Inhibition by mepacrine and amylase secretion from intact and permeabilized rat pancreatic acini.

In intact rat pancreatic acini, the phospholipase A2 inhibitor mepacrine did not affect basal amylase release but dose-dependently inhibited the carbachol (IC50 65 microM) and CCK-8 (IC50 210 microM)-stimulated amylase release. In permeabilized acini, mepacrine shifted the dose-response curve for calcium to the right by a factor 2 and inhibited the release of amylase stimulated by GTPrS. From these results we conclude that carbachol, CCK-8 and GTPrS probably activate a phospholipase A2 closely coupled to exocytosis.     

Reversible acetaldehyde inhibition of A23187-stimulated amylase secretion from isolated rat pancreatic acini

The Ca2+ ionophore, A23187, stimulated amylase secretion from isolated rat pancreatic acini in a dose-dependent manner with a maximal effect at 6 microM. Acetaldehyde, a metabolite of ethanol, caused a reduction in the magnitude of ionophore-stimulated secretion with no evidence of competitive inhibition. Furthermore, 6 microM ionophore-stimulated amylase secretion was dose-dependently inhibited by acetaldehyde. This inhibitory effect of acetaldehyde, however, was reversible on washing and reincubating acetaldehyde-treated acini. These results suggest that acetaldehyde reversibly inhibits intracellular components mediating stimulated secretion and this inhibition requires a continuous chemical interaction between acetaldehyde and intracellular component(s) regulating stimulated enzyme secretion.     

Calcium dependence of proteinase-activated receptor 2 and cholecystokinin-mediated amylase secretion from pancreatic acini

Pancreatic acini secrete digestive enzymes in response to a variety of secretagogues including CCK and agonists acting via proteinase-activated receptor-2 (PAR2). We employed the CCK analog caerulein and the PAR2-activating peptide SLIGRL-NH(2) to compare and contrast Ca(2+) changes and amylase secretion triggered by CCK receptor and PAR2 stimulation. We found that secretion stimulated by both agonists is dependent on a rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and that this rise in [Ca(2+)](i) reflects both the release of Ca(2+) from intracellular stores and accelerated Ca(2+) influx. Both agonists, at low concentrations, elicit oscillatory [Ca(2+)](i) changes, and both trigger a peak plateau [Ca(2+)](i) change at high concentrations. Although the two agonists elicit similar rates of amylase secretion, the rise in [Ca(2+)](i) elicited by caerulein is greater than that elicited by SLIGRL-NH(2). In Ca(2+)-free medium, the rise in [Ca(2+)](i) elicited by SLIGRL-NH(2) is prevented by the prior addition of a supramaximally stimulating concentration of caerulein, but the reverse is not true; the rise elicited by caerulein is neither prevented nor reduced by prior addition of SLIGRL-NH(2). Both the oscillatory and the peak plateau [Ca(2+)](i) changes that follow PAR2 stimulation are prevented by the phospholipase C (PLC) inhibitor U73122, but U73122 prevents only the oscillatory [Ca(2+)](i) changes triggered by caerulein. We conclude that 1) both PAR2 and CCK stimulation trigger amylase secretion that is dependent on a rise in [Ca(2+)](i) and that [Ca(2+)](i) rise reflects release of calcium from intracellular stores as well as accelerated influx of extracellular calcium; 2) PLC mediates both the oscillatory and the peak plateau rise in [Ca(2+)](i) elicited by PAR2 but only the oscillatory rise in [Ca(2+)](i) elicited by CCK stimulation; and 3) the rate of amylase secretion elicited by agonists acting via different types of receptors may not correlate with the magnitude of the [Ca(2+)](i) rise triggered by those different types of secretagogue.     

Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.     

Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]methylscopolamine binding. Their relationship with calcium 45 efflux and amylase secretion

N-[3H]Methylscopolamine (NMS) binding, amylase secretion, and 45Ca efflux from dispersed rat pancreatic acini were investigated in parallel, in the presence or absence of 4 muscarinic agonists and 3 muscarinic antagonists. Scatchard analysis of [3H]NMS saturation isotherms gave a KD of 0.9 nM and an average binding capacity of 24,000 sites per cell. Binding competition curves with the antagonists atropine, dexetimide, and NMS gave KD values of 3.5, 3.5, and 0.5 nM, respectively. With the 3 full agonists oxotremorine, muscarine, and carbamylcholine, the receptor population could be divided into two classes of binding sites: a minor one (15%) with high affinity (KD = 20-35 nM) and a major one (85%) with low affinity (KD = 3-65 microM). There was a receptor reserve of about 50% with respect to carbamylcholine-stimulated amylase secretion. Further analysis of dose-effect curves suggests that low affinity binding sites were involved in the secretory response to muscarinic stimulation. Pilocarpine, like muscarinic antagonists, recognized all binding sites with the same affinity but acted as a partial agonist on amylase secretion and 45Ca efflux.     

Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Effect of cytochalasin D on secretion by rat pancreatic acini

Using the model of isolated acini the effect of cytochalasin D (CD) on rat pancreatic secretion in vitro was studied. The influence of CD (0.01-10 micrograms/ml = 0.02-19.7 microM) on amylase secretion and 3H-pulse-labelled protein release was measured under two sets of conditions: (a) basal, and (b) stimulated by 77pM caerulein. Basal secretion was not altered, but stimulated secretion of amylase and 3H-labelled proteins were similarly inhibited by up to 45%. It is concluded that CD affects only exocytosis of zymogen granules and not intracellular transport.     

Cholecystokinin-induced secretion and synthesis of amylase and cationic trypsinogen by pancreatic acini isolated from rats given an ethanol diet

Effects of chronic alcohol intake on secretion and synthesis of amylase and cationic trypsinogen (CT) were studied with pancreatic acini isolated from male Sprague-Dawley rats fed a Lieber-DeCarli ethanol or control diet for 30 days. Pancreatic acini were incubated in a media containing increasing concentrations of cholecystokinin octapeptide (CCK-8: 0-1000pM) followed by addition of [3H]leucine. Amylase and CT secreted in the media and those labeled in acini were quantitated. Basal and CCK-stimulated secretion of CT was not different in alcoholic and control groups. On the other hand, a dose-response curve of CCK-stimulated amylase secretion from alcoholic acini was markedly reduced with both basal and maximal secretion decreased to only 40% of controls. Basal incorporation of [3H]leucine into amylase was reduced by 70% in alcoholic acini compared to controls while that into CT was not different in the two groups. CCK-8 exhibited a biphasic effect on [3H]leucine incorporation into both enzymes in alcoholic acini: low concentrations of CCK-8 (less than 100pM) increased the incorporation whereas high concentrations (greater than 100pM) decreased it. However, in control acini, CCK-8 induced progressive suppression of the incorporation into these enzymes, the pattern of which was similar to that previously observed in fasted rats (Am. J. Physiol. 241:G116-G112, 1981). This difference in the response pattern resulted in significantly higher rates of CCK-8 induced incorporation into CT in alcoholic acini. These results suggest that the differences observed may possibly be attributable not only to alcohol intake but also to the differences in carbohydrate intake and in temporal patterns of diet consumption.     

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.     

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     

Loss of Munc18-1 long splice variant in GABAergic terminals is associated with cognitive decline and increased risk of dementia in a community sample

Background

Presynaptic terminals contribute to cognitive reserve, balancing the effects of age-related pathologies on cognitive function in the elderly. The presynaptic protein Munc18-1, alternatively spliced into long (M18L) or short (M18S) isoforms, is a critical modulator of neurotransmission. While subtle alterations in Munc18-1 have been shown to cause severe neuropsychiatric disorders with cognitive impairment, little information is known regarding the specific roles of Munc18-1 splice variants. We first investigated functional and anatomical features evidencing the divergent roles of M18L and M18S, and then evaluated their contribution to the full range of age-related cognitive impairment in the dorsolateral prefrontal cortex of a large sample of participants from a community-based aging study, including subjects with no-(NCI, n?=?90), or mild-(MCI, n?=?86) cognitive impairment, or with clinical dementia (n?=?132). Finally, we used APP23 mutant mice to study the association between M18L/S and the time-dependent accumulation of common Alzheimer’s disease pathology.

Results

Using isoform-specific antibodies, M18L was localized to the synaptosomal fraction, with a distribution matching lipid raft microdomains. M18S was found widely across cytosolic and synaptosomal compartments. Immunocytochemical studies identified M18L in perisomatic, GABAergic terminals, while M18S was broadly distributed in GABAergic and glutamatergic terminals. Using regression models taking into account multiple age-related pathologies, age, education and sex, global cognitive function was associated with the level of M18L (p?=?0.006) but not M18S (p?=?0.88). Mean M18L in dementia cases was 51 % lower than in NCI cases (p?<?0.001), and each unit of M18L was associated with a lower likelihood of dementia (odds ratio?=?0.68, 95 % confidence interval?=?0.50–0.90, p?=?0.008). In contrast, M18S balanced across clinical and pathologically diagnosed groups. M18L loss may not be caused by age-related amyloid pathology, since APP23 mice (12- and 22-months of age) had unchanged cortical levels of M18L/S compared with wild-type animals.

Conclusions

M18L was localized to presynaptic inhibitory terminals, and was associated with cognitive function and protection from dementia in an elderly, community-based cohort. Lower M18L in inhibitory presynaptic terminals may be an early, independent contributor to cognitive decline.

     

Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini

Treatment of intact rat pancreatic acini with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA) resulted in a time- and concentration-dependent translocation of phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK) from the soluble fraction. Redistribution of PL/Ca-PK was concurrent with stimulation of amylase secretion by TPA-treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca-PK completely inhibited TPA-induced amylase secretion. These findings are consistent with a role for PL/Ca-PK in the regulation of pancreatic exocrine secretion.     

A new splice variant of glial fibrillary acidic protein,GFAP epsilon,interacts with the presenilin proteins

We describe a new human isoform, GFAP epsilon, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP epsilon mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP epsilon has a new C-terminal protein sequence. This provides GFAP epsilon with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP epsilon is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP epsilon should be taken into consideration when studying neurodegenerative diseases.     

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.     

Phorbol ester inhibits cholecystokinin octapeptide-induced amylase secretion and calcium mobilization, but is without effect on secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in rabbit pancreatic acini

The effect of prolonged protein kinase C activation on cholecystokinin octapeptide (CCK-8)-induced amylase secretion from rabbit pancreatic acini was studied by means of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). The phorbol ester itself increased basal amylase secretion but inhibited completely the secretory response to relatively low concentrations of CCK-8. The inhibitory action of TPA on CCK-8-induced amylase secretion was paralleled by inhibition of CCK-8-induced calcium mobilization but not by inhibition of CCK-8-induced breakdown of 32P-labelled phosphatidylinositol 4,5-bisphosphate. The results presented suggest that protein kinase C, or one of its phosphorylated products, inhibits the CCK-8-stimulated pathway leading to secretion at a level beyond the secretagogue-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inhibition of the initial, inositol 1,4,5-trisphosphate-mediated and extracellular calcium-independent, increase in free cytosolic calcium concentration, together with the findings of others, suggests that the efficacy of this inositol-phosphate to release calcium is reduced.