Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Cholecystokinin-stimulated tyrosine phosphorylation of PKC-delta in pancreatic acinar cells is regulated bidirectionally by PKC activation

PKC-delta is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-delta TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-delta TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-delta TYR-P by activation of the low affinity CCK(A) receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-delta TYR-P but instead, increased it. A23187 did not stimulate PKC-delta TYR-P. Wortmannin and LY 294002 did not alter CCK-stimulated PKC-delta TYR-P. GF 109203X, at low concentrations, increased PKC-delta TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, G? 6976 and safingol, caused a similar increase in TPA- and CCK-stimulated PKC-delta TYR-P. These results demonstrate that CCK(A) receptor activation causes PKC-delta TYR-P through activation of only one of its two receptor affinity states. This PKC-delta TYR-P is not directly influenced by changes in [Ca(2+)](i); however, the resultant activation of PKC-alpha has an inhibitory effect. Therefore, CCK activates both stimulatory and inhibitory PKC cascades regulating PKC-delta TYR-P and, hence, likely plays an important role in regulating PKC-delta degradation and cellular abundance.     

Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10^?8 to 10^?5 M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10^?8?10^?7 M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10^?6?10^?5 M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10^?3 M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion. © 1994 Wiley-Liss, Inc.     

PKC-delta and -epsilon regulate NF-kappaB activation induced by cholecystokinin and TNF-alpha in pancreatic acinar cells

Although NF-kappaB plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-kappaB activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-alpha, which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-kappaB activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-kappaB activation. Both CCK-8 and TNF-alpha activated the novel isoforms PKC-delta and -epsilon and the atypical isoform PKC-zeta but not the conventional isoform PKC-alpha. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-kappaB activation induced by CCK-8 and TNF-alpha. NF-kappaB activation by CCK-8 and TNF-alpha required translocation but not tyrosine phosphorylation of PKC-delta. Activation of PKC-delta, PKC-epsilon, and NF-kappaB with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-alpha they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-alpha initiate NF-kappaB activation by different PLC pathways that converge at the novel PKCs (delta and epsilon) to mediate NF-kappaB activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

Roles of Munc18-3 in amylase release from rat parotid acinar cells

Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.     

BAY-K-8644-stimulated amylase secretion from pancreatic acinar cells

Pancreatic acinar cells do not contain depolarization-sensitive calcium channels. Nonetheless, in the current study, the calcium channel activator, BAY-K-8644, was found to stimulate a time- and concentration-dependent increase in the spontaneous release of amylase. Secretion was dependent on the presence of extracellular calcium in the incubation medium. Racemic BAY-K-8644 and (or) its S(-)optical isomer did not enhance the secretory response to either carbachol or cholecystokinin octapeptide; however, when co-applied with either phorbol ester, vasoactive intestinal peptide, or forskolin, they potentiated amylase secretion. Nifedipine and the R(+)isomer of BAY-K-8644, which are both calcium channel antagonists, did not alter basal or forskolin-stimulated amylase secretion, and [3H]nitrendipine did not bind to acinar cell membranes. Neither atropine nor dibutyryl cGMP, inhibitors of cholinergic and cholecystokininergic receptors, respectively, affected BAY-K-8644-induced amylase secretion. While BAY-K-8644 stimulated concentration-dependent cGMP synthesis in acinar cells, it had no effect on basal or forskolin-stimulated cAMP formation. The data suggest that BAY-K-8644 may bind to acinar cell sites that are not functional calcium channel proteins but are coupled nevertheless to the secretory response, and that calcium channel antagonists do not bind to these sites. The mechanism of the secretagogue action of BAY-K-8644 remains to be elucidated.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Regulation of calcium handling by rat parotid acinar cells

Summary Salivary gland fluid secretion following neurotransmitter stimulation is Ca²⁺-dependent. We have studied the control of cellular Ca²⁺ following secretory stimuli in rat parotid gland acinar cells. After muscarinic-cholinergic receptor activation, cytosolic Ca²⁺ is elevated 4–5 fold, due to both intracellular Ca²⁺ pool mobilization and extracellular Ca²⁺ entry. Fluid movement ensues due to the Ca²⁺-activated enhancement of membrane permeability to K⁺ and Cl^–. Basal cytosolic Ca²⁺ levels are tightly controlled at 150–200 nM through the action of high affinity and high capacity ATP-dependent Ca²⁺ transporters in the basolateral and endoplasmic reticulum membranes. Activity of these Ca²⁺ transporters can be modulated to facilitate rapid responsiveness and a sustained fluid secretory response necessary for alimentary function.     

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with ³⁵S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl β-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.     

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular mileu within minutes. The accessability to these markers seemed independent of the ionised Ca²⁺ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10^?9 M Ca²⁺, but typically 4% was released when the Ca²⁺ level was subsequently raised to 10^?5M levels, the EC₅₀ for Ca²⁺ being 2 μM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca²⁺ concentration. No amylase was released at 10^?7M Ca²⁺ when permeable cells were exposed to cyclic 3′,5′-AMP or cyclic 3′,5′-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca²⁺ in the presence of the phorbol ester 12-O-tetradecanoyl phorbol 13 acetate. These results suggest that when “functionally normal” isolated acinar cells are rendered permeable, Ca²⁺ — but not cyclic nucleotides — acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

Melatonin modulates Ca2+ mobilization and amylase release in response to cholecystokinin octapeptide in mouse pancreatic acinar cells

In the present work, we have evaluated the effect of an acute addition of melatonin on cholecystokinin octapeptide (CCK-8)-evoked Ca²⁺ signals and amylase secretion in mouse pancreatic acinar cells. For this purpose, freshly isolated mouse pancreatic acinar cells were loaded with fura-2 to study intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Amylase release and cell viability were studied employing colorimetric methods. Our results show that CCK-8 evoked a biphasic effect on amylase secretion, finding a maximum at a concentration of 0.1 nM and a reduction of secretion at higher concentrations. Pre-incubation of cells with melatonin (1 μM–1 mM) significantly attenuated enzyme secretion in response to high concentrations of CCK-8. Stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, followed by a decrease towards a constant level. In the presence of 1 mM melatonin, stimulation of cells with CCK-8 resulted in a smaller [Ca²⁺]_c peak response, a faster rate of decay of [Ca²⁺]_c and lower values for the steady state of [Ca²⁺]_c, compared with the effect of CCK-8 alone. Melatonin also reduced the oscillatory pattern of Ca²⁺ mobilization evoked by a physiological concentration of CCK-8 (20 pM), and completely inhibited Ca²⁺ mobilization induced by 10 pM CCK-8. On the other hand, Ca²⁺ entry from the extracellular space was not affected in the presence of melatonin. Finally, melatonin alone did not change cell viability. We conclude that melatonin, at concentrations higher than those found in blood, might regulate exocrine pancreatic function via modulation of Ca²⁺ signals.     

Regulation by clozapine of calcium handling by rat submandibular acinar cells

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.     

Development of secretagogue response in rat pancreatic acinar cells

Two to 3 days prior to birth, acinar cells of the rat pancreas acquire morphologic and biochemical characteristics of the adult gland. To determine if differentiation of the secretory apparatus coincides temporally with the capacity of the cell to respond to secretory stimuli, lobules of embryonic, neonatal, and adult rat pancreas were compared for their ability to respond to secretagogues presumed to act directly via hormone receptors [caerulein and carbamylcholine (carbachol)] or indirectly (cyclic nucleotide analogs and the Ca²⁺ ionophore A23187). Of all agents tested, only dibutyryl cAMP elicited discharge of secretory proteins at day 20 in utero and preceded hormone stimulation by 1 day. A23187 elicited discharge by Day 21 in utero; its action was near adult levels in contrast to hormonal stimuli whose effect was maximal only at birth. All secretagogues required Ca²⁺ and energy to induce discharge. Pulse-chase autoradiography of lobules from Day 20 embryonic glands indicated that the acinar cells were capable of transporting [³H]leucine-labeled proteins to zymogen granules at rates roughly equivalent to those in adult glands. SDS gel electrophoretograms confirmed that the bulk of ¹⁴C-amino acid incorporation into proteins at a given age was primarily into exportable proteins. The results indicate that acinar cells synthesize and package secretory proteins into zymogen granules about 2 days before they are capable of responding to hormonal stimuli and to intracellular effectors.     

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin

Summary Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 ug x kg^-1 x h^-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU x kg^-1 x h^-1) and infusion period (1–24 h), except an increased number of coated vesicles in duct cells.Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/15-1)     

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin

Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 micrograms X kg-1 X h-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU X kg-1 X h-1) and infusion period (1-24 h), except an increased number of coated vesicles in duct cells. Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.     

Effect of thapsigargin on cytosolic Ca2+ level and amylase release in rat parotid acinar cells.

At concentrations greater than 0.01 microM, thapsigargin (ThG) dose-dependently caused an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in rat parotid acinar cells, as measured by the fluorescent Ca(2+)-indicator fura-2. In the absence of extracellular Ca2+, a transient increase in [Ca2+]i by ThG was observed, and subsequent addition of carbachol (CCh) did not produce a further [Ca2+]i response, suggesting that ThG released Ca2+ from the CCh-sensitive intracellular Ca2+ pool. Since ThG did not stimulate formation of inositol phosphates, the ThG-induced Ca2+ mobilization is independent of phosphoinositide breakdown. High concentrations (greater than 0.1 microM) of ThG induced amylase release from rat parotide acini, but the effect was very poor as compared with that of CCh or the protein kinase C activator, PMA (phorbol 12-myristate 13-acetate). Combined addition of ThG and PMA modestly potentiated amylase release induced by PMA alone. These results support the view that amylase release by muscarinic stimulation is mediated mainly by activation of protein kinase C rather than a rise in [Ca2+]i, although Ca2+ may modulate the secretory response.     

Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells

We have tested the effects of alcohols differing by their alkyl chain length on the membrane channels and amylase secretion of rat pancreatic acinar cells. In intact acini, alcohols with a chain of seven, eight, or nine carbons (C-7, C-8, and C-9) induced dye uncoupling and increased basal amylase release. These effects were readily reversible after alcohol removal. By contrast, an alcohol with a chain of 15 carbons (C-15) and several alcohols with chains of fewer than six carbons (C-2, C-4, and C-6) did not uncouple acinar cells and had no effects of amylase secretion. Neither did alkanes and oxidized derivatives of C-7 and C-8 alcohols did not affect dye coupling. Double patch-clamp experiments on pairs of acinar cells, under conditions of strong cytosolic Ca2+ and pH buffering, showed that C-7, C-8, and C-9 alcohols blocked completely and reversibly the electrical conductance of junctional channels. Furthermore, studies of single voltage-clamped acinar cells revealed that the uncoupling alcohols did not affect the resting nonjunctional membrane conductances. Thus the alcohols that did not affect acinar cells coupling did not affect amylase secretion, whereas the alcohols that caused uncoupling increased secretion. The latter effect was not mediated by changes in the conductance of nonjunctional membrane, cytosolic Ca2+, and pH and, as revealed by an immunological hemolytic plaque assay for amylase, had a time course consistent with the rapid (within 1 min) inhibition of coupling. These data provide new support for the view that the regulation of cell-to-cell communications is correlated with that of digestive enzyme secretion.