Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.

Isolated pancreatic acini were prepared by a new method from mouse and rat pancreases by digestion with purified collagenase and chymotrypsin followed by mechanical shearing. Acini were structurally similar to those of the intact pancreas, having a normal luminal structure but with the basal acinar cell membranes exposed to the incubation medium. Amylase release in response to both cholinergic analogues and the cholecystokinin analogues caerulein and pentagastrin was comparable to that of the intact pancreas, but was much greater than previously reported for isolated acinar cells. Cholinergic-stimulated release was inhibited by atropine with a Ki value of 1.4 nM which is comparable to other muscarinic receptors. All agonists tested, when added at supramaximal concentrations, produced a submaximal release of amylase even though ATP levels and the release of slowly exchanging 45Ca2+ were normal or increased. Acini releasing amylase submaximally after being exposed to supramaximal concentrations of carbachol failed to respond to a maximal amount of caerulein or to the Ca2+ ionophore A23187. It is concluded that the decreased response (desensitization) is a postreceptor phenomenon and possibly mediated by Ca2+ itself.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Ca++ and pancreatic amylase release.

The effects of Ca'++ on release of amylase by mouse pancreas in vitri was studied.The cholinergic agonist bethanechol was depressed about 50% in O Ca'++ medium.When pancreatic fragments were superfused with O Ca'++ medium, bethanechol still produced a normal stimulation of amylase release after half of the total tissue Ca'++ was washed out. This Ca'++ loss included both extracellular and some intracellularCa'++. Uptake and equilibration of '45Ca'++ into pancreatic fragments was multiphasic, with total equilibration with stable Ca'++ still not reached after 3 h. Addition of bethanechol had no effect on the rate of '45Ca uptake and equilibration. '45Ca'++ efflux was not influenced by superfision in O Ca'++ medium, while thestimulation of '45Ca'++ efflux by bethanechol was enhanced. It is concluded that extracellular Ca'++ and/or Ca'++ influx is not of major importance in triggering pancreatic enzyme release, but that extracellular Ca'++ may regulate the release process thus accounting for the parallel changes in unstimulated and stimulated amylase release.     

Calcium fluxes in isolated pancreatic acini: effects of secretagogues

45Ca2+ exchange and total calcium content were measured in isolated mouse pancreatic acini. 45Ca2+ uptake could be described as the sum of a constant and a single exponential kinetic component; about 60% of total acinar calcium was exchangeable. Stimulation by bethanechol increased 45Ca2+ uptake, but the time course of uptake could be fit only by the addition of a more rapid kinetic component without any change in the total exchangeable Ca2+. 45Ca2+ washout after 1-h loading could be fit as the sum of two exponential components. Stimulation increased the rate of 45Ca2+ washout with the appearance of a third and more rapid kinetic component. There was not, however, a good correspondence between the exponential constants measured in uptake and washout protocols in unstimulated acini. Exponential constants were also affected by the concentration of calcium in the medium, further indicating the presence of nonlinearities in 45Ca2+ exchange. The dose-response relationships were similar for bethanechol stimulation of 45Ca2+ uptake and amylase release, whereas stimulation of 45Ca2+ washout reached a maximum at a higher concentration of bethanechol. Both 45Ca2+ uptake and analytical measurement of total Ca2+ showed a rapid drop in acinar Ca2+ content followed by a gradual reuptake on stimulation by bethanechol. It is concluded that the initial primary effect of secretagogues is to increase Ca2+ efflux, which is interpreted to be the result of release of sequestered calcium into the cytosol.     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice.

Fluxes of 45Ca2+ were studied in beta-cell rich islets of non-inbred ob/ob-mice, using LaCl3 to wash out extra-cellular and superficially bound 45Ca2+. The ionophore A-23187 (10 microM) increased the 45Ca2+ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of 45Ca2+ than did A-23187. The ionophore enhanced the unidirectional efflux of 45Ca2+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced 45Ca2+ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced 45Ca2+ uptake. The results suggest that A-23187 acts by catalyzing Ca2+ fluxes across the beta-cell plasma membrane. The different effects of D-glucose and A-23187 on 45Ca2+ fluxes suggest that the two agents act through different mechanisms in the beta-cells.     

Pancreatic acinar cells: effects of lanthanum ions on amylase release and calcium ion fluxes

1. The effect of La(3+) on amylase release and Ca(2+) fluxes in mouse pancreatic fragments in vitro was studied.2. Amylase release was increased by 0.1 mM-La(3+) and progressively inhibited by 1.0-10 mM-La(3+). Non-stimulated and bethanecol stimulated secretion were altered in an identical manner. Inhibition of amylase release was rapid and reversible.3. Uptake of (45)Ca(2+) was multiphasic with equilibrium with stable Ca(2+) still not complete after 2 hr. La(3+) (10 mM) limited uptake of (45)Ca(2+) to the extracellular space and slightly decreased total Ca(2+) content. Lower concentrations of La(3+) affected (45)Ca(2+) uptake and total Ca(2+) content in a biphasic manner which paralleled effects on amylase release.4. La(3+) restricted washout of (45)Ca(2+) to isotope in the extracellular space and abolished the bethanecol-stimulated increase in (45)Ca(2+) efflux.5. Uptake of (45)Ca(2+) into intracellular space, as measured by the ;lanthanum' method, was not affected by bethanecol.6. Tissue ultrastructure and Na(+) and K(+) content were not affected by La(3+).7. It is concluded that an influx of extracellular Ca(2+) is not important for triggering of secretion and that La(3+) may inhibit amylase release by acting on the release process rather than on Ca(2+) influx.     

Expression of Ca2+-dependent synaptotagmin isoforms in mouse and rat parotid acinar cells

Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.     

Protein synthesis inhibitors enhance secretagogue sensitivity of in vitro rat pancreatic acini

Isolated rat pancreatic acini were treated with cycloheximide and amylase release was measured. This agent increased the sensitivity to both synthetic octapeptide of cholecystokinin (CCK8) and carbamylcholine, the major secretagogues known to utilize Ca2+ as a second messenger. The mechanism of the cycloheximide effect was via inhibition of protein synthesis, as indicated by the following: 1) the concentration of cycloheximide used inhibited leucine incorporation by greater than 90%; 2) this effect was not instantaneous but increased up to a 2-h pretreatment; and 3) a similar effect was obtained with puromycin, a chemically different inhibitor of protein synthesis. Cycloheximide acted on the steps by which secretagogues mobilize cellular Ca2+ because the dose-response curve for 45Ca2+ efflux was shifted to the same extent as that for amylase release, whereas the dose-response curve for amylase release induced by the Ca2+ ionophore A23187 was not altered. The results suggest, therefore, that a rapidly turning-over protein present in pancreatic acinar cells exerts an inhibitory influence on Ca2+ mobilization by secretagogues.     

Actions of peptides isolated from amphibian skin on pancreatic acinar cells.

In dispersed acinar cells prepared from guinea pig pancreas, peptides isolated from amphibian skin (caerulein, bombesin, litorin, and physalaemin) as well as eledoisin, a peptide isolated from the posterior salivary gland of a Mediterranean octopod, increased outflux of 45Ca, release of bound 45Ca, accumulation of cyclic GMP, and release of amylase. In addition, bombesin, litorin, physalaemin, and eledoisin each increased the initial uptake of 45Ca by dispersed acinar cells, whereas C-terminal octapeptide of porcine cholecystokinin (CCK-OP) and carbamylcholine did not increase the initial uptake of 45Ca but, rather, abolished the increase caused by the other agents. None of the actions of these amphibian peptides was altered by concentrations of atropine sufficient to abolish the effects of muscarinic cholinergic agents. None of the amphibian peptides altered cellular cyclic AMP or the increase caused by secretin or porcine vasoactive intestinal peptide (VIP). Acinar cells preincubated with 45Ca plus bombesin showed the same rate of release of 45Ca as did control cells and this rate was not altered by adding bombesin but was increased fivefold by adding CCK-OP. In terms of their chemical structures as well as the potency and efficacy with which they alter acinar cell function, the amphibian peptides plus CCK-OP can be grouped into three pairs: caerulein with CCK-OP, bombesin with litorin, and physalaemin with eledoisin.     

Stimulus-permeability coupling in rat lacrimal gland

Incubation of isolated cells wtih 10(-2) M ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid or cobalt inhibits 22Na and 45Ca uptake stimulated by carbachol. The artificial introduction of Ca into the cytosol by the cation ionophore A23187 also initiates the 22Na uptake. Amiloride (10(-5) M) partially inhibits 22Na uptake induced by carbachol, but has no effect on receptor-stimulated 45Ca uptake or 86Rb release. Tetrodotoxin (TTX) has no effect on 22Na uptake stimulated by carbachol, whereas methoxyverapamil (D 600) produces a small but significant decrease in both 22Na and 45Ca uptake. This effect of D 600 may be related to a block of receptor activation and not to a block of Ca channel activation. Incubation in high K (56 mM) does not prevent the change in membrane permeability to Ca, K, and Na initiated by carbachol. It is concluded that carbachol stimulates the influx of Ca; the rise in the cytosolic Ca concentration then couples receptor activation to a change in membrane permeability to K and Na. The permeability mechanisms for Ca, K, and Na that are activated by carbachol appear to be specific for each of the three cations and appear to be dissimilar to permeability mechanisms in excitable tissue that carry the same ions.     

Cholinergic agents inhibit sodium transport across the isolated toad bladder.

Acetylcholine and carbamylcholine (carbachol) inhibited sodium transport across the toad bladder. This effect was blocked by atropine. Carbachol increased the uptake of 45Ca by isolated toad bladder epithelial cells. This increased 45Ca uptake was blocked by atropine, pentobarbital, or lanthanum chloride. The inhibitory effect of cholinergic agents on sodium transport was dependent on external calcium concentration and was abolished by decreasing external calcium from 2 to 0.2 mM or by agents (pentobarbital, lanthanum chloride) which prevent the movement of calcium into cells. Carbachol increased modestly, but significantly, the levels of cyclic GMP in isolated toad bladder epithelial cells. This effect was blocked by atropine. Whether the decrease in sodium transport produced by cholinergic agents is the result of increased intracytoplasmic calcium levels, increased cyclic GMP levels, or a combination of both remains to be established.     

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+. The increment in 45Ca2+ uptake caused by TRH was dependent on the concentration of Ca2+ in the medium. In contrast to the effect of 50 mM K+, which also causes Ca2+ influx, TRH caused 45Ca2+ efflux and TSH release from TtT cells even when the concentration of Ca2+ in the medium was lowered below 100 micro M. TRH stimulated TSH release during perifusion in medium in which the free Ca2+ concentration was lowered to approximately 0.02 micro M, and reintroduction of Ca2+ into the medium simultaneously with TRH markedly increased TSH release. We suggest that TRH may affect Ca2+ metabolism in TtT cells by both extracellular Ca2+-independent and -dependent mechanisms and that this dual mechanism of action serves to augment further TSH secretion induced by TRH.     

Stimulation of amylase and sialic acid releases from dog submandibular gland slices by pilocarpine or high K+ medium: a possible role of calmodulin for their releases

The mechanism of amylase and sialic acid releases stimulated by pilocarpine or high K+ medium was investigated in the slices of dog submandibular glands. The release of both amylase and sialic acid was dose-dependently increased by pilocarpine and a considerable release was observed at pilocarpine concentrations of more than 1 microM. Similar effects were observed when K+ concentration in the medium was increased and the maximal response was observed at 75 mM K+. The release of amylase and sialic acid by pilocarpine or K+ considerably decreased by removing Ca2+ from the medium and the slices. The release of amylase in the Ca2+-deficient slices was nearly recovered by the addition of 2.5 and 5.0 mM Ca2+, whereas that of sialic acid was recovered by only 60-75%. Ca2+ inhibitors, La3+ and verapamil, and calmodulin inhibitors, trifluoperazine, prenylamine, and W-7, significantly inhibited the release of amylase and sialic acid induced by the stimulants. These results suggest that the release of amylase and sialic acid stimulated by pilocarpine or K+ is dependent on the presence of Ca2+, and that the activation of calmodulin is involved in the process of the release.     

Phorbol ester desensitization of clonal insulin-releasing cell response to carbachol involves depletion of an intracellular calcium pool

The mechanism by which 12-o-tetradecanoylphorbol-13-acetate (TPA) desensitizes carbachol mobilization of glucose-incorporated calcium (Ca2+) was studied in clonal insulin-releasing cells (RINm5F) using colour indicators and dual wavelength spectrophotometry. The net uptake of Ca2+ stimulated by 20 mM glucose reached saturation after 19 +/- 2 min when it corresponded to 1.21 +/- 0.09 mmol calcium kg-1 protein. Carbachol then induced a release of 0.21 +/- 0.03 mmol calcium kg-1 protein. Half of the remaining Ca2+ was liberated by antimycin A and the rest with the Ca2+ ionophore A-23187. When 0.1 microM TPA was added initially, the cells lost 0.29 +/- 0.08 mmol calcium kg-1 protein within 10 min. The subsequent addition of glucose resulted in a sluggish uptake of only 0.58 +/- 0.09 mmol calcium kg-1 protein reaching equilibrium after 35 +/- 3 min. Carbachol now failed to induce any Ca2+ release. The actions of TPA were essentially unchanged by previous exposure to glucose, removal of Na+ from the medium and even when some of the glucose-incorporated Ca2+ had been liberated with carbachol. The results indicate that TPA desensitization of carbachol-induced mobilization of Ca2+ in the RINm5F cells is due to the disappearance of Ca2+ from the sensitive pool, an effect which may depend on stimulated extrusion of Ca2+ from the cells by the (Ca2+-Mg2+)-ATPase.     

Characterization of an ATP-dependent Ca2+ uptake system in mouse pancreatic microsomes

The uptake of 45Ca2+ was studied in microsomes prepared from isolated mouse pancreatic acini. These microsomes accumulated 45Ca2+ in the presence of ATP; uptake was potentiated by addition of oxalate. Sequestered microsomal 45Ca2+ was only gradually removed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) but was readily released by the divalent cation ionophore A23187. Inhibitors of mitochondrial oxidation and mitochondrial calcium transport had little effect on microsomal 45Ca2+ uptake. A separate subcellular fraction enriched in plasma membranes took up 45Ca2+ poorly compared with the microsomal fraction. Half-maximal 45Ca2+ uptake by the microsomal fraction was observed at a free Ca2+ concentration of 1.1 microM. 45Ca2+ uptake was dependent on Mg-ATP and showed a pH optimum at 6.8-7.0. Subfractionation of the total microsomes into "heavy" and "light" microsomal fractions indicated higher 45Ca2+ uptake activity associated with the heavy fraction. A Ca2+-activated, Mg2+-dependent ATPase was demonstrated in this fraction. Stimulation of pancreatic acini with the cholecystokinin analogue caerulein prior to homogenization increased the subsequent rate of 45Ca2+ uptake by the microsomal fraction.     

Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.     

Pharmacologic modulation of the IgE or Ca2+ ionophore A23187 mediated Ca2+ influx, phospholipase activation, and histamine release in rat basophilic leukemia cells

The stimulation of the rat basophilic leukemia cells (RBL-2H3) mediated either through the IgE receptor or by the Ca2+ ionophore A23187 results in 45Ca2+ influx, phospholipase activation, and histamine release. This study compared in parallel the effects of pharmacological agents on 45Ca2+ influx, phospholipase activation as measured by the release of [14C]-arachidonic acid, and histamine release. Microtubule-depolymerizing agents (demecolcine, colchicine, and vinblastine sulfate) did not affect 45Ca2+ influx, but blocked the IgE- or A23187-mediated [14C]-arachidonic acid and histamine release (e.g., IC50 for vinblastine sulfate = 10 nM). In contrast, a microtubule-stabilizing agent (taxol) blocked the IgE- and A23187-mediated 45Ca2+ influx and [14C]-arachidonic acid and histamine release (IC50 = 20 microM). Microfilament-disrupting agents (cytochalasin B, C, D, and E) enhanced 45Ca2+ influx and [14C]-arachidonic acid and histamine release in the same dose-dependent fashion (e.g., EC50 for cytochalasin B = 0.4 microM). Other pharmacological agents such as a metabolic inhibitor (antimycin A), calmodulin inhibitors (W-7, trifluoperazine, and chlorpromazine), and protease inhibitors (TPCK and TLCK) blocked the IgE- and A23187-mediated 45Ca2+ influx and [14C]-arachidonic acid and histamine release. Therefore, the coupling of Ca2+ influx and the phospholipase activation step requires a functioning microtubule system. Other inhibitors act at sites prior to the Ca2+ influx step in the release process.     

Alteration of expression of Ca2+ signaling proteins and adaptation of Ca2+ signaling in SERCA2+/- mouse parotid acini

PURPOSE: The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.     

Binding of45Calcium to isolated rat mast cells in connection with histamine release

The time courses for histamine release and uptake of⁴⁵Ca were compared on isolated rat mast cells after stimulation under different experimental conditions with antigen, compound 48/80, ATP, or the ionophore A23187. Except for ATP the results did not show a time-dependent correlation between histamine release and⁴⁵Ca uptake.The uptake of⁴⁵Ca under conditions where membrane-bound calcium is utilized for optimal anaphylactic histamine release did not differ from that of controls, whereas the presence of⁴⁵Ca in the incubation medium led to a substantial uptake without influence on the histamine release.The uptake of⁴⁵Ca induced by antigen or compound 48/80 was completely inhibited by antimycin A as confirmed by the use of two different methods. In addition, an energy-dependent restitution of the permeability properties of the plasma membrane seemed to follow histamine release. Antimycin A partly reduced the uptake of⁴⁵Ca after stimulation with ATP, and did not affect binding following exposure of the cells to the ionophores A23187 or X537A.The ionophore A23187 was able to reduce the⁴⁵Ca content of mast cells previously loaded with the isotope. Mast cells pretreated with A23187 in the absence of extracellular calcium did, after washing, accumulate substantial amounts of⁴⁵Ca without release of histamine.The results suggest that only small amounts of calcium are required to trigger histamine release and that studies with⁴⁵Ca do not distinguish between specific uptake of calcium and nonspecific equilibration secondary to morphological secretory changes.The present results were in part presented at the Sixth Meeting of The Histamine Club, London, April 1977.     

Development of secretory mechanisms in rat pancreas.

Mechanisms and development of secretory function were studied in rat pancreas in vitro. Amylase release from term fetal pancreas was refractory to stimulation by carbamylcholine chloride (carbachol) and cholecystokinin-octapeptide (CCK-OP), but was significantly augmented by calcium ionophore (A23187), DBcAMP, 8-Br-cGMP, and theophylline. The latter agent when combined with either cyclic nucleotide analogue further increased secretory responses. At 1 day and 8 days postnatally, responsiveness to carbachol and CCK-OP had been acquired because amylase secretion stimulated by these agents was brisk and at a level comparable to that found in mature tissue. Increasing extracellular calcium concentrations from 1.23 to 5.28 mM had no effect on basal amylase release in either the fetal or 8-day pancreas. No changes in intracellular cAMP concentrations were found at any age under experimental conditions used. Similarily, in fetal tissue, no changes in cGMP concentrations were found in response to carbachol or A23187. However, at 8 days of age, both agents produced two- to four-fold increases in tissue cGMP levels at 1, 2, and 5 min of incubation. These studies confirm that responsiveness to carbachol and CCK-OP is a maturational process in the pancreas that lags behind the development of intracellular processes involved in stimulus-secretion coupling.