Multiple effects of Na+ removal on pancreatic secretion in vitro

Summary The effects of removing Na⁺ from the incubation medium on basal and secretagogue induced zymogen release by pancreatic fragments and isolated pancreatic acini were studied by both morphological evaluation and measurement of amylase release. In both fragments and isolated acini, removal of Na⁺ led to an increased basal secretion of zymogen granule contents from acinar cells via exocytosis; secretory material, however, accumulated in acinar and ductular lumina as a result of the lack of fluid secretion necessary to wash out the enzymes. In studies with fragments, after Na⁺ removal there was no significant increase in amylase release into the medium; isolated acini, in contrast, showed an increased amylase release consistent with the shorter distance from the acinar lumen to the bathing medium. Stimulation with either bethanechol or caerulein led to a further depletion of zymogen granules in both preparations; in the absence of Na⁺ secretory product accumulated in intracellular lakes as well as in duct lumens. The hypothesis that Na⁺ influx is important in stimulus-secretion coupling to release intracellular Ca²⁺ was directly tested by measuring ⁴⁵Ca²⁺ efflux. No effect of removing Na⁺ on ⁴⁵Ca²⁺ efflux was seen. It was concluded, therefore, that while Na⁺ is essential for pancreatic fluid secretion, it is not necessary for the secretion of zymogen granule contents into acinar lumina.Supported by NIH grant GM-19998 from the United States Public Health Service     

Pancreatic acinar cells: use of Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling

The Ca⁺⁺ ionophore A23187 had no effect on the release of amylase by mouse pancreas fragments in the absence of Ca⁺⁺ but when Ca⁺⁺ was re-added to the medium amylase release was observed in a pattern which mimicked that produced by normal stimulants. Uptake of ⁴⁵Ca⁺⁺ by pancreatic fragments was increased by A23187. Tetracaine and dinitrophenol at concentrations which block cholinergic stimulated enzyme release blocked ionophore induced release whereas atropine did not. None of the inhibitors studied affected the ionophore induced Ca⁺⁺ uptake.     

The effect of the ionophore A23187 on amylase release,cellular integrity and ultrastructure of mouse pancreatic acini

Summary The effects of the Ca²⁺ ionophore A 2317 on pancreatic amylase and lactate dehydrogenase (LDH) release, cellular electrolyte balance and ultra-structure were studied with the use of incubated pancreatic fragments. A 23187 (0.3 M) in the presence of Ca²⁺, increased amylase release but at higher concentrations (1–10 M) also increased LDH release and increased uptake of ¹⁴C-sucrose with concomitant loss of tissue K⁺ and gain in Na ⁺. The ultrastructure of the majority of acini appeared normal and showed depletion of zymogen granules. Microtubules and microfilaments which have been implicated in the release process were normal or increased in number. In the absence of Ca⁺ the ionophore had no effect on secretion, cellular integrity or ultrastructure. It is concluded that A 23187 in the presence of Ca²⁺ increases amylase release by a mechanism comparable to the terminal steps in stimulussecretion coupling induced by physiological secretagogues. This provides further evidence that amylase release is mediated by a rise in cell Ca²⁺ although the mechanisms of the ionophore- and physiological secretagogue-induced rise in Ca⁺ are probably different. High concentrations of ionophore (> 1 M) also induce Ca²⁺ dependent damage in a fraction of the cells.Supported by grants from the NIH (GM 19998) and the Cystic Fibrosis FoundationI am indebted to Drs. Douglas Chandler and John Heuser for discussion and advice and to M. Lee and E. Roach for technical assistance     

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.     

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.     

Effects of membrane stabilizers on pancreatic amylase release

Summary Compounds with membrane stabilizing activity were studied as to their ability to affect pancreatic amylase release and the steps in the stimulus-secretion coupling process. Chlorpromazine, propranolol, and thymol were all found to inhibit bethanechol-stimulated amylase release and at slightly higher concentrations to induce release regardless of the presence of the secretagogue. This biphasic effect was similar to that found previously for the local anesthetic tetracaine. Release by high concentrations of propranolol and tetracaine was accompanied by ultrastructural evidence of cell damage. Membrane stabilizers at concentrations which inhibited amylase release were shown to block bethanechol-induced depolarization and stimulation of⁴⁵Ca⁺⁺ efflux although the drugs alone partially depolarized pancreatic cells. Release of amylase induced by Ca⁺⁺ introduced by the ionophore A23187 was also abolished. These findings indicate that membrane stabilizers independently inhibit the steps leading to a rise in intracellular Ca⁺⁺ and the subsequent Ca⁺⁺-activated amylase release.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

IP3 Receptor Type 2 Deficiency Is Associated with a Secretory Defect in the Pancreatic Acinar Cell and an Accumulation of Zymogen Granules

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca²⁺. These aberrant Ca²⁺ elevations are triggered by release of Ca²⁺ from apical Ca²⁺ pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca²⁺ release channel (IP3R2^−/−). Using live acinar cell Ca²⁺ imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca²⁺ signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2^−/− and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca²⁺ signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.     

Effects of Ca ionophore A23187 and Ca deficiency on pancreatic phospholipids and amylase release in vitro

The Ca²⁺ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinositol breakdown are dependent on medium Ca²⁺. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca²⁺ in the pancreas.     

Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

Amylase release from rat parotid glands I. General characteristics

The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca²⁺ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K⁺ concentration, reduction of the H⁺ concentration, La³⁺, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced by dibutyryl cyclic AMP, K⁺, and reduction of H⁺, but had no effect on La³⁺, caffeine or tolbutamide-stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K⁺, reduction of H⁺ or caffeine.The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La³⁺, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux of both amylase and lactate dehydrogenase.The differential involvement of Ca²⁺, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase.     

Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

Calcium and cyclic nucleotide involvement in exocrine pancreatic enzyme secretion studied with the ionophore A-23187.

The ionophore, A-23187, was an effective pancreatic secretagogue. The response to A-23187 was Ca²⁺-dependent; Mg²⁺ reduced the secretory response to the ionophore. A-23187-stimulated enzyme release was potentiated by dibutyryl cyclic AMP; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The time-course for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Ca²⁺-dependent production of pancreatic cyclic GMP which preceded the onset of the secretory response. A-23187 did not significantly alter basal or carbachol-stimulated ⁴⁵Ca efflux from isotope preloaded glands; yet in Ca²⁺-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Ca²⁺. Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Ca²⁺. The data suggest that the pancreatic cholinergic receptor acts as a Ca²⁺-ionophore and that extracellular Ca²⁺ is utilized in the synthesis of cyclic GMP.     

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.     

Bcl-2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin-Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca²⁺-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca²⁺ ([Ca²⁺]_i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca²⁺ diminished somewhat the size of the initial increase of [Ca²⁺]_i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La³⁺ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca²⁺ chelator, inhibited the peak [Ca²⁺]_i caused by thapsigargin but did not inhibit the sustained elevation of [Ca²⁺]_i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto-oncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca²⁺]_i or the elevation of [Ca²⁺]_i induced by thapsigargin. Our results suggest that abnormal Ca²⁺ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca²⁺ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca²⁺ release in GT1-7 neuronal cells.     

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca²⁺ release induced with 0.18 μM IP₃ is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP₃, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca²⁺ uptake into intracellular stores, does not influence IP₃ induced Ca²⁺ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP₃ to the same cellular preparations was investigated. We found that binding of IP₃ to a high affinity receptor site (K_d approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP₃ binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP₃ with the same affinity (K_d approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP₃ was displaced from these subcellular membranes with similar kinetics by unlabelled IP₃ and decavanadate. The data suggest that the inhibitory action of decavanadate on IP₃ induced Ca²⁺ release is a consequence of its effect on binding of IP₃ to its receptor.     

Inhibition of ruthenium red-insensitive mitochondrial Ca2+ release and its pyridine nucleotide specificity

Isolated, intact rat liver mitochondria, without extraneous substrates but loaded with Ca²⁺ (20 nmol/mg), can be observed to release Ca²⁺ when treated with ruthenium red. Such release can be inhibited by 0.33 mM dlisocitrate but not by 10 mM dl-β-hydroxybutyrate. Assays of NADP⁺, NADPH, NAD⁺, and NADH revealed that only the reduction of NADP⁺ can be linked with such inhibition of Ca²⁺ release, not that of NAD⁺. Since ruthenium redinsensitive Ca²⁺ release is a physiological (but normally masked) process, this experimental approach avoids some potential problems ascribed to strong pyridine nucleotide oxidation. It is suggested that specific NADP⁺:NADPH dependent reactions are part of a physiological mechanism regulating Ca²⁺ release/retention.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Summary The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.Supported by NIH grant GM-19998 from the United States Public Health Service. — We are indebted to Dr. John Heuser for advice throughout this project and assistance in the preparation of rapidly frozen acini, and to M. Lee for technical assistance     

Ruthenium red-sensitive and Ruthenium Red-insensitive release of calcium by mitochondria isolated from rat liver and from rat heart

EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca²⁺ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca²⁺ transport. The EGTA-induced release of Ca²⁺ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl^?, inhibited the Ruthenium Red-sensitive Ca²⁺ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca²⁺ induced by acetoacetate in liver and heart mitochondria and by Na⁺ in heart mitochondria.