 3. Aswas observed with histamine,12 InsP2 and InsP3

showed early increases of 1.5- to 1.8-fold that peakedat 1 min, while InsP, increased in a sustained fashionfor at least 30 min. Similar patterns of InsP,, InsP2,and InsP3 formation were observed following treat-ment with carbachol or bombesin (data not shown).

Bombesin belongs to a family of structurally relatedpeptides, including GRP, litorin, and neuromedin B.These also were tested for the ability to stimulate inosi-tol phosphates in NPE (Fig. 4) Twenty nanomolar

1000

Total

IP's/cpm\l"STJ

800

600

400

200

Neuromedin B

oo 10 9 8 7 6 5

-log [PEPTIDE] (M)

Fig. 4. Effect of bombesin-related peptide concentrations on totalinositol phosphate stimulation. Cells were labeled with [3H] myo-inositol for 24 hr and then exposed to 10 mM LiCl and variousconcentrations of peptide for 30 min at 37°C. The incubations wereterminated and inositol phosphates analyzed as in the legend toFigure 1. Litorin (0), GRP (•), and neuromedin B (A).



litorin caused a 2.6-fold stimulation of total inositolphosphate formation, with an EC50 of 0.8 nM. GRPgave a maximal stimulation of 1.9-fold at 10 nM, withan EC50 of 1.8 nM, whereas neuromedin B gave amaximal stimulation of 2.6-fold at 10 /JM, with anEC50 of 0.2 nM. Regarding EC50, the rank order was:bombesin < litorin < GRP < neuromedin B. Withrespect to maximal stimulation, the order was: litorin= neuromedin B > bombesin > GRP. These peptidesmay stimulate inositol phosphates via the same recep-tor, because treatment of cells with 10 nM bombesinplus 10 nM neuromedin B or 10 nM litorin causedstimulation of inositol phosphates roughly equivalentto that caused by bombesin alone (see Table 1).

Agonist and Antagonist Studies

Acetylcholine antagonists were employed to estab-lish rank order of effectiveness in inhibiting the carba-chol-specific stimulation of inositol phosphates. Pir-enzepine, an M,-specific antagonist,22 methoctra-mine, an M2-specific antagonist,23 and 4DAMP, anM3-specific antagonist,24 were used. Figure 5 showsthat 4DAMP was the most potent antagonist tested,with an IC50 (half maximal inhibitory concentration)of 3 nM and complete inhibition of carbachol stimula-tion at 100 nM. Pirenzepine showed an IC50 of 3.5 /xMwith complete inhibition at 100 fxM, and methoctra-mine showed inhibitory effects only at concentrationsabove 60 ^M. The rank order of IC5Os for the carba-chol response therefore was 4DAMP < pirenzepine< methoctramine.

Three antagonists of vasopressin then were testedfor their ability to inhibit inositol phosphates forma-

1200r

1000

TotalIP's 800

/cpm\ 6 0 0

Vml )

400

200

Methoctramine

4 DAMP

10

-log [ANTAGONIST] (M)

Fig. 5. Effect of cholinergic antagonists on inositol phosphatestimulation by carbachol. Cells were labeled 24 hr with [3H] myo-inositol and then treated for 30 min with 10 raM LiCl and 1 mMcarbachol plus various concentrations of the following: 4DAMP(A); pirenzipine (•); or methoctramine (O). The incubations wereterminated and inositol phosphates analyzed as described in thelegend to Figure 1. Control (LiCl alone) values were 418 ± 16cpm/ml.

tion by vasopressin. d(CH2)5 [Tyr(Me)2]AVP, a V, se-lective antagonist;25 d(CH2)5 [Tyr(Et)2,Val4]AVP, amixed V,/V2 antagonist;26 and d(CH2)5[Ile

2,Val4]-AVP, a V2-selective antagonist,27 were added in in-creasing concentrations to cells simultaneously given100 nM vasopressin. The results of such an experi-ment (Fig. 6a) show that the IC5Os of all three antago-nists were roughly the same (20-40 nM). In all cases,vasopressin-stimulated inositol phosphate formationwas completely inhibited by 10 MM antagonist.

Two agonists of vasopressin also were tested (Fig.6b). Deamino [Arg8] dAVP, a potent V2-specific ago-nist in animals,28 PIOVP, a V! agonist in animals,29

and vasopressin were compared for their ability tostimulate NPE inositol phosphates. dVAP stimulatedwith an EC50 of 30 nM, about five times lower thanthat of vasopressin (EC50 = 150 nM in this experi-ment), with a maximum attained by both peptides at1 )LiM. PIOVP, in contrast, showed a maximal stimula-tion of 1.4 fold compared to a maximal stimulationby vasopressin and dAVP of 2.3 fold. An EC50 of ap-proximately 20 nM was observed with this partial ago-nist. The rank order of potency therefore was dAVP> vasopressin, with PIOVP a submaximal stimulator.These data present a paradox, because vasopressin re-ceptor subtypes have been defined by second messen-ger linkages (V1 to cAMP formation and V2 to intra-cellular Ca2+ elevation 30). This is addressed in theDiscussion.

Bombesin stimulation of inositol phosphate forma-tion was probed with three bombesin antagonists:peptide A* ([D-Arg1, D-Pro2, D-Trp7-9, Leu11] sub-stance 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 Dwas shown to be five-fold more potent than peptide Ain blocking bombesin effects.31 Figure 7 shows the re-sults of adding various concentrations of each antago-nist with 1 nM bombesin to NPE cells. Peptide Dblocked stimulation of inositol phosphates by bombe-sin with an IC50 of 0.4-0.7 nM, with maximal inhibi-tion achieved at 3 fiM. Peptide A inhibited with anIC50 of 1.4 ixM, with maximal inhibition at 10 ixM.Thus, peptide D was about 3.5 times more effectivethan peptide A. Spantide inhibited with an IC50 of0.2-0.6 /iM with total inhibition at 3 ^M. The rankorder of potencies therefore was spantide ^ peptide D> peptide A.

Additivity Studies

The responsiveness of NPE to several neurotrans-mitters and neuropeptides suggested the possibilitythat in vivo NPE might be stimulated by combina-tions of these effectors. Numerous examples exist ofneurotransmitters and neuropeptides contained


No. 5 INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook ond Polonsky 1711

Fig. 6. Effect of vasopres-sin antagonists and agonistson inositol phosphate stimu-lation by vasopressin. Cellswere labeled with [3H]myoinositol for 24 hr andthen treated as follows, (a)Antagonists: cells weretreated for 30 min with 10mM LiCl and 100 nM vaso-pressin, plus various con-centrations of d(CH2)5D-IleVAVP (•); d(CH2)Tyr(Me)-AVP (O); or d(CH2)5Tyr(Et)VAVP (V). The incubationswere terminated and inosi-tol phosphates analyzed asin the legend to Figure 1. (b)Agonists: cells were treatedfor 30 min with 10 mM LiCland various concentrationsof vasopressin (O), dAVP(•), or PIOVP (V). The in-cubations were then termi-nated and inositol phos-phates analyzed as in the leg-end to Figure 1.

a. b.1000r

TotalIP's

/cpm\\ml /

100nM Vasopressin +Od(CH2)Tyr(Me)AVPad(CH2)D-IleVAVPvd(CH2)Tyr(Et)VAVP

500-

400-

1600

1400

1200

1000

800

600

400

PIOVP

Vasopressin

10

-log [ANTAGONIST] (M) -log [AGONIST] (M)

within the same neuron and presumably released to-gether upon depolarization.32"34 Moreover, ciliaryprocesses are known to be supplied with both adrener-

600

500Total

IP's/cpm\\ml / 400

300

250-

oo

1nM Bombesin +o Peptide A

D Peptide D

v Spantide

7 6 5

-log [ANTAGONIST] (M)

Fig. 7. Effect of bombesin antagonists on inositol phosphate stim-ulation by bombesin. Cells were labeled with [3H] myoinositol for24 hr and then treated for 30 min with 10 mM LiCl and 1 nMbombesin, plus various concentrations of the following: spantide(V); peptide D (•); or peptide A (O). The incubations were termi-nated and inositol phosphates analyzed as in the legend to Figure 1.Control (LiCl alone) values were 346 ± 17 cpm/ml.

gic and cholinergic nerves, as well as nerves contain-ing a variety of neuropeptides (for review, see refer-ence 1). Accordingly, experiments were conducted inwhich various combinations of carbachol, vasopres-sin, bombesin, and histamine were added to cells andthe inositol phosphate response ("observed") wascompared to the responses of the sum of each agonistadded individually ("predicted"). The ratio of ob-served to predicted will be 1 if the stimulation bymore than 1 effector is equivalent to the sum of theresponses of each effector added alone.35 Maximallystimulating concentrations of each effector were usedin each case to simplify interpretation of the data. Ta-ble 1 shows that in all cases treatment with combina-tions of effectors (lines 5-15) resulted in inositol phos-phate stimulations greater than observed with eachagonist alone. In most cases, the stimulations werewithin 10-15% of that predicted on the basis of pureadditivity, which is consistent with the concept thateach effector stimulates inositol phosphates via a dif-ferent receptor.36 No profound inhibitions or stimula-tions of one member of a group by the others wereobserved, which suggests that the receptors for eacheffector probably act independently of one another.Stimulation by combinations of the related peptidesbombesin, neuromedin B, and litorin, which stimu-late to a similar degree when added independently(Fig. 4), were not additive (lines 16-17). Bombesinstimulation was not further increased by neuromedinB or litorin, which is consistent with the concept thatthese peptides stimulate inositol phosphates via thesame receptor.


1712 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol. 33

c.Bomb GP EGTA

1 U

Orr<

LLJo<rr

100 200 300 400 500

200-

100-

300 0 100 200 300

TIME (sec)

Fig. 8. Stimulation of intracellular Ca2+ by vasopressin, carbachol, and bombesin. (a) Vasopressin. Cell suspensions prepared from cellsconfluent for 7 d and treated with indo-1-AM as described in Materials and Methods were given 1 fiM vasopressin, and the change influorescence measured and converted to molar concentrations, (b) Carbachol. Cells prepared as described in (a) were given 1 mM carbacholand intracellular Ca2+ was measured, (c) Bombesin. Cells prepared as described were given 100 nM bombesin and intracellular Ca2+ wasmeasured 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 MMvasopressin 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 inositolphosphates in many systems has been linked to subse-quent increases in intracellular Ca2+,37 we evaluatedthe effect of vasopressin, carbachol, and bombesin onintracellular Ca2+ levels in NPE cells using the fluores-cent calcium probe, indo-1-AM.21 Figure 8 shows fluo-rescence tracings from cells treated with the three ef-fectors. Determination of Fmax (addition of Genapolx-080) and Fmin (addition of EGTA), which allowedconversion of fluorescence units to Ca2+ concentra-tions, are also shown (Fig. 8a-c). From a resting levelof 95 nM, 1 fiM vasopressin evoked a rapid rise to 180nM, about a two-fold increase in intracellular Ca2+

(Fig. 8a). Intracellular Ca2+ then declined to a plateauthat was maintained for at least 8 min. One millimo-lar carbachol (Fig. 8b) elicited a smaller response, a1.5-fold rise to 142 nM, whereas 100 nM bombesin(Fig. 8c) gave a two-fold elevation to 211 nM intracel-lular Ca2+. These responses occurred within 5-15 secfollowing introduction of an agonist into the cuvette.The biphasic Ca2+ response exhibited by stimulatedNPE is characteristic of electrically nonexcitable cells.After an initial spike, the Ca2+ level fell to a new, sus-tained level.38

To determine whether extracellular Ca2+ is re-quired for the intracellular Ca2+ flux to occur, cellswere suspended in indo buffer either lacking CaCl2 orcontaining 1 mM CaCl2, and the responses to 100 nM

bombesin were compared (Fig. 8d). The initial peak,although slightly reduced, was present in cells lackingCaCl2 compared to cells with CaCl2. In the absence ofCaCl2, however, intracellular Ca2+ fell rapidly to base-line instead of establishing a second plateau. Thus, theinitial Ca2+ spike did not require extracellular Ca2+,while the second phase did.

Dose-response curves for carbachol, vasopressin,and bombesin elevations of intracellular Ca2+ areshown in Figure 9. The EC50 for intracellular Ca2+

elevation by carbachol was 100 j*M (Fig. 9a), similar

-~ 250r

Bombesin

u

13

o

2

200-

150-

100-

10

Vasopressin

8 7 6 5 4-log [AGONIST] (M)

Fig. 9. Effect of agonist concentration on intracellular Ca2+ stimu-lation. Cells prepared and treated with indo-1-AM as in Figure 8awere aliquotted, given various concentrations of vasopressin (D),carbachol (O), or bombesin (A), and peak fluorescence was con-verted into molar concentrations. Each point represents an averageof three to six determinations.


No. 5 INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook and Polansky 1713

200-

Fig. 10. Augmentation and antagonismof carbachol-stimulated intracellular Ca2+

levels, (a) Effects of subsequent addition ofvasopressin. Cells prepared as in Figure 8awere given 1 mM carbachol. When thepeak of the Ca2+ response had beenreached, 1 /*M vasopressin was added andintracellular Ca2+ levels monitored as inFigure 1 a. (b) Effect of 4DAMP. Cells stim-ulated with 1 mM carbachol as in (a) weregiven 100 nM 4DAMP where indicated,and intracellular Ca2+ monitored as in (a).

+CO

O

CO<

LUo<cc

100-

Carbachol 4 DAMP

Carbachol

f Vasopressin

100 200 300 400 500 0

TIME (sec)

100 200 300

to that observed for inositol phosphates formation(Fig. 2). Vasopressin gave an EC50 of 180 nM, with amaximum at 1 nM. These values are also similar tothose for inositol phosphates stimulation (Fig. 2).Bombesin gave an EC50 of 10 nM, or 40-fold greaterthan the EC50 observed for inositol phosphate stimula-tion (0.25 nM). Maximum Ca2+ levels were attainedat 100 nM bombesin.

To determine whether the presence of a receptorantagonist would cause the reversal of intracellularCa2+ stimulation by an agonist, we tested the effect ofthe muscarinic M3 antagonist 4DAMP on carbacholstimulation of intracellular Ca2+ (Fig. 10a). Additionof 4DAMP after stimulation of intracellular Ca2+ with1 mM carbachol resulted in an immediate drop ofCa2+ to levels near baseline. This suggests that thecontinued occupation of an M3 receptor by carbacholis required for maintenance of elevated intracellularCa2+. We also sought to determine whether increasedinositol phosphate stimulation response to multipleeffectors would be reflected in intracellular Ca2+ levelsby testing the sequential addition of 1 mM carbacholand 1 nM vasopressin to NPE cells. Figure 10b showsthat the maximal elevation attained by 1 mM carba-chol, 160 nM, was surpassed by the subsequent addi-tion of 1 IJM vasopressin, which gave 230 nM Ca2+. Inthis case, therefore, intracellular Ca2+ levels may beinfluenced by the presence of more than one agonist.

Discussion

The present data describe the responsiveness ofNPE, via the inositol phosphate arm of the IP/DAGpathway, to vasopressin, carbachol, and bombesin.The physiological responses of the NPE to these effec-

tors remain to be determined, but the possibility existsthat they or other agents that regulate the IP/DAGpathway could influence aqueous humor secretion.Vasopressin and carbachol have been reported to af-fect aqueous inflow and intraocular pressure,2-39 al-though the mechanisms underlying these effects areunclear. Few studies have been performed on bombe-sin in the eye,40 but like vasopressin, it is a neuropep-tide known to regulate autonomic functions.

The increase in responsiveness to histamine, carba-chol, and vasopressin observed when NPE are main-tained in a confluent monolayer over several daysraises the possibility that, at least in tissue culture, anondividing state may be a prerequisite for optimalresponsiveness. DNA synthesis in NPE cells decreasesfor about 4 d in confluent culture before reaching aminimal level (RBC, GM Lui, and JRP, manuscriptin preparation). This is approximately when signifi-cant responses to some effectors begin to appear. Thedecline in responsiveness of inositol phosphate for-mation to bombesin after NPE reach confluency isthe exception to the pattern of observed increases.The reason for this decline is not known, but bombe-sin is a potent mitogen in several cell lines29'41 andresponses to mitogens have been observed to decreasewhen dividing cells cease cell division.42 Besideschanges in the extent of responsiveness, time in mono-layer culture had no other effect on NPE responses toneuropeptides and neurotransmitters described in thepresent report. Whether bombesin might be a mito-gen for NPE cells is unknown.

Measurement of InsPl5 InsP2, and InsP3 levels afterexposure to vasopressin or other agonists under studyshowed early increases in InsP2 and InsP3 followed bya sustained rise in InsP,. This pattern is similar to our


1714 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol. 33

findings for histamine12 and conforms to the generallyheld view that InsP3 is the cleavage product of recep-tor-activated phospholipase C hydrolysis of phospha-tidylinositol 4,5 bisphosphate, and therefore appearsfirst, with InsP2 and InsPj subsequently generated bysequential dephosphorylations of InsP3.

14 The func-tion of InsP3 is thought to be to bind to specific siteson the endoplasmic reticulum, causing the release ofsequestered Ca2+, which transiently elevates intracel-lular Ca2+ levels.1443 That vasopressin, carbachol, andbombesin each elevated intracellular Ca2+ in NPEcells is consistent with this view. Wax and collabora-tors also have reported stimulation of inositol phos-phates10 and intracellular Ca2+n by vasopressin, car-bachol, and several other agents in SV40-transformedNPE cells.

Intracellular Ca2+ Studies

The intracellular Ca2+ dose-response data showthat carbachol and vasopressin stimulated at concen-trations similar to those for inositol phosphate stimu-lation. This is consistent with the concept that InsP3

stimulation leads to a rise in intracellular Ca2+. Withbombesin, however, significantly greater concentra-tions were required to achieve EC50 and maximal val-ues relative to inositol phosphate stimulation. Thiscould be interpreted to mean that Ca2+ stimulationoccurs by means other than InsP3 elevation with thisagent. However, we cannot rule out the possibilitythat treatment of cells with trypsin to obtain suspen-sion cultures resulted in modified bombesin recep-tors, with the result that higher concentrations of thispeptide than normal were required to stimulate. Fur-ther experiments will be needed to confirm dose-de-pendent differences between inositol phosphate andintracellular Ca2+ stimulations in NPE cells.

The rapid decline in intracellular Ca2+ after stimula-tion by agonists in the absence of extracellular Ca2+

suggests that the sustained phase of the calcium re-sponse is a result of influx of extracellular calciumthat replenishes depleted intracellular stores, as de-scribed in other systems.4445 The calcium responsesreported in the study generally occurred after 5-10 secand peaked at 10-15 sec after the addition of agonist(Fig. 8). InsP3 in these and prior studies12 could beseen at 30-45 sec, but the experimental protocol inthe present studies did not allow for sufficiently shortincubation times (1-5 sec) that would permit theorder of appearance of changes in InsP3 and Ca2+ tobe determined. Although current belief is that an ele-vation of InsP3 precedes an elevation of cytosolicCa2+, evidence that contradicts this sequence has beenreported.46

Many cellular responses to neuronal stimuli occurover millisecond time periods rather than seconds or

minutes. Methods such as patch clamping and inte-gration of membrane fragments into lipid bilayers willbe needed to accurately measure such responses.

Agonist and Antagonist Studies

Pharmacological studies of receptors for vasopres-sin, carbachol, and bombesin using agonists and an-tagonists allowed preliminary characterization ofthese receptors. In animals, two types of peripheralreceptors are known for vasopressin. V, receptorsmodulate pressor and glycogenolytic responsesthrough a Ca2+-dependent pathway, whereas V2 re-ceptors modulate antidiuretic responses via a cAMP-dependent pathway in renal tubules.30 With this crite-rion, the vasopressin receptor on NPE cells is V,. How-ever, our finding that the V2-specific agonist dAVPwas 4-5 times more effective than vasopressin in stim-ulating inositol phosphates, whereas the V,-specificagonist PIOVP only stimulated half as well, is consis-tent with a V2 subtype. This apparent contradictionmay be resolved by reports that vasopressin antago-nist and agonist responses vary among species.47"49

Thus, agonist and antagonist data derived from ani-mal studies may not apply to human vasopressin re-ceptors. It is noteworthy that SKF 101926, a strongantidiuretic antagonist in rats, behaves in man as aV,/V2 mixed antagonist,47 which is consistent withour antagonist data. Except for platelet recep-tors,48>50>51 vasopressin receptors have not been wellcharacterized in human tissue. The present study,therefore, defines the vasopressin receptor on NPEcells as V, based on linkage to intracellular Ca2+. How-ever, the present study also provides evidence that thepharmacological characteristics of this receptor maybe different from V\ receptors in other species.

Cholinergic receptors are either of the nicotinic ormuscarinic subtypes. Nicotinic receptors are locatedin autonomic ganglionic synapses and striated mus-cles. Muscarinic receptors are found in smooth mus-cle and glands.38 Muscarinic receptors are subdividedpharmacologically into Ml5 M2, and M3 subtypes.24

The antagonist rank order determined for the NPEcholinergic receptor was: 4DAMP > pirenzepine> methoctramine. Tubocurarine, a nicotinic receptoragonist, had no effect on NPE cells at concentrationsof up to 1 mM (data not shown). The greater effi-ciency (by three logs) of 4DAMP compared to piren-zepine or methoctramine is consistent with the NPEreceptor being of the M3 subtype. This also is consis-tent with results using SV40-transformed human cili-ary epithelial cells.10 Recombinant DNA methods re-cently have been used to detect four genes for musca-rinic receptor proteins,52 so pharmacological analysismay not be distinguishing all subtypes. Further workwill be needed to determine whether more than one


No. 5 INOSITOL PHOSPHATES AND CALCIUM IN HUMAN NPE CELLS / Crook ond Polansky 1715

class of muscarinic receptor is present in human NPEcells.

Bombesin receptor antagonist studies gave a rankorder of potencies of spantide > peptide D, with pep-tide A appearing to be a partial agonist. The potenciesof peptides D and A are similar to those observed inSwiss 3T3 cells and human small cell lung cancerSCLC cells.30 However, spantide was considerablyless potent in those studies than in the present experi-ments, suggesting that differences exist between theNPE receptor and those on Swiss 3T3 or small celllung cancer cells. For example, the substance P deriva-tives used in the present study, which possess the high-est specificity reported to date,30 also bind to vaso-pressin receptors.30'53 The bombesin effect on NPEcells is probably not achieved through binding to va-sopressin receptors, however, because additivity ex-periments showed that inositol phosphate stimulationby maximal concentrations of bombesin plus vaso-pressin given together caused a response nearly equalto the sum of the responses caused by either effectoralone. Such a result suggests separate sets of receptorsfor vasopressin and bombesin.35 If bombesin actedthrough vasopressin receptors, the combination ofbombesin plus vasopressin would have been expectedto give maximal stimulation between that of vaso-pressin or bombesin alone. The same argument ap-plies to carbachol and histamine and suggests thateach effector acts via receptor population distinctfrom the others.

The cells used in the present study were of fetalorigin. Thus, there could be differences between theresponses reported here and those of adult cells. Fur-thermore, responses observed in cells maintained intissue culture may differ from responses of cells invivo, because of alterations of neuronal and tonic envi-ronments. Based on these cautions, the responsive-ness of cultured NPE cells to vasopressin, carbachol,and bombesin, in addition to histamine, raises the pos-sibility that these bioactive compounds might influ-ence aspects of ciliary epithelium physiology in vivo.We recently have found that stimulation of the DAG-protein kinase C arm of the IP/DAG pathway causesalterations in K+ transport in NPE (RBC, DK vonBrauchitsch, and JRP, in preparation). Further studyof this signalling pathway in the ciliary epitheliummay provide information about physiological mecha-nisms that play a regulatory role in ion transport. Thiscould be relevant to the study of aqueous humor in-flow.Key words: neuropeptides, carbachol, ciliary epithelium,inositol phosphates, calcium

AcknowledgmentsThe authors wish to thank Drs. Nejat Duzgunes, Deme-

trios Papahadjopoulos, and Arthur Weiss for the use of their

fluorimeters; Drs. Mark Goldsmith and Dennis Alperts forinstruction in calcium measurements; Ms. Tracy Hydornfor ECM preparation; and Richard Dye for manuscriptword processing.

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