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.     

Evaluation of the role of calcium in cytotoxic injury in isolated rat pancreatic acini

The role of extracellular Ca2+ in pancreatic acinar membrane damage (cellular injury) by nicotine, membrane-active agents (mellitin, snake venom and Ca2+ ionophore A23187) and secretagogues (CCK-8 and secretin) was investigated. Freshly isolated dispersed pancreatic acini from 18 h fasted adult rats were incubated with one of the aforementioned agents, in the absence and presence of Ca2+. Cellular injury was assessed by measuring the release of pulse-labeled 51Cr and LDH. In addition, release of amylase, trypsinogen and chymotrypsinogen was also determined. In the absence of Ca2+ nicotine (6 mM) caused a profound release of 51Cr and LDH as well as amylase, trypsinogen and chymotrypsinogen from the isolated pancreatic acini. Release of these enzymes and 51Cr decreased sharply with addition of increasing concentrations (0.25-5 mM) of Ca2+. Release of 51Cr and amylase by snake venom (50 micrograms/ml) was found to be 100 and 25% higher, respectively, in the absence of Ca2+ than in its presence. On the other hand, the Ca2+ ionophore A23187 (7 micrograms/ml) was found to be effective in releasing 51Cr and amylase only in the presence of Ca2+. CCK-8, (0.25nM), secretin (1 microM) and mellitin (0.5 microgram/ml) although significantly stimulated amylase secretion (225-350%) in the presence of Ca2+, none of the agents induced 51Cr release from acini, either in the absence or in the presence of extracellular Ca2+. It is concluded that the extracellular Ca2+ plays no specific role in cytotoxic injury in isolated pancreatic acini.     

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)- conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

The effect of human pancreatic polypeptide (HPP) on rat pancreatic acini has been studied. It was found that HPP stimulated amylase and lipase release from the acini. The secretory response of acini to HPP was dose-dependent in a sigmoidal fashion. Between 10(-9) M and 10(-8) M concentration of HPP there was a slow increase of enzyme release to about 40-60% over basal release. At concentrations of HPP above 10(-8) M there was a rapid increase of enzyme release, amounting to 4-6 times over basal release at 10(-6) M concentration of HPP. The potency of HPP compared to other secretagogues at 10(-7) M concentration was 45% of CCK, 60% of carbachol and 75% of secretin. HPP did not inhibit the effect of CCK, secretin and carbachol on amylase release. The amylase release stimulated by HPP was accompanied by an increase in 45Ca2+ efflux. Atropine or dibutyryl cyclic GMP did not influence the effect of HPP. It is concluded that HPP stimulates the release of enzymes from rat pancreatic acini and that Ca2+ may be a mediator for this secretion.     

Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.     

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

1,2-Diacylglycerol, protein kinase C, and pancreatic enzyme secretion

To determine the role of 1,2-diacylglycerol (1,2-DAG) and protein kinase C in pancreatic enzyme secretion, we measured the effect of various pancreatic secretagogues on the cellular mass of 1,2-DAG and amylase release in dispersed pancreatic acini from the guinea pig. In addition, we measured the effect of a recently described protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry 23, 5036-5041), on secretagogue-stimulated amylase release from the acini. Cholecystokinin-octapeptide (CCK-OP), cholecystokinintetrapeptide, and carbachol each increased 1,2-DAG 2-3-fold but the increases occurred only with concentrations of these secretagogues that were supramaximal for amylase release and that had an inhibitory effect on stimulated amylase release. Supramaximal concentrations of bombesin stimulated only a small increase in 1,2-DAG and did not cause inhibition of stimulated amylase release. When the action of carbachol was terminated with atropine or CCK-OP with dibutyryl cyclic GMP, stimulated amylase release ceased immediately but cellular 1,2-DAG required at least 15 min to return to the basal level. Increasing cytosolic free Ca2+ with the Ca2+ ionophore, A23187, in Ca2+-containing incubation media augmented amylase release stimulated by 4 beta-phorbol 12-myristate 13-acetate but inhibited amylase release stimulated by CCK-OP, carbachol, and bombesin without decreasing the cellular content of 1,2-DAG. H-7 inhibited protein kinase C activity in a pancreatic homogenate but augmented amylase release from acini stimulated by either CCK-OP, carbachol, or 4 beta-phorbol 12-myristate 13-acetate. These findings indicate that 1,2-DAG and protein kinase C do not have a stimulatory role in pancreatic stimulus-secretion coupling but may have an inhibitory one.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. I. Ca2+ channel blockers

The effects of Ruthenium red and tetracaine, which inhibit Ca2+-induced Ca2+ release from the isolated sarcoplasmic reticulum (e.g., Ohnishi, S.T. (1979) J. Biochem. (Tokyo) 86, 1147-1150), on several types of Ca2+ release in vitro were investigated. Ca2+ release was triggered by several methods: (1) addition of quercetin or caffeine, (2) Ca2+ jump, and (3) replacement of potassium gluconate with choline chloride to produce membrane depolarization. The time-course of Ca2+ release was monitored using stopped-flow spectrophotometry and arsenazo III as a Ca2+ indicator. Ruthenium red inhibited all of these types of Ca2+ release with the same concentration for half-inhibition C1/2 = 0.08-0.10 microM. Similarly, tetracaine inhibited these types of Ca2+ release with C1/2 = 0.07-0.11 mM. Procaine also inhibits both types of Ca2+ release induced by method 2 and 3 with C1/2 = 0.67-1.00 mM. These results suggest that Ruthenium red, tetracaine and procaine interfere with a common mechanism of the different types of Ca2+ release. On the basis of several pieces of evidence we propose that Ruthenium red and tetracaine block the Ca2+ channel of sarcoplasmic reticulum.     

Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.     

Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin

We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha- amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events.     

Mechanism of action of bombesin on amylase secretion. Evidence for a Ca2+-independent pathway

The mode of action of bombesin on amylase secretion was investigated in rat pancreatic acini. Bombesin induced a dose-dependent increase in inositol 1,4,5-trisphosphate and cytosolic free Ca2+. The threshold concentration capable of inducing both effects was 0.1 nM and the half-maximal dose of the peptide for Ca2+ mobilization was approximately 0.6 nM. By contrast, amylase release was approximately 30 times more sensitive than inositol 1,4,5-trisphosphate production and Ca2+ mobilization to bombesin action, with 1 pM being the first stimulatory concentration and a half-maximal effect at approximately 20 pM. The ability of low bombesin doses to trigger enzyme secretion was unaffected by chelation of extracellular Ca2+ with EGTA. In order to test whether the stimulation of amylase release was truly a Ca2+-independent response, the intracellular Ca2+ stores were depleted by pretreating acini with EGTA plus ionomycin, the Ca2+ ionophore. Under these conditions bombesin was still capable of eliciting a significant twofold enhancement of the secretory activity. These results indicate that bombesin, an agonist thought to activate secretion mainly through mobilization of Ca2+ from intracellular stores, elicits amylase release at low concentrations, independently of a concomitant rise in cytosolic free Ca2+. The relevance of these findings to the physiological regulation of pancreatic exocrine secretion is discussed.     

Isolation and functional characterization of rat pancreatic acini.

The Authors describe an optimized procedure for the isolation of rat pancreatic acini and the preliminary results concerning the functional characterization of the cells. Isolation is carried out by two sequential digestive steps in a KREBS modified medium containing collagenase, separated by an intermediate step in which acini separation is fostered by incubation in a Ca++ free medium containing the Ca++ chelator EDTA. Final separation is obtained through the application of mechanical forces by aspirating the suspension in plastic pipettes. The choice of the collagenase, the duration and the entity of the mechanical dissociation strength are the main modifications to the classic procedure and allow to obtain a very high yield of cells maintaining their ability to secrete enzymes for a long period (6-7 hours). Functional characterization is completed mainly by the determination of the amylase release stimulated by increasing doses of cholecystokinin.     

Neurotransmitter release

Axon terminals release more than one physiologically active substance. Synaptic messengers may be stored in two different types of vesicles. Small electron-lucent vesicles mainly store classical low molecular weight transmitter substances and the larger electron-dense granules store and release proteins and peptides. Release of the two types of substances underlies different physiological control. Release of messenger molecules from axon terminals is triggered by influx of Ca2+ through voltage sensitive Ca2+ channels and a rise in cytosolic Ca2+ concentrations. Neither the immediate Ca2+ target(s) nor the molecular species involved in synaptic vesicle docking, fusion and retrieval are known. It is, however, likely that steps involved in the molecular cascade of transmitter release include liberation of vesicles from their association with the cytonet and phosphorylation by protein kinase C of proteins which have the ability to alter between membrane bound and cytoplasmic forms and thus facilitate or initiate the molecular interaction between synaptic vesicles and the plasma membrane.     

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.     

Rat pancreatic acini permeabilised with streptolysin O secrete amylase at Ca2+ concentrations in the micromolar range, when provided with ATP and GTP gamma S.

The aim of this study was to define the conditions required for exocytosis in pancreatic acini permeabilised with the bacterial toxin streptolysin O. Treatment of a suspension of acini with streptolysin O caused the release of both the cytoplasmic enzyme lactate dehydrogenase and the zymogen granule enzyme amylase. The release of amylase occurred more quickly than that of lactate dehydrogenase and was smaller in magnitude. In addition, a component of amylase release occurred only in the presence of Ca2+ (at concentrations in the micromolar range), ATP and GTP gamma S. We conclude that this component represents an exocytotic event, but that the release of lactate dehydrogenase occurs through toxin-generated lesions. The concentrations of Ca2+, ATP and GTP gamma S causing half-maximal exocytosis were 0.7 microM, 0.2 mM and 10 microM, respectively. This system should permit a study of the mechanisms underlying regulated exocytosis in this cell type.     

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tetracaine inhibition of electron transport in pea chloroplasts is coupled to calcium displacement by the local anesthetic

Chloroplasts isolated from pea seadlings grown on water containing 45Ca2+ were treated with local anesthetic tetracaine. Addition of tetracaine inactivated the electron transport activity of donor side photosystem II. This inhibition was accompanied by 45Ca2+ release from the chloroplast membranes as the whole and destroyed by osmotic shock. No such effect was observed when Tris or hydroxylamine were used to inhibit the donor side photosystem II. Upon thermal inactivation of chloroplasts 45Ca2+ release occurred but at temperatures above 80 degrees. The functional role of Ca2+ in photosystem II is discussed.     

Measurement of cytosolic free Ca2+ in individual pancreatic acini

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.