The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas

1. The role of extracellular and intracellular Ca(2+) in pancreatic enzyme secretion has been assessed by correlating the exchange of (45)Ca with amylase secretion in the isolated uncinate pancreas of baby rats.2. The rate coefficient of (45)Ca efflux from pre-loaded glands declined continually (indicating that (45)Ca is retained in several different pools) and probably reflects changes in the concentration of cytoplasmic free (45)Ca, which is determined by the rate at which (45)Ca is released from intracellular organelles into the cytoplasm.3. The rate coefficient of (45)Ca release was not influenced by extracellular Ca(2+) or Mg(2+) concentrations.4. Cholecystokinin-pancreozymin (CCK-PZ) and acetylcholine accelerated the release of both (45)Ca and amylase in a dose-dependent fashion, even when extracellular Ca(2+) was reduced to 0.1 mM, but did not affect the initial rate of (45)Ca uptake by the tissue.5. In Ca(2+)-free media (containing 0.5 mM-EGTA) basal amylase secretion slowly declined and stimulated secretion was virtually abolished, but the accelerated release of (45)Ca was maintained.6. These observations indicate that natural stimuli of pancreatic enzyme secretion alter (45)Ca distribution in the cell by a process which is independent of extracellular Ca(2+) and which is associated with amylase secretion provided that the plasma membrane has not been depleted of Ca(2+).7. Secretin, glucagon and insulin did not influence (45)Ca release. Secretin slightly increased amylase secretion, but this may have been a washout effect.8. Replacement of extracellular Na(+) by Li(+) increased the release of (45)Ca and amylase, but only in the presence of extracellular Ca(2+). Li(+)-substitution also increased (45)Ca uptake. Thus, under special conditions, secretion may be stimulated when increased amounts of Ca(2+) are made available from extracellular sources.9. Hyperosmolarity (known to increase (45)Ca release in muscle) also accelerated (45)Ca release and amylase secretion.10. 2,4-Dinitrophenol markedly accelerated (45)Ca efflux but did not stimulate amylase secretion, indicating that a rise in cytoplasmic Ca(2+) will not initiate secretion if energy metabolism is impaired.11. CCK-PZ slightly increased the rate coefficient of (42)K release, indicating a changed membrane permeability.12. The stimulatory effects of CCK-PZ and acetylcholine were suppressed during Na(+)-substitution by Li(+), suggesting that the Na(+) concentration gradient across the membrane is important in secretion.13. It is concluded that the primary action of CCK-PZ and acetylcholine may be to increase the influx of Na(+) into the cell by changing membrane permeability. This in turn is responsible for the release of Ca(2+) from intracellular stores (probably endoplasmic reticulum), leading to a rise in Ca(2+) concentration close to the structures involved in enzyme secretion. Secretion then follows provided that ATP is available and the plasma membrane is not depleted of Ca(2+).     

Pancreatic acinar cells: ionic dependence of the membrane potential and acetycholine-induced depolarization

1. Intracellular recordings of membrane potentials have been made in vitro from the exocrine acinar cells of the mouse pancreas using glass micro-electrodes.2. The mean membrane potential of the acinar cells during superfusion with Krebs-Henseleit solution was -39.2 mV. Increasing [K](o) tenfold decreased the membrane potential by 28 mV when [K](o) was above 10 mM. This depolarization was not affected by atropine (1.4 x 10(-6)M). Strophanthin-G (10(-3)M) slowly depolarized the cells at about 10 mV hr(-1).3. Brief exposure to acetylcholine (ACh), 5.5 x 10(-5)M, or pancreozymin resulted in a short lasting depolarization of the acinar cells. Atropine (1.4 x 10(-6)M) blocked the depolarizing action of ACh but not that of pancreozymin. Adrenaline (5.5 x 10(-5)M) or cyclic AMP (10(-3)-10(-4)M) did not influence the membrane potential.4. The amplitude of the ACh-induced depolarization was not dependent on the presence of CO(2)/HCO(3) in the bathing fluid, but it was closely dependent on the extracellular Na concentration. However, ACh was still able to evoke a small depolarization even after prolonged exposure of the tissue to a Na-free solution.5. During exposure of the tissue to a Ca-free solution the resting membrane potential was decreased and the ACh-induced depolarization was significantly reduced. Some substances which are known in other tissues to inhibit membrane Ca(2+) currents, i.e. La(3+), D-600 and tetracaine, were able to reduce, but never abolish, the ACh-induced depolarization.6. These results suggest that the effect of ACh on the pancreatic acinar cell is to increase the permeability of the membrane to commonly occurring ions with a consequent Na-influx and a small Ca-influx.     

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

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

Interaction of substance P with dispersed pancreatic acinar cells from the guinea pig. Stimulation of calcium outflux, accumulation of cyclic GMP and amylase release

In dispersed acinar cells from the guinea pig pancreas, substance P (SP) was found to stimulate outflux of 45Ca, cellular accumulation of cyclic GMP, and release of amylase. Maximal effects on accumulation of cyclic GMP and release of amylase were obtained with 3 x 10(-8) M of SP, 10(-7) M of Sp caused maximal outflux of 45Ca. These effects corresponded to 30-50% of the maximal effects obtained with caerulein, a cholecystokinin-like decapeptide. The concentrations of SP required for stimulation of 45Ca outflux, accumulation of cyclic GMP, and release of amylase correspond well with those which affect binding of 125I-tyr8-SP to pancreatic acinar cells.     

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

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

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

Effect of extracellular magnesium on nerve-mediated and acetylcholine-evoked in vitro amylase release in rat parotid gland tissue

In this study the effects of changes in extracellular magnesium ([Mg(2+)](o)) and calcium ([Ca(2+)](o)) concentrations on basal and on nerve-mediated and acetylcholine (ACh)-evoked in vitro amylase release and calcium mobilization were investigated in rat parotid gland tissue. In the presence of a normal (2.56 mM) [Ca(2+)](o), both zero (0 mM) and an elevated (10 mM) [Mg(2+)](o) significantly attenuated basal and ACh-evoked amylase release compared to the response obtained in normal (1.1 mM) [Mg(2+)](o). During electrical field stimulation (EFS) of parotid tissues, only elevated [Mg(2+)](o) reduced amylase release. In a Ca(2+)-free medium, both basal and ACh-evoked amylase output were markedly reduced compared to the responses obtained under similar conditions in normal [Ca(2+)](o). Again, the ACh-induced amylase release in a Ca(2+)-free solution was larger in normal [Mg(2+)](o) than when the [Mg(2+)](o) was either zero or was elevated to 10 mM. Perturbation of [Mg(2+)](o) had no significant effect on basal intracellular free calcium concentration ([Ca(2+)](i)) in parotid acinar cells loaded with the fluorescent Ca(2+) indicator fura-2. Both zero Mg(2+) and an elevated [Mg(2+)](o) significantly reduced the ACh-induced rise in the peak and the plateau phase of the Ca(2+) transient that was seen in normal [Mg(2+)](o). In parotid acinar cells loaded with the fluorescent Mg(2+) indicator magfura-2, ACh elicited a gradual decrease in intracellular free Mg(2+) concentration ([Mg(2+)](i)) to below the basal level. The results indicate that both hypo- and hypermagnesaemia may reduce both basal and ACh-evoked amylase secretion from the salivary gland. As far as the ACh-evoked response is concerned, the effect may be exerted by a decrease in cellular Ca(2+) transport.     

Pancreatic acinar cells: the role of calcium in stimulus-secretion coupling.

1. Segments of mouse or rat pancreas were placed in a flow cell through which physiological salt solutions of varying composition were pumped at a constant rate. Intracellular recordings of membrane potential, resistance and electrical time constant were made from the acini using fine glass micro-electrodes. In some experiments two micro-electrodes were inserted into two acinar cells within the same acinus to assess directly cell to cell coupling. The concentration of amylase in the effluent was measured continuously. 2. Electrical coupling between two acinar cells was observed when the tips of the two micro-electrodes were less than 50 mum from each other. The coupling ratio was close to 1. Acetylcholine (ACh) always evoked depolarization of exactly the same amplitude in two coupled cells and reduced the amplitude of current-pulse induced membrane potential changes in both cell simultaneously. 3. Stimulation with ACh caused an immediate increase in amylase output. Replacement of superfusion fluid Na by Tris or Cl by sulphate abolished ACh-evoked increase in amylase release, but the subsequent reintroduction of Na or Cl caused an increase in amylase release of a magnitude similar to what was normally observed following stimulation. 4. Omitting Ca from the superfusion fluid and adding EGTA rapidly depolarized the acinar cell membrane, reduced the input resistance and caused a marked reduction in amylase secretion. During exposure to a Ca-free, EGTA containing solution a marked increase in amylase release occurred following maximal ACh stimulation. 5. Addition of small amounts of Mg, Ca or Mn to a Ca-, Mg-free solution caused an increase in membrane potential, input resistance and electrical time constant and markedly increased amylase release. The effect on the electrical parameters was reversed in the absence of extracellular Na while extracellular Na was of no importance for the effect on amylase release. 6. The effect of ACh on amylase was enhanced during superfusion with a fluid containing 20 mM-Ca. The presence of Mn (5 mM) in an otherwise normal control had no effect on ACh-evoked release. 7. These results show that ACh acts on the acinus by reducing the surface cell membrane resistance. It is suggested that the ACh-receptor interaction causes a release of Ca from the surface cell membrane and that the concentration of Ca in the surface cell membrane determines the specific membrane resistance particularly for Na. The release of Ca to the cytosol activates exocytosis while the Na influx is of importance for acinar fluid secretion. The effect of ACh on amylase secretion can be mimicked by agents displacing membrane-bound Ca (Mg, Ca, Mn).     

Influences of ionic environments on ACh-induced secretory responses in isolated perfused pancreas of rats

The influence of extracellular Ca2+ concentration, [Ca2+]o, on the secretory response to acetylcholine (ACh) was analyzed in isolated perfused rat pancreas. The decrease of [Ca2+]o strongly diminished the amylase output and pancreatic juice flow in response to continuous stimulation with 5 X 10(-8) M ACh. A quantitative relation was found between the amount of amylase release by 5 X 10(-8) M ACh and the [Ca2+]o over a range of 0.1--2.5 mM. The partial replacement of NaCl with LiCl produced a diminution in both amylase output and pancreatic juice flow. A quantitative relation existed between the amount of ACh-induced amylase release and the [Na+]o over a range of 86--157 mM. The partial replacement of KCl with NaCl produced falls in both amylase output and pancreatic juice flow. Again, a quantitative relation existed between ACh-induced amylase release and [K+]o over a range of 1.0--5.6 mM. These results are compatible with the view that both the amylase output and the juice flow induced by 5 X 10(-8) M ACh are proportional to the amount of carrier-Ca complex and that the inward movement of the complex may be linked closely to the activation of Na pumps on the pancreatic acinar cell. A dose-response relation was found between the concentration of ACh and the amylase output. The relation was shifted to the left when 1 mU/ml cholecystokinin-pancreozymin (CCK-PZ) was added. A similar shift was observed when 1 mU/ml secretin was added. These results support the view that ACh, CCK-PZ, and secretin may activate the common cellular process in stimulus-secretion coupling, although these secretagogues may severally act on the different receptor sites.     

Effect of extracellular K+ concentration on resting potential,caerulein-induced depolarization and amylase release from mouse pancreatic acinar cells

Summary Acinar cell membrane potentials and amylase release were measured from in vitro preparations of mouse pancreas. The effect of a 10-fold increase of the extracellular K⁺ concentration (to 47 mM) was studied on the resting membrane potential and amylase release as well as on the membrane depolarization and amylase release induced by the cholecystokinin-pancreozymin analogue, caerulein. In the presence of atropine (to exclude the effect of a possible release of endogenous acetylcholine), the increased K⁺ concentration depolarized the cells from –45 to –20 mV without influencing the rate of the unstimulated release of amylase. Under these conditions, the depolarizing effect of caerulein was almost abolished, while the caerulein-induced amylase was not. It is concluded that caerulein-induced enzyme secretion from pancreatic acinar cells is independent of the level of the membrane potential as well as extracellular K⁺ concentration in the range from 4.7–47 mM.     

Interaction between secretin and cholecystokinin-octapeptide in the exocrine rat pancreas in vivo and in vitro.

This study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2+ and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10(-8) mol kg-1 h-1) or staurosporine (10(-8) mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10(-11) M) or secretin (10(-11) M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10(-6) M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10(-11) M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10(-11) M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK8. In superfused pancreatic segments CCK8 (10(-11) M) evoked a net efflux of Mg2+ whereas secretin (10(-11) M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.     

Effect of acetylcholine stimulation on calcium and sodium uptake by the isolated rat pancreas.

1. The role of Ca2+ and Na+ ions in 'stimulus-secretion' coupling in the isolated uncinate pancreas of 4-week-old rats has been examined using radioisotope tracer techniques. The amount of 45Ca2+ and 22Na+ (mumole/g wet wt.) taken up by unstimulated glands was compared to that of glands in which amylase release was stimulated by ACh(10(-5) M) at various incubation times. 2. The amount of 45Ca taken up by the glands within 60 min of incubation was not found to be increased by the presence of ACh(10(-5) M). In fact, during short incubation periods the 45Ca uptake was significantly less in the stimulated glands than the unstimulated glands. 3. Presence of ACh(10(-5) M) did cause a significant initial increase in 22Na uptake lasting up to 20 min from onset of stimulation. 4. These results indicate that the rise in intracellular Ca2+ concentration which is suggested by an increase in 45Ca efflux during the action of pancreatic secretagogues, is not a consequence of increased 45Ca uptake by the pancreas; but they indicate that an initial action of ACh could be to elicit an increase in Na+ influx and that Na+ ions are likely to be involved in the action of ACh on pancreatic acinar cells.     

Studies of radiocalcium efflux in single barnacle muscle fibres: effects of procaine and external divalent cations

1. (45)Ca efflux from single barnacle muscle fibres loaded with radio-calcium by microinjection was studied.2. The (45)Ca washout curve consisted of three exponential phases with half-times of 4.8, 12.6 and 111.1 min.3. Removal of external Ca(2+) reduced (45)Ca efflux by 65%. The (45)Ca efflux recovered upon restoring external Ca(2+), the magnitude of the recovery being dependent upon the external Ca(2+) concentration. 10 mM procaine was found to reduce the magnitude of the recovery.4. Removal of external Mg(2+) resulted in a 38% increase in (45)Ca efflux.5. External application of procaine at pH 7.8 caused a dose-dependent inhibition of (45)Ca efflux. The magnitude of the inhibition was reduced in the presence of low external Ca(2+) concentrations. 10 mM procaine at pH 9.3 caused a biphasic effect: inhibition was followed by stimulation.6. Microinjection of 0.5 M procaine caused only inhibition of (45)Ca efflux, whereas microinjection of 1.5 M procaine caused stimulation followed by inhibition. These effects were observed at an external pH of 7.8 and 9.3.7. Injection of 100 mM-EGTA abolished the stimulatory but not the inhibitory effect produced by procaine injection.8. These results are interpreted as indicating that a major fraction of the (45)Ca efflux involves Ca-Ca exchange which is inhibited by the charged form of procaine in a non-competitive manner at the external surface of the muscle fibre. The stimulatory action is attributed to release by procaine of Ca(2+) from internal binding sites.     

Effect of lanthanum on pancreatic protein synthesis in streptozotocin-diabetic rats

The role of extracellular Ca2+ in mediating the stimulatory effect of cholecystokinin octapeptide (CCK8) on [3H]phenylalanine incorporation into protein was studied in isolated pancreatic acini from streptozotocin-diabetic rats. The stimulatory effect of CCK8 (10(-10) M) on [3H]phenylalanine incorporation was completely abolished by preincubating acini with either 10(-4) M lanthanum or 10(-3) M manganese. At these concentrations neither compound altered the basal rate of amino acid incorporation, and both compounds inhibited CCK-mediated Ca2+ influx without affecting either basal or CCK-mediated 45Ca2+ efflux. Lanthanum (10(-4) M) also blocked the stimulatory effect of the cholinergic analogue carbachol (10(-5) M) on amino acid incorporation but did not alter the stimulatory effect of insulin (1.67 x 10(-8) M). Vasoactive intestinal polypeptide and 12-O-tetradecanoyl-phorbol-13-acetate failed to increase the incorporation of [3H]phenylalanine into acinar protein. These findings suggest that CCK and other pancreatic secretagogues that act via Ca2+ enhance protein synthesis by increasing cell membrane permeability to Ca2+ and provide additional evidence that this may be an important mechanism by which CCK regulates pancreatic exocrine function.     

Calcium fluxes and contractility in isolated guinea pig atrium: effect of A23187.

The Ca2+ ionophore A23187 (10(-6) to 3 X 10(-5) M) increased the force of contraction is isolated guinea pig atria. In individual twitches, peak tension, maximum rate of tension development, time to peak tension, and total twitch duration were all increased by A23187. Tripelennamine, indomethacin, and atropine did not significantly alter the inotropic effect of A23187. Serotonin produced changes in individual twitches that differed qualitatively and quantitatively from those of A23187. Therefore, the inotropic action of A23187 is probably not mediated by release of endogenous histamine, prostaglandins, acetylcholine, or serotonin. 45Ca influx and efflux were increased by A23187. The enhanced 45Ca efflux exceeded that which would be predicted if the ionophore acted only to increase the passive Ca2+ permeability of the myocardial cell membrane. These results suggest that A23187 facilitates the entry of extracellular Ca2+ into the myocardial cell and the release of intracellular Ca2+ stores into the myoplasm. The resultant increase in intracellular Ca2+ activity could account for the positive inotropic action of A23187.     

Histamine-evoked amylase secretion is associated with small changes in calcium mobilization in isolated guinea-pig pancreas.

An investigation was made of the effect of histamine on amylase secretion and calcium mobilization in isolated guinea-pig pancreatic segments. The effect of acetylcholine (ACh) was also examined for comparison. Histamine evoked a dose-dependent increase in amylase output from superfused segments. It had small transient effects on both 45Ca influx and efflux, and elevated cytosolic free Ca2+ concentration in acini. The equivalent dose of ACh evoked a greater amylase output and greater changes in Ca2+ mobilization. The results suggest that histamine may have a physiological role in exocrine secretion of the guinea-pig pancreas but is less potent than ACh.     

Calcium-dependent amylase release and electrophysiological measurements in cells of the pancreas

1. Transmembrane potential, effective membrane resistance, and amylase output were recorded from acinar cells of rat pancreas perfused in vitro.2. Both pancreozymin and acetylcholine hyperpolarized the acinar cells, increased effective membrane resistance, and augmented amylase output.3. The omission of calcium from the perfusion medium increased effective membrane resistance and potential, and abolished the increase in amylase output in response to the drugs.4. A quantitative relation was found between the amount of amylase released by pancreozymin and the concentration of calcium in the perfusion medium at values below the normal 2.5 mM calcium. Excess magnesium did not inhibit the increase in amylase output in response to the drug.5. It is concluded that the release of amylase from the pancreas depends on the entry of calcium into the acinar cells. The entry of calcium seems to be mediated by a carrier in the membrane and may be considered as a ;facilitated diffusion'.6. The electrophysiological findings taken together with morphological evidence provided by scanning electron microscopy favour the view that pancreatic zymogens are released from the granules in the acinar cells to the lumen by the process known as exocytosis.     

Ca2+]i oscillations induced by high [K+]o in acetylcholine-stimulated rat submandibular acinar cells: regulation by depolarization,cAMP and pertussis toxin

Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.     

On the functional relationship between 45Ca2+ release and prolactin secretion in cultured rat pituitary tumour (GH3) cells

We have evaluated the role of cellular Ca2+ transport associated with stimulus-secretion coupling in prolactin (PRL) producing rat pituitary adenoma cells (GH3 cells). The action of different substances, known to modify PRL secretion, on release of 45Ca2+ from preloaded cells were examined. Surface-bound 45Ca2+ was removed by pretreatment with trypsin in EDTA buffer. During the first 6 min, basal efflux of 45Ca2+ occurred at a constant rate (0.24 min-1) at 37 degrees C. Addition of TRH (5 X 10(-7) M) resulted in an immediate enhancement of 45Ca2+ release representing about 20% of the remaining cellular 45Ca2+. In the same experiments PRL secretion increased by 45%. The EDTA in the external medium reduced the basal rate of 45Ca2+ release by 60%, but did not apparently affect the TRH-stimulated release. Somatostatin (10(-6) M) and verapamil (5 X 10(-5) M) inhibited both basal and TRH-stimulated PRL secretion, whereas high extracellular concentration of K+ (5 X 10(-2) M) had a stimulatory effect. However, neither of these treatments changed cellular 45Ca2+ release. Interference with energy-dependent Ca2+ transport by using metabolic inhibitors (iodoacetate, 6 X 10(-3) M; and antimycin, 2 X 10(-6) M) or by replacing Na+ in the medium by choline or by lowering the incubation temperature from 37 to 25 degrees C, had no effect on TRH-stimulated 45Ca2+ release although basal and TRH-stimulated PRL secretion were reduced. Thus, TRH apparently releases 45Ca2+ from calcium binding sites in the cell membrane.