THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. …hibited by M atropine, sodium substitutes, or lo-‘ M tetrodotoxin. Exposure of lobules to elevated concen- trations of potassium chloride

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255.. No. 10, h u e of May 25, pp. 4918-4927, 1980 Printed ut U.S. A.

Potassium- and Ionophore A23187-induced Discharge Protein in Guinea Pig Pancreatic Lobules ROLE OF EXTRACELLULAR CALCIUM*

of Secretory

(Received for publication, January 15, 1979, and in revised form, March 10, 1980)

George Scheele and Asher Haymovits From the Rockefeller Uniuersity, New York, New York a n d the State University of New York, Downstate Medical Center, Brooklyn, New York

Elevated concentrations of potassium chloride (50 to 120 mM) in the incubation medium stimulated in vitro discharge of secretory protein from guinea pig pancreatic lobules. The effect of potassium was not inhibited by M atropine, sodium substitutes, or lo-‘ M tetrodotoxin. Exposure of lobules to elevated concentrations of potassium chloride did not increase the release of tissue lactic dehydrogenase and resulted in the appearance of exocytotic images detected by electron microscopy. The time course and extent of discharge due to 75 m~ KC1 were similar to those caused by the ionophore A23187 and the secretory effect of both agents depended on extracellular calcium and intracellular energy reserves. Potassium chloride stimulation of 75 mM increased the influx of extracellular calcium by 498, as measured by net 4SCa uptake. Opti- mal carbamylcholine chloride or pancreozymin stimulation consistently showed a greater effect on discharge than optimal KC1 or A23187 stimulation and the additional effect depended on the ability of these physiological secretagogues to recruit calcium from intracellular sources. Potassium chloride stimulation did not result in cyclic GMP elevations in the presence of atropine and those elevations due to A23187 stimulation were small (21 to 30%) and dissimilar both in character (calcium dependence) and time course compared to those resulting from the physiological secretagogues. These findings allow us to define two interrelated pathways which couple hormonal stimulation and discharge of secretory protein in the exocrine pancreas.

The sequence of events which couple secretagogue stimulation to the discharge of exportable protein in the exocrine pancreas has drawn considerable attention in recent years (summarized in References 1 and 2). Despite this attention, a number of critical questions remain to be answered. First, although cholinergic and hormonal stimulation have been shown to result in depolarization of the acinar cell membrane (3-8), attempts to cause pancreatic enzyme secretion by potassium-induced membrane depolarization have not been successful (7, 9-11). Second, while the effect of the ionophore A23187 (7, 11, 12-17) resulting in discharge of secretory protein requires the presence of extracellular calcium,’ the results

* This research was supported in part by Grants AMDD 18532 and AMDD 11431 from the National Institutes of Health and by a grant from the CIBA-GEIGY Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ Names of all metals indicate their use in ionic form.

of studies with 45Ca (5, 14, 18-25) and chlortetracycline (26, 27) have suggested that physiological stimulation is associated with translocation of calcium from intracellular sources. Using guinea pig pancreatic lobules we have undertaken a critical reevaluation of these discrepant findings. In this paper we examine in detail the kinetics of discharge of secretory protein and tissue levels of cGMP resulting from elevated concentrations of KC1, ionophore A23187, and carbamylcholine as a function of the absence and presence of atropine, calcium, calcium analogues, and energy inhibitors in the incubation medium. The associated and dissociated findings presented here and elsewhere (28) allow us to arrange in probable sequence a number of events in two coordinated pathways which mediate the stimulus-secretion process. A preliminary report of these findings has been presented (29).

EXPERIMENTAL PROCEDURES

Materials

Carbonyl cyanide m-chlorophenylhydrazone, ethylene glycol bis(P- aminoethyl ether)N,N,N’,N’-tetraacetic acid, and tetrodotoxin were from Sigma. Carbamylcholine and ~-[4,5-’H]leucine were obtained from Schwarz Mann Biochemicals and Radiochemicals, Orangeburg, N. Y. Lanthanum chloride was from Fisher Scientific Co. Tritiated inulin (2.37 Ci/mM) and 45Ca (2.16 mCi/ml) were obtained from Amersham Radiochemical Centre, England. Reagents for the radioimmunoassay of cGMP were obtained from New England Nuclear, Boston, Mass. All other reagents were of analytical grade. Cholecys- tokinin-pancreozymin, 500 Ivy Dog Units/mg, was from Dr. Mutt, Karolinska Institutet, Stockholm, and ionophore A23187 was from Dr. R. Hosley of Eli Lilly.

Methods

Incubation of Pancreatic Lobules Guinea pig pancreatic lobules were prepared according to Scheele

and Palade (30) and incubated in vitro in a physiological salt solution containing (except where indicated) 125 mM NaCl, 5 mM KCl, 2.53 mM CaC12, and 1.16 mM MgS04 buffered with either 25 mM Hepes’/ NaOH, pH 7.4, or 25 mM Tris-HCl, pH 7.4, and supplemented with physiological levels of glucose, 1 mg/ml, and essential and nonessential amino acids (31). Incubations with 6 lobules/flask were carried out at 37°C with appropriate secretagogues and inhibitors for varying periods of time under 100% 0 2 atmosphere. The addition of a circular piece of nylon mesh, Nitex 114p (1.5 cm in diameter), allowed efficient transfer of lobules from one flask to another since lobules adhered to the mesh. In experiments where the potassium chloride concentration was elevated, sodium chloride concentrations were appropriately diminished to maintain isotonicity. In experiments where atropine, CCCP, or Nz atmosphere was used, pancreatic lobules were exposed

The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera- zine ethanesulfonic acid CCCP, carbonyl cyanide m-chlorophenylhydrazone; carbachol, carbamylcholine chloride; EGTA, ethylene glycol bis(B-amino ethyl ether)N,N,N’,N’-tetraacetic acid.

_ _ _ _ _ _

4918

Potassium- and Ionophore A23187-induced Pancreatic Secretion 4919

to these agents 10 min prior to stimulation with carbamylcholine, calcium ionophore, or elevated concentrations of potassium chloride.

When pancreatic lobules were incubated in the absence of calcium several precautions were taken in the preparation of calcium-free medium and in the rapid reduction of calcium contained in the extracellular space of pancreatic lobules. In the preparation of calcium-free medium, calcium contamination was measured in all stock reagents, using atomic absorption spectroscopy, and individual stock reagents were chosen accordingly (28): ( a ) MgS0, contained 25 to 50 times less calcium than MgC12, ( b ) commercial preparations of essential and nonessential amino acids which contained 3.15 to 7.73 p~ calcium at physiological levels were omitted from the medium, and ( c ) bovine serum albumin (1 X crystallized from Sigma) which contained 2 pmol of calcium/g of bovine serum albumin was exhaustively dialized first against two changes of 500 mM KC1 and second against two changes of double distilled water (final calcium concentration 5 nmol/g of bovine serum albumin). Using these precautions, "calcium- free medium" contained calcium levels ~ 1 0 - ~ M. In order to achieve the rapid reduction of extracellular calcium in the tissue, ( a ) pancreatic lobules were rinsed in a 1000-fold volume of calcium-free medium compared to the extracellular space volume (60 to 70% of ['HH]sucrose, a marker of the extracellular space, was removed from pancreatic lobules using this rinse technique), and (6) introduced into experimental flasks containing a -1000-fold volume of calcium-free incubation medium. When EGTA was added to calcium-free incubation medium, EGTA stock solutions were first neutralized to pH 7.0 with NaOH. In experiments where strontium chloride or barium chloride was substituted for calcium chloride, MgCL was used in place of MgSO, to prevent the formation of insoluble SrS04 or BaS0, crystals and lo-, M EGTA was used to remove the calcium contaminating the MgC12 and BaC12 solutions.

When pancreatic lobules were incubated in the presence of the calcium ionophore A23187, the following steps were taken. Lobules were rinsed in a 1000-fold volume of calcium- and magnesium-free medium prior to their introduction at time zero into the same incubation medium containing the calcium ionophore. At the 20-min time point calcium was added to the incubation medium. This procedure allowed adequate time for uptake of ionophore into pancreatic membranes prior to stimulation with calcium added to the medium (12, 15).

Radioactive Protein Assay of Pancreatic Secretion Secretory proteins were radiolabeled by the following pulse-chase

protocol. Pancreatic lobules were pulsed with 0.44 /AM ~-[4,5-'HH]leucine (20 pCi/ml) for 15 min during which time approximately 95% of the trichloroacetic acid-insoluble radioactivity was incorporated into secretory protein as judged by the two-dimensional gel electrophoresis procedure of Scheele (32). Pancreatic lobules were then washed, transferred, and incubated in a second flask containing physiological quantities of nonradioactive essential and nonessential amino acids for 90 min, during which time the majority of the radiolabeled secretory proteins were transported to the zymogen granule pool. The lobules were then transferred to the experimental flask, which contained additional chase medium (5 ml) with the appropriate reagents under study. Sequential aliquots of the medium were withdrawn during the incubation period. Tissue was taken at the end of incubation and homogenized in 1% Triton X-100 and 25 mM Tris-HC1 at pH 9. Medium and tissue aliquots (100 pl), which were processed for trichloroacetic acid-insoluble radioactivity (33), contained between 250 and 4000 cpm. The data were expressed as cumulative discharge of labeled protein into the incubation medium as percentage of total labeled protein (tissue plus medium) as a function of time. During basal discharge or discharge stimulated by physiological secretagogues, carbachol or pancreozymin, the data derived from this pulse- chase protocol closely agreed with those derived from assay of amylase.

At early times during potassium chloride stimulation (0 to 20 min) we frequently observed a lag in the discharge of pulse-labeled proteins (see Figs. 4 and 6 ) relative to amylase activity. The presence of this lag was found to depend on the interval of chase of pulse-labeled proteins prior to the stimulation period. It progressively declined during chase intervals longer than 60 to 90 min. Carbachol stimulation resulted in a similar lag, but less pronounced since it occurred only after short chase intervals, 0 to 30 min. Since discharge of amylase represents the release of secretory proteins from all granules and discharge of radioactivity represents predominantly the discharge of proteins from recently formed granules, these observations indicate that cytoplasmic mixing of zymogen granules, formed at varying

times, is less efficient during potassium chloride stimulation than during carbachol Stimulation.

Flux Studies Cellular content was measured in lobules incubated under

steady-state conditions. Lobules were incubated at 37°C under 95'% 02, 5% C02 in Krebs Ringer bicarbonate buffer, containing 2.5 mM CaCL, 1.0 pCi/ml of [45Ca]C12, and 0.5 pCi/ml of ['Hlinulin. Following an equilibration period of 60 min, the lobules were transferred to experimental flasks containing identical concentrations of '"Ca, and [3H]inulin. Experimental incubations were terminated at 5, 10, 20, and 40 min by rapid (10 s) placement of lobules on a Millipore filter and rapid (-25 s ) wash with ice cold saline under gentle suction. The lobules were then homogenized in 1% Triton X-100 and 20 mM Tris-HCI, pH 9.0. Duplicate aliquots were analyzed for and "H using double-label scintillation spectrometry and protein by the method of Lowry et al. (34). Tissue content of was corrected for the amounts of contained in the extracellular space using the 45Ca/JH ratios in the incubation media and tissue homogenates. The net calcium uptake was then expressed in nanomoles/mg of protein.

Extraction and Measurement of Cyclic GMP Timed incubations were terminated by removal of the nylon mesh

disc with the adherent lobules and instant freezing of the tissue in liquid NZ. The frozen tissue was suspended and homogenized in 3 ml of ice cold 6% trichloroacetic acid. The homogenate was centrifuged at 5oOO x g for 15 min. The precipitate was used for the determination of DNA content by the diphenylamine method of Burton (25). Cyclic nucleotides were extracted from the supernatant as described by Haymovits and Scheele (36) and cyclic GMP was measured by the specific radioimmunoassay of Steiner et al. (37) as modified by Frandsen and Krishna (38). Tissue nucleotide levels were expressed as picomoles/pg of DNA/sample.

Determination of Enzyme Activities Amylase-The procedure of Bernfeld (39) was used with soluble

starch as substrate. Activity was expressed as porcine amylase equivalents, using Worthington amylase AABBA, 900 units/mg as standard.

Lactate Dehydrogenase-The oxidation of NADH2 by lactate dehydrogenase in the presence of sodium pyruvate was followed by the initial rate of decrease in absorbance at 340 nm (40).

Determination of Acetyl Choline Levels Acetyl choline was measured in the incubation medium and the

tissue homogenate by Dr. P. Szilagyi in the Department of Neurology at the Mount Sinai School of Medicine, City University of New York, New York, using the coupled gas-liquid chromatography-mass spectroscopy method (41). Diisopropyl fluorophosphate (5 X M ) was added to the incubation medium during the experiment to inhibit cholinesterase activity. Diisopropyl fluorophosphate in concentrations up to lo-" M had no effect on the discharge of secretory protein.

Electron Microscopy Pancreatic lobules were fxed in 2.58 glutaraldehyde in 0.1 M

cacodylate buffer for 1 h at 3°C and postfixed in 1% oso4 in veronal acetate buffer for 1 h at 3°C. Lobules were stained with 0.5% uranyl acetate in veronal acetate buffer before dehydration and Epon embed- ding. Sections were cut on a Porter-Blum MT2-B ultramicrotome (DuPont Instruments, Sorvall Operations, Newtown, Conn.) equipped with a diamond knife (DuPont Instruments, Wilmington, Del.). They were stained with uranyl acetate (42) and lead citrate (43) and viewed with a Hitachi electron microscope at 80 kV.

RESULTS

Postassium Chloride-stimulated Discharge of Secretory Pro- teins from Pancreatic Lobules

Dose-Response Study-Fig. 1 shows the effect of elevated concentrations of potassium chloride in the incubation medium on discharge of pulse-labeled secretory protein from pancreatic lobules. Incubation of lobules in 25 mM KC1 showed no stimulation of discharge over that in 5 m~ KCl, which represents basal discharge in the presence of physiological levels of extracellular potassium. Concentrations of potassium

4920 Potassium- and Ionophore A23187-induced Pancreatic Secretion

70 t

Incubation time (min)

FIG. 1. Kinetics of discharge of pulse-labeled proteins in pancreatic lobules stimulated with potassium chloride; effect of potassium chloride concentration, atropine, and energy inhibitors; comparison to carbamylcholine stimulation. Guinea pig pancreatic lobules were labeled with [”Hlleucine according to the pulse-chase protocol described under “Methods.” Pulse-labeled proteins were chased in this study for 120 min prior to the experimental period shown in the figure. Pancreatic lobules were then incubated in the presence of varying quantities of potassium chloride, 5 mM KC1 representing the physiological level (e), 25 mM KC1 (X) , 50 mM KC1 minus atropine (A), 50 mM KC1 plus M atropine (A), 75 mM KC1 minus atropine (v), 75 mM KC1 plus M atropine (r); 100 mM KC1 (a), and 120 mM KC1 (M); incubation of pancreatic lobules in the presence of 75 mM KC1 and N, atmosphere (0) or 75 mM KC1 and 10 ‘’ M CCCP (*). Stimulation by potassium chloride is compared to 1 0 ‘’ M carbachol stimulation (O), M carbachol plus M atropine was identical with basal discharge described above (a) and 10 ’ M carbachol plus M CCCP was identical with 75 mM KC1 and 10 ‘ M CCCP (+). Similar findings were observed in two additional experiments for 50 mM KC1 and 26 additional experiments for 75 mM KCI.

chloride from 50 to 120 mM clearly stimulated discharge; 75 mM KC1 showed the optimal stimulatory effect and resulted in discharge of 36% of pulse-labeled proteins over the 120-min incubation period as compared to basal discharge of 7.5%. Higher concentrations of potassium chloride, 100 mM and 120 mM, resulted in decreased levels of protein discharge compared to that seen with 75 m~ KCI.

Effect of Atropine-The addition of M atropine to the incubation medium, which caused complete inhibition of M carbachol-induced discharge, had little or no effect on potassium-induced discharge a t potassium concentrations of 50 or 75 mM (Fig. 1). These data indicate that potassium chloride exerts its effect directly on the pancreatic acinar cell and not indirectly through the release of acetylcholine from nerve terminals present in the tissue. Atropine, M, was routinely added in subsequent experiments when the secretory effects of elevated concentrations of potassium chloride were studied.

Effect of Energy Inhibitors-Inhibition of oxidative phosphorylation with M CCCP, an uncoupler of oxidative phosphorylation, or in the presence of 100% Na markedly inhibited the secretory effects of both KC1 stimulation and carbachol stimulation (Fig. 1). As seen previously for carbachol (30), the inhibitory effect on KC1 stimulation is more complete with CCCP than with N2.

Effect of Sodium Substitutes and Tetrodotoxin-In order to determine if the effect of potassium chloride depended on the influx of sodium ions into the pancreatic acinar cell, pancreatic lobules were stimulated with 75 mM KC1 in the

absence of sodium and in the presence of alternative mono- valent cations, LiC1, choline chloride, or tetramethylammo- nium chloride, at a concentration of 50 mM. In another study, pancreatic lobules were stimulated with 75 m~ KC1 in the presence of 50 m~ NaCl and lo-’ to M tetrodotoxin, a natural toxin from the Japanese Puffer fish, which has been shown to block voltage-sensitive sodium channels in the giant squid axon (44). At the time points tested (30,60, and 90 rnin), no significant changes were seen with any of these agents in the potassium-induced discharge of pulse-labeled protein.

Release of Lactic Dehydrogenase Actiuity-Two studies were carried out to evaluate the effect of potassium chloride stimulation on release of lactic dehydrogenase from pancreatic lobules. Incubation of lobules over 120 min gave the following results: basal discharge, 4.1% of tissue lactate dehydrogenase released into the medium; M carbachol stimulation, 2.5% release; and 75 mM KC1, 4.0% release. These data along with those concerning the effect of inhibitors of oxidative phosphorylation indicate that elevated concentrations of KC1 do not result in the nonspecific disruption of cellular compartmen- tation.

Electron Microscopy-Fig. 2a shows the morphological effect of 75 mM KC1 on guinea pig pancreatic lobules. Acinar cells and duct cells were intact by electron microscopic evaluation. At the luminal membrane of acinar cells exocytotic images were seen. In this photomicrograph two exocytotic images in neighboring cells are indicated by the arrows. The limiting membrane of each discharging granule shows no microvillar structures and is surrounded by a narrow margin of microfiiamentous material. Several membrane fragments are seen in the luminal space. Fig. 2, b and c, show caerulein ( M)-stimulated and unstimulated tissue, respectively.

Comparison of Potassium Chloride Stimulation and Car- bamylcholine Stimulation-Fig. l illustrates a quantitative comparison of optimal potassium chloride stimulation (75 mM) and optimal carbachol stimulation M); 75 mM KC1 stimulation resulted in discharge of 36% of pulse-labeled proteins over 120 min of incubation as compared to discharge of 51% by M carbachol. The secretory response to 75 mM KC1 ( n = 27) varied between experiments from 50 to 80% of that seen with optimal carbachol stimulation. The kinetics of discharge of secretory protein due to KC1 stimulation were similar to those due to carbachol stimulation in that (a) the response was sustained during the period of stimulation, and (b) discharge rates were largest at the beginning of stimulation and decreased with time.

Effect of Calcium Addition to the Incubation Medium on Potassium Chloride-stimulated Discharge of Secretory Pro-

tein Fig. 3 shows the effect of stimulation of pancreatic lobules

with 75 mM potassium chloride in the absence of calcium and in the presence of log increases in calcium added to the incubation medium. Calcium-free medium was prepared using the procedures described under “Methods.” Under these conditions, calcium-free medium completely inhibited discharge stimulated by 75 mM KC1;’

Log increases in the calcium concentration of the incubation medium containing 75 mM KC1 caused stepwise increases in the discharge of pulse-labeled proteins. Supraphysiological levels of calcium, 10 m, resulted in no greater stimulation of

When these procedures were not followed, as for example when KC1 stimulation was carried out in calcium-free medium containing essential and nonessential amino acids, calcium levels contaminating these amino acids led to 12.5 to 17% discharge of pulse-labeled proteins by the 120-min time point.

Potassium- and Ionophore A23187-induced Pancreatic Secretion 492 1

,,*'

\. r.." L

FIG. 2. Exocytotic images during 75 mM KCI-induced discharge in guinea pig pancreat ic lobules . a, pancreatic lobules were incubated in 75 mM KCI, 50 mM NaCI, 2.5 mM CaCI?, 1.16 mM MgSO.,, 25 mM Tris HCI, pH 7.4, and 10 .' M atropine for 15 min, at which time they were immersed into a fixation solution containing 2.58 glutaraldehyde in 0.1 M cacodylate buffer at 3°C and tissue was further processed by the procedures given under "Methods." Exocy- totic images are marked by the two arrows. Magnification X 15,000. b shows an exocytotic image (arrow) occurring during incubation of pancreatic lobules with 10." M caerulein. c shows pancreatic tissue in the absence of stimulants.

discharge than physiological levels of calcium, 2.5 mM. These data indicate that potassium chloride-stimulated discharge of secretory proteins from pancreatic lobules is entirely dependent on the calcium present in the extracellular fluid. They also indicate that our procedure for the rapid reduction of extracellular calcium in pancreatic lobules to minimum levels is adequate.

Effect of Calcium Analogues and Lanthanum on Potassium Chloride-stimulated Discharge of Secretory Protein

Fig. 4 shows the effect of magnesium, strontium, or barium substitution for calcium during 75 mM potassium chloride stimulation. Incubation of lobules in calcium-free medium

30 60 90 120 Incubation time (min)

FIG. 3. Kinetics of discharge of pulse-labeled proteins from pancreat ic lobules s t imulated with 75 mM potassium chloride in varying concentrat ions of medium calcium. Pulse-labeled proteins were chased in this study for 120 min prior to the experimental period shown in the figure. Pancreatic lobules were rinsed with a 1000-fold volume of calcium-free medium prior to transfer into experimental flasks containing 75 mM KC1 and the following calcium concentrations: no calcium (V), 0.01 mM Ca" (A) , 0.1 mM Ca" (A), 1.0 mM Ca" (A), and 2.5 mM or 10 mM CaL' (A). Basal discharge in the presence of physiological levels of KC1 (5 mM) in the presence or absence of 2.5 mM calcium is defined by 0. Atropine ( 1 0 I M ) was present in all experimental flasks. Similar findings were observed in two additional experiments.

35 -

30 - 0,

0 P '0 2 5 - ._ '0 m

20- 2 al a 0, > 0

15- ._ - c

= 10- s 0

5 -

a

30 60 90 120 Incubation time (min)

FIG. 4. Substi tution of magnesium, s t ront ium, or bar ium for calcium during potassium chlor ide s t imulat ion. Pulse-labeled proteins were chased for 90 min prior to the experimental period shown in the figure. Pancreatic lobules were rinsed in a 1000-fold volume of calcium-free medium prior to transfer into experimental flasks which contained 75 mM KC1 in the presence of 2.5 mM Ca" (A), or 75 mM KC1 minus Ca'+ (V), 75 mM KC1 minus Ca" plus 2.5 mM MgCl? or 2.5 mM Ba" (V), or 75 mM KC1 minus Ca" plus 2.5 mM Sr" (A). Incubation of pancreatic lobules in 75 mM KC1 f 2.5 mM Ca", and either 3 mM or 10 mM La'' is also defined by V. Basal discharge in the presence of physiological levels of KC1 (5 mM) in the presence or absence of 2.5 mM calcium is defined by 0. Atropine (lo" M), ECTA M), and MgSCI, (1.16 m) were present in all experimental flasks. Similar findings were observed in two additional experiments.


containing 2.5 mM MgC1, or 2.5 mM BaCI2 showed complete inhibition of potassium chloride-induced discharge of pulse- labeled proteins. In contrast, 2.5 mM strontium added to calcium-free medium fully supported potassium chloride-induced discharge. Lanthanum chloride, a calcium-displacing agent, in concentrations of 3 mM or 10 mM, caused complete inhibition of potassium-stimulated discharge in the presence of calcium in the incubation medium. As indicated in Refer- ence 28, lanthanum may well have additional pharmacological effects beyond its inhibitory effect on calcium influx.

4sCa Flux Studies Cellular “Ca content under steady-state conditions was

measured in basal and 75 mM stimulated pancreatic lobules as described under “Methods.” After correction for the extracellular 45Ca present in the tissue, 75 mM KC1 stimulation, in the presence of M atropine, resulted in a significant ( p < 0.005) increase in intracellular 45Ca content a t all time points tested (see Table I). This increase in tissue content of radioactive calcium represents a net influx of calcium ranging between 5.6 and 19.9 nmol of calcium/mg of protein, with a mean value of 11.8 nmol/mg, which constitutes an increase of 49% over the mean calcium content of unstimulated lobules.

Effect of Cholinergic or Hormonal Stimulation on the Dis- charge of Secretory Protein from Pancreatic Lobules Incu- bated in 75 m M KCI: Role of Intracellular Calcium Stores

It was of interest to determine if potassium chloride-stimulated discharge and carbachol-stimulated discharge modu- lated identical or separate intermediate steps involved in the stimulus-secretion process. Two observations suggested that potassium chloride and carbachol compete for similar or identical steps: ( a ) additive effects were seen when pancreatic lobules were stimulated with suboptimal levels of potassium (50 mM) and carbachol (3 X 10” mM), (b ) a t optimal levels of potassium and carbachol the secretory effect of the combined agents was no greater than that of carbachol alone (data not shown). However, carbachol stimulation modulates a component of the secretion process not shared by potassium stimulation (see Fig. 5). Potassium chloride stimulation in calcium- free medium resulted in discharge kinetics similar to those of basal discharge. When M carbachol or 0.1 u n i t / d of pancreozymin was added to the incubation medium at the 5- min time point, discharge of pulse-labeled protein increased at a markedly accelerated rate over the subsequent 15-min period. Rates of discharge decreased thereafter to levels comparable to basal discharge. The kinetics of carbachol- and pancreozymin-stimulated discharge resembled closely that component of carbachol-induced discharge demonstrated previously to be independent of calcium added to the incubation

TABLE I ’Ca content in guinea pig pancreatic lobules during stimulation

with 75 mM KC1 Measurements were made under conditions of 4sCa equilibration

between Dancreatic tissue and incubation medium. The data are

4‘

expressed as nanomoles of intracellular calcium per mg of protein. Time Basal 75 mM KC1 Increment min

5 26.7 32.3 +5.6“ 10 22.7 42.6 +19.9” 20 24.9 33.1 +8.2“ 40 22.2 35.9 +13.7”

Mean f S.D. 24.1 & 1.8 35.9 2 4.1 +11.8“ (49%)

“ p < 0.005.

Incubation time (min 1 FIG. 5. Cholinergic and peptide secretagogue stimulation of

pancreatic lobules in the presence of 75 n m potassium chloride, role of intracellular calcium stores. Pancreatic lobules were labeled by the pulse-chase protocol as indicated in the legend to Fig. 1 except that the chase period was 90 min. Following a rinse in a 1ooO- fold volume of calcium-free medium, lobules were then incubated in 75 mM KC1 in calcium-free medium (0). At the 5-min time point indicated by the arrow, M carbachol was added to one of the flasks (A), and 0.1 Ivy dog unit/ml of pancreozymin was added to another (V). A similar addition of carbachol was made to a set of pancreatic lobules in which tissue levels of calcium had been partially depleted during a 30-min preincubation in calcium-free medium (0). Basal discharge in the presence of physiological levels of KC1 (5 mM) was identical with the curve defined by 0. Similar findings were observed in one additional experiment.

medium, component I-stimulated discharge (28). Component I of carbachol-stimulated discharge was shown to depend on adequate intracellular stores of calcium and could be inhibited by preincubation of pancreatic lobules for 30 min in calcium- free medium. In order to confirm that the unique component of carbachol stimulation over that of potassium chloride stimulation shown in Fig. 5 is due to the recruitment, by carbachol stimulation, of calcium from intracellular stores, pancreatic lobules were partially depleted of tissue calcium (-55%), during a 30-min preincubation in calcium-free medium prior to their introduction into calcium-free medium containing 75 mM KC1. When M carbachol was added 5 min later, no acceleration in discharge of pulse-labeled proteins was observed (Fig. 5).

Effect of Potassium Chloride Stimulation on the Discharge of Secretory Proteins in Rat Pancreatic Lobules

Experiments ( n = 3) were carried out to evaluate the effect of elevated potassium chloride concentrations on amylase discharge in rat pancreatic lobules. The representative findings were as follows. At the 60-min incubation time point basal discharge caused 4% discharge of amylase from pancreatic lobules; M carbachol in the presence of medium calcium caused 38% discharge; 75 m potassium chloride, in the absence of atropine caused 20% amylase discharge and in the presence of M atropine caused 18.4% discharge. 75 mM KC1 stimulation in calcium-free medium caused 5.2% discharge of amylase from pancreatic lobules. These data indicate that elevated concentrations of potassium chloride stimulate directly discharge of secretory protein from rat pancreatic lobules. As seen in the guinea pig, this stimulatory effect in rat pancreatic lobules is dependent on calcium added to the incubation medium.

Ionophore A231 87-stimulated Discharge of Secretory Protein from Pancreatic Lobules; Comparison with Potassium Chlo-

ride and Carbamylcholine Stimulation Fig. 6 shows the kinetics of discharge of pulse-labeled pro-

tein from guinea pig pancreatic lobules stimulated with


60 -

50 - 0

F - % 40- a

[ 30- "

.- I -

5 - f 4 25-

V

IO -

Incubation time (min) FIG. 6. Kinetics of discharge of pulse-labeled proteins from

pancreatic lobules stimulated with the calcium ionophore, A23187; effect of calcium added to the incubation medium and effect of the energy inhibitor, CCCP; comparison to potassium chloride stimulation and carhamylcholine stimulation. Pan- creatic lobules were labeled by the pulse-chase protocol as indicated in the legend to Fig. 1. (The chase period was 90 rnin.) Lobules were then rinsed in a 1000-fold volume of calcium- and magnesium-free medium and transferred to experimental flasks containing the same medium plus M ionophore. At the 20-min time point indicated by the arrow the following additions were made: no additions (A), 2.5 nm Ca" (A), 2.5 mM Ca" plus IO-5 M CCCP (V), 2.5 mM Mg'+, 2.5 Sr2+ or 2.5 m Ba2+ gave identical results (V). In the remaining flasks no ionophore was added and 2.5 mM calcium was present from zero

change representing basal discharge (a), transfer of lobules to medium time. At the 20-min time point the following changes were made: no

containing 75 m KC1 plus 2.5 mM CaZ' (A), or the addition of M carbachol (0). Similar findings were made in four additional experiments.

M ionophore A23187.4 When 2.5 mM calcium was added after a 20-min incubation of lobules in the presence of ionophore, discharge of protein was stimulated in a quasi-linear manner for the ensuing 120 min of incubation. In the presence of calcium, ionophore caused the discharge of 38.5% of pulse- labeled proteins from pancreatic lobules compared to a basal discharge of 7.5%. In the absence of added calcium, discharge kinetics resembled closely those of basal discharge. The addition of 2.5 mM MgSO.,, 2.5 rrm SrC12, or 2.5 a BaCL to the incubation medium containing ionophore A23187 resulted in no significant stimulation of the discharge of secretory protein. The addition of calcium in log increases of concentration at the 20-min time point resulted in stepwise increases in secretory activity in the presence of ionophore (data not shown). CCCP, M, added a t 10 min of incubation, followed by 2.5 mM calcium added at 20 min blocked the effect of ionophore and calcium on the discharge of secretory protein.

Data presented in Fig. 6 also indicate that calcium ionophore stimulation and potassium chloride stimulation are similar in the kinetics and extent of discharge of pulse-labeled proteins. Carbachol, M, stimulation consistently resulted in more effective discharge at comparable time points. The increased efficiency seen with carbachol stimulation over that with either ionophore or potassium stimulation occurs pri- marily in the early time periods, 0 to 20 min.

Higher concentrations of ionophore (3 X lo-" and 10"" M ) were not used since their effects were frequently nonspecific, i.e. caused the release of pulse-labeled protein in the presence of magnesium alone or following calcium addition in the presence of M CCCP. Similar dose related toxic effects due to ionophore A23187 have been reported by others (12, 15).

Cellular Cyclic GMP Levels in Pancreatic Lobules Stimu- lated by Potassium Chloride and Carbamylcholine in the

Presence and Absence of Atropine Fig. 7 shows a kinetic analysis of the cyclic GMP levels in

pancreatic lobules stimulated with M carbachol or 75 mM potassium chloride in the absence and presence of atropine sulfate M. While carbachol-induced discharge (inset) was fully inhibited by atropine, potassium chloride-induced discharge was unaffected by the presence of atropine. The main panel in Fig. 7 indicates that the tissue cyclic GMP response to carbachol stimulation like the discharge of secretory protein was fully inhibited by atropine. Potassium chloride stimulation in the absence of atropine raised tissue cyclic GMP levels to a modest extent. The integrated area under the curve due to KC1 stimulation represents approximately 25% that of the curve due to carbachol stimulation. In the presence of atropine, however, cyclic GMP levels were no greater than basal levels during KC1 stimulation. In the case of carbachol stimulation, both the cyclic GMP response and the secretory response were inhibited by atropine. In the case of potassium chloride stimulation, the effect of atropine on cGMP response (inhibition) was dissociated from its effect on the secretory response (no inhibition). This suggests that while the cGMP response to KC1 stimulation alone is due to an indirect effect via acetylcholine release from nerve terminals in the lobule preparation, the secretion of pulse-labeled proteins is due to the direct effect of KC1 on the pancreatic acinar cell.

Release of Acetylcholine from Pancreatic Lobules Stimu- lated with Elevated Concentrations of Potassium Chloride

Two experiments were carried out to examine the effect of elevated potassium chloride concentrations on the release of acetylcholine from nerve terminds contained in pancreatic lobules. Diisopropyl fluorophosphate, 5 X M, was added to the incubation medium to inhibit acetylcholinesterase activity. Using the coupled gas-liquid chromatography-mass spectrometry procedure (41), the basal release of acetylcholine into medium containing 5 lll~ KC1 was 3.5 pmol/mg of tissue.

l4 r

0 . 4 A 'v\\ ".-.-,-o E+ - 1 " A x

I 2 3 4 5 6 7 8 9 1 0 20 30 60 lncubatlon tlme (mm)

FIG. 7. Kinetic changes in cellular cyclic GMP levels in pancreatic lobules stimulated by M carbachol and 75 lILM potassium chloride in the presence and absence of M atropine. Inset shows the kinetics of discharge of pulse-labeled proteins in response to these stimuli at the early time points chosen for six cGMP measurements. The experimental conditions are represented as follows: basal or unstimulated (X) , M carbachol ( W ,

M carbachol plus M atropine (A), 75 mM KC1 (v), and 75 mM KC1 plus lo-' M atropine (A). Similar findings were observed in three additional experiments.


Incubation of lobules in 75 m~ KC1 resulted in the release of 10.5 pmoI/mg of tissue of acetylcholine at the 2-min time point and 22.6 pmol/mg of tissue at the 10-min time point. The data indicate that elevated concentrations of KC1 do stimulate the release of acetylcholine from nerve terminals in pancreatic tissue.

Cellular Cyclic GMP Levels in Pancreatic Lobules Stimu- lated with the Ionophore A23187

Fig. 8 compares the effect of ionophore A23187 (1O"j or M) and M carbachol, each in the presence or absence of calcium in the medium, on cellular cGMP levels. The cGMP response to carbachol stimulation is maximal, 57 times the basal level, a t the 2-min time point. Tissue levels in response to the cholinergic agent fall rapidly to a plateau level approximately 4 times the basal level beyond the 10-min time point. The cGMP response to carbachol stimulation in the absence of calcium, plus or minus M EGTA, is approximately 80% that in the presence of calcium. When pancreatic lobules were exposed to lo-" or M ionophore A23187 in calcium-free medium for a 20-min preincubation period followed by the addition of 2.5 mM calcium at zero time, the kinetics and extent of the cGMP response were distinctly different from those observed during carbachol stimulation. With ionophore

Incubation lime (min)

FIG. 8. Kinetic changes in cellular cyclic GMP levels in pancreatic lobules stimulated by M carbachol or ionophore A23187. Solid symbols represent experiments carried out in the presence of 2.5 mM calcium. open symbols represent experiments carried out in the absence of calcium. Carbachol M) stimulation was carried out in the presence of 2.5 mM calcium (A) and in the absence of calcium with (A) or without M EGTA (Q). Preincu- bation of lobules in medium containing M A23187 for a 20-min period followed by: ( a ) the addition of 2.5 nm calcium (at time zero in the figure) is indicated by 0 or (b ) no addition of calcium (0). A similar preincubation study was carried out with M A23187 followed by calcium addition as indicated by (*); or no calcium addition (n). Preincubation in medium containing 10"' M A23187 for a 5-min period of time prior to calcium addition is represented by B. In a different experimental design (17), lobules were incubated in the presence of 2.5 m~ calcium (V), or in the absence of calcium with (V) or without EGTA (0) for a 5-min period of time, followed by the addition of M A23187 (at time zero in the figure). The illustrated data represent results from three experiments.

stimulation maximal levels were observed at the 3-min time point and these levels were 21% and 3076, respectively, compared to maximal levels during carbachol stimulation. Follow- ing the point of maximal response, cGMP levels decreased more slowly with A23187 stimulation than with carbachol stimulation. Atropine sulfate at M, which inhibits fully the cGMP response to carbachol or potassium chloride stimulation, had no effect on cGMP levels in response to A23187 stimulation. Pancreatic lobules preincubated in 10"' or M ionophore in calcium-free medium for the 20-min period of time and not followed by the addition of calcium at zero time showed no rise in cGMP levels, in marked contrast to the case with carbachol stimulation. Shorter preincubation times with A23187, e.g. 5 min, prior to the addition of 2.5 mM calcium resulted in a decrease in the calcium-dependent cGMP response. In an alternative experimental approach, similar to that used by other investigators (17) lobules were preincubated in medium with or without 2.5 mM calcium for 5 min, followed by the addition of IO-' M A23187 at time zero. The cellular cGMP response observed under these conditions was also dependent on the presence of calcium in the incubation medium and amounted, at the 2-min time point, to levels -4 times that of basal.

DISCUSSION

Elevation of potassium chloride in the extracellular medium results in depolarization of the cell membrane as fiist demonstrated in the giant squid axon (summarized in Ref. 45). Since that time potassium chloride has been used in the study of several secretory systems to examine coupling of membrane depolarization with release of neurotransmitters, hormones, and enzymes. The release of norepinephrine from adrenal medullary cells (46), thyroid-stimulating hormone (47), folli- cle-stimulating hormone (48), luteinizing hormone (49), adren- ocorticotrophic hormone (50), and growth hormone (51) from the anterior pituitary gland, and insulin from pancreatic /I cells (3) have been shown to result from the direct action of potassium chloride on these cells. In the salivary gland, however, amylase secretion resulting from high potassium stimulation was believed to be secondary to release of endogenous catecholamines from nerve endings (52).

Argent et al. (9), using the isolated cat pancreas perfused in situ, found that while elevated potassium concentrations of 30 to 120 mM increased amylase secretion, this effect was inhibited by atropine sulfate. Later Benz et al. (10) and Poulsen and Williams (7) made similar observations on pancreatic fragments from guinea pig and mouse, respectively. Recently, observations on dissociated pancreatic acinar cells from the guinea pig and mouse indicated that a 10-fold increase in extracellular potassium (47.5 mM) was without effect on amylase release (11). These results have suggested that the effect of potassium on the exocrine pancreas, both in situ and in vitro, was mediated by release of acetylcholine from nerve endings. Direct measurement of the membrane potential in these fragments indicated that the potassium treatment, with or without atropine, depolarized the acinar cell membrane at least 20 mV (3,4, 7,53), an effect which exceeded the maximal depolarizing effect of carbachol, 12 to 15 mV (3, 6, 7). Taken together, these observations have suggested that membrane depolarization is not a critical step in the sequence of events responsible for stimulus-secretion coupling.

The results reported here on guinea pig pancreatic lobules demonstrate that increased concentrations of potassium in the extracellular medium can directly stimulate release of enzymes and zymogens from the exocrine pancreas. Atropine at a concentration, M, capable of completely blocking optimal carbachol, M, induced secretion had little or no


effect on potassium-induced secretion. A similar observation was made on rat pancreatic lobules stimulated with 75 mM KC1 in the absence and presence of M atropine sulfate. These findings demonstrate that the major effect of elevated concentrations of KC1 on pancreatic lobules prepared from the guinea pig or rat are clearly unrelated to the release of acetylcholine from the nerve endings in the tissue.

Potassium chloride-stimulated discharge of secretory protein was independent of the presence of sodium in the incubation medium. This finding supports the idea that elevated concentrations of KC1 result in depolarization of the cell membrane according to the ionic parameters described by the Nernst equation, a mathematical formulation describing the ionic factors which determine membrane potential. In addition, the changes in secretory rates measured here correlate with the changes in membrane potential reported elsewhere (3) in response to potassium chloride stimulation. For example, no response was achieved in either parameter until potassium chloride concentrations exceeded 25 m ~ .

Potassium chloride-stimulated secretion depends in its entirety on the presence of calcium in the extracellular medium. Our ability to demonstrate this finding in a conclusive manner has depended on following a number of precautions for both the preparation of calcium-free medium and the rapid reduction of extracellular calcium present in pancreatic lobules. These precautions are detailed here under “Results” and “Methods” and elsewhere (28). The addition of log increases in extracellular calcium concentration up to 2.5 mM in the presence of 75 mM KC1 is associated with a stepwise increase in the discharge of secretory protein. Magnesium and barium do not substitute for calcium, and lanthanum, a calcium- displacing agent, blocks potassium-induced discharge in the presence of calcium.

Several observations suggest that the secretory effect of KC1 is not due to a general disruption of cellular compart- ments but rather to a specific coupling effect between membrane depolarization and exocytosis: (a) Evaluation by electron microscopy indicates that the general morphological integrity of acinar cells is retained and a number of exocytotic images are seen (see Fig. 2a); (6) release of lactic dehydrogenase, an enzyme normally confined to the cytoplasmic space, from pancreatic lobules is not increased during potassium chloride stimulation compared to that observed with basal or carbachol-induced discharge; (e) the secretory effect is blocked by N2 atmosphere or CCCP, an uncoupler of oxidative phosphorylation; and (d) the effect is blocked by incubation in calcium-free medium.

Measurement of intracellular 45Ca content under steady- state conditions with respect to radioactive calcium has indicated that 75 mM KC1 stimulation results in a significant influx of calcium from the extracellular medium to the intracellular space. This finding provides an adequate explanation for the dependence of potassium-induced discharge on extracellular calcium. Using similar radioactive techniques, others have defined a supportive role for extracellular calcium during carbachol (54)- and ionophore A23187 (13)-induced secretion.

We believe that the successful demonstration of the direct effect of potassium on the discharge of secretory protein from acinar cells in pancreatic lobules is due to the efficient ex- change of solutes in the lobule system. For example, pancreatic lobules show a greater response to carbachol stimulation (6 to 10 times the resting level) than do pancreatic fragments or slices (1.5 to 3.0 times the resting level) (10, 55- 57). While potassium treatment of pancreatic fragments would be expected to depolarize acinar cells near the surface of the tissue fragment, those cells monitored in the microelectrode studies, the effect on the t,otal tissue fragment may be negli-

gible except for that on penetrating nerve endings. The ex- change of solutes should, of course, be most efficient using dispersed pancreatic cells. However, while the use of dispersed cells has several advantages (the disassembly of both intersti- tial and intraluminal spaces), they show consistently the following functional deficiencies: (a) dispersed cells require a 10- fold greater concentration of cholinergic agents to stimulate amylase secretion than do pancreatic lobules or fragments (11, 17,58,59), (b) at optimal levels of cholinergic agents, discharge of protein is increased 2.5- to 3.0-fold over basal discharge in dispersed cells compared to 6- to 10-fold increases in pancreatic lobules (11, 17,59), and (e ) caerulein, which stimulates the intact pancreas in the mouse, shows no stimulatory activity on dispersed cells prepared from the same tissue (1 1). The lack of response of dispersed pancreatic cells to KC1 stimulation raises the possibility that the necessary enzyme treatment during tissue preparation has altered the basic integrity of the cell membrane with regard to electrical and ionic properties (see also Ref. 1). No independent measure of this membrane property, such as the electrophysiological measurements of the membrane potential, has been carried out on dispersed pancreatic cells. While the use of dispersed cells should not necessarily be discouraged, it seems unwise to make detailed studies of this preparation without control studies on the preparation itself. The use of pancreatic lobules has the distinct advantage that active proteases, phospholipases, and chelators are not used during the tissue preparation. Studies with dispersed pancreatic cells should therefore include control studies with pancreatic lobules (or their equivalents).

The effect of ionophore A23187 on the discharge of secretory protein is similar to that of KC1 stimulation since the stimulatory effect of the ionophore is (a) unchanged by the presence of atropine, (b) dependent on intracellular energy stores, ( e ) dependent on calcium added to the incubation medium, (d) proportional to log increases in intracellular calcium, ( e ) accompanied by an influx of calcium from the extracellular medium to the intracellular space (13), and ( f ) specific for calcium since magnesium and barium are unable to substitute. Strontium chloride, an effective substitute for calcium during KC1-induced discharge, showed no capacity to substitute during A23187 (or carbachol) stimulation. Furthermore, as seen in Fig. 6, the kinetics and extent of discharge of secretory proteins are remarkably similar for both 75 mM KC1 and ionophore A23187. The requirements for extracellular calcium and intracellular energy reserves strongly suggest that the secretory effects of both high potassium and calcium ionophore are mediated through the final steps involved in stimulus-secretion coupling since these requirements are also necessary for cholinergic or peptide hormone-induced secretion (28-30, 60).

As demonstrated in Figs. 1 and 6, carbachol-stimulated discharge of secretory protein is consistently more efficient than discharge stimulated by either ionophore or elevated concentrations of KC1. The data presented in Fig. 5 indicate that the increased efficiency of carbachol stimulation is due to its capacity to recruit calcium from intracellular stores during the early periods of stimulation, 0 to 20 min. This component corresponds closely in extent and character to that component of carbachol-induced discharge which is independent of calcium added to the incubation medium, described elsewhere (28) as Component I of carbachol-stimulated discharge. That component of carbachol-induced discharge, which was shown to depend on calcium added to the incubation medium, described elsewhere as Component I1 (28), corresponds closely in extent and character to discharge induced by either KC1 stimulation or ionophore stimulation. Thus the data presented here and elsewhere (28) suggest strongly that


there are two pathways involved in stimulus-secretion coupling in the exocrine pancreas, one determined by events which presumably involve intracellular membranes5 and the release of calcium from intracellular stores (carbachol- or pancreozymin-induced discharge independent of medium calcium, Component I), and the other determined by events in the extracellular membrane, possibly including membrane depolarization and definitely involving the influx of calcium from the extracellular medium (KC1-induced discharge, ionophore-induced discharge, and carbachol- or pancreozymin- induced discharge dependent on medium calcium, Component 11).

The earliest events associated with stimulus-secretion coupling in the exocrine pancreas (see Fig. 9) are believed to be (a) the secretagogue-receptor interaction for both cholinergic and hormonal stimulants at the cell membrane (1, 2, 17, 36, 62), and ( b ) the stimulation of a cellular guanylate cyclase leading to a rise in intracellular cGMP (63, 36, 17, 64). While secretion stimulated by elevated concentrations of KC1 was associated with a modest rise in cGMP, 20 to 25% compared to that resulting from carbachol stimulation, in the presence of atropine at levels which inhibit the action of acetylcholine released from nerve endings in the tissue, no rise in cGMP occurred. Thus, in the presence of KCl, atropine caused complete dissociation of the cGMP response (inhibition) from the secretory response (no inhibition). From this finding we con- clude that cGMP does not mediate potassium-induced discharge.

The cyclic GMP response to ionophore A23187 is clearly distinguished and separate from that due to cholinergic or hormonal stimulation since (a ) the response to A23187 is dependent on the presence of calcium added to the incubation medium (see Fig. 8) while that due to physiological secretagogues is largely independent of medium calcium (see Fig. 8 and References 17 and 28), and (b ) the rise in cGMP due to A23187 is small (21 to 30%) and dissimilar in time course compared to that resulting from optimal carbachol or pancreozymin stimulation (36). These data are at variance with those published by Christophe et al. (17) on dispersed guinea pig pancreatic cells where the tissue cGMP response to 5 x

M A23187 stimulation was independent of the presence of calcium added to the incubation medium and similar in extent and time course to that resulting from M CCK-octapep- tide or lo-" M carbachol. These investigators concluded that cGMP elevations due to cholinergic or hormonal stimulation were mediated by the release of calcium from intracellular sources. Our results, along with those indicating that the cGMP response to optimal doses of carbachol or pancreozymin is maintained in the absence of both extracellular and available intracellular stores of calcium (28) clearly indicate that, under physiological conditions, the majority of the tissue cGMP response is coupled to the secretagogue-receptor interaction and not to either early or late events resulting in. calcium translocation. The small rise in cGMP observed with. ionophore stimulation was dependent, however, on calcium in the extracellular medium. The physiological significance of this small rise in cGMP will require further study. Despite coupling of the cGMP response to the secretagogue-receptor' interaction, there is no evidence to date that cGMP is directly involved in the mediation of stimulus-secretion coupling in the exocrine pancreas. In contrast, the evidence presented here and elsewhere (28) suggest that calcium mediates in its entirety the secretagogue-stimulated discharge of secretory protein.

Other possibilities to be considered are that intracellular calcium is released from the plasma membrane itself or from calcium-binding proteins present in the cytosol (61).

~ T m n s l o ~ o m n of C O I C I Y ~ llom CIlrOCellYlor I lorel

~COmpDnenln 1

t t 75mM 10-'M

FIG. 9. Schematic diagram which illustrates the proposed sequence of events which couple cholinergic and peptide hormonal stimulation to discharge of secretory protein in the acinar cell of the exocrine pancreas. This sequence of events is resolved into two interrelated pathways, I and 11, as proposed elsewhere (28). Our results suggest that elevated concentrations of KC1 and ionophore A23187 act at two distinct sites in pathway 11. The details are presented in the text.

KC1 A23187

The schematic diagram in Fig. 9 arranges the sequence of events suggested here and elsewhere (28) for each of the proposed pathways which appear to couple cholinergic and peptide stimulation to discharge of secretory proteins in guinea pig pancreatic lobules. These two pathways apparently diverge following the secretagogue-receptor interaction and converge at the point where cytoplasmic free calcium levels are elevated. In the presence of physiological secretagogues the two pathways apparently work in concert to augment the effect of each individual pathway.

Acknowledgments-We wish to thank Jessica Pash, Morris Mil- man, Peony Lam, and Henrich Lutcke for their expert technical assistance.

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Documents

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. …hibited by M atropine, sodium substitutes, or lo-‘ M tetrodotoxin. Exposure of lobules to elevated concen- trations of potassium chloride