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.     

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.     

The relationship between acetylcholine-evoked Ca2+-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.     

Effect of extracellular magnesium on secretagogue-evoked amylase secretion in the isolated rat parotid gland segments.

This study investigates the effect of perturbation of extracellular magnesium [Mg2+]o on basal and acetylcholine (ACh), noradrenaline (NA) and phenylephrine (PHE)-evoked amylase secretion from isolated rat parotid gland segments. Both zero (0 mM) and elevated (5 mM and 10 mM) [Mg2+]o can significantly (P < 0.05) inhibit basal and secretagogue-evoked amylase secretion compared to the responses obtained in normal (1.1 mM) [Mg2+]o. The inhibitory effect of zero [Mg2+]o was more pronounced compared 10 mM [Mg2+]o. A concentration of 5 mM, [Mg2+]o was less effective at a secretagogue concentration of 10(-5) M but more pronounced in inhibiting amylase secretion when the concentration of the secretagogue was reduced to 10(-6) M. The results indicate that both hypo and hypermagnesaemia are associated with reduced salivary amylase secretion and both conditions may be associated with 'the dry mouth syndrome'.     

Accumulation of cAMP evoked by acetylcholine stimulation in rat submandibular acinar cells: observation of exocytosis, fluid secretion and [Ca2+]i

The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 microM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 microM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 microM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.     

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.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.     

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca2+, 15-75 min of exposure to anoxia increased 45Ca influx by 46-129% (P<0.001) and Ca2+ content by 20-50% (P<0.001). Mg2+ (11.2 mM) reduced the anoxia-induced increase in 45Ca influx by 43% (P<0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P<0.001). Increasing [Ca2+]o to 5 mM induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2=0.55, P<0.001). In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self-amplifying process of cell membrane leakage.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

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).     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

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.     

Transport of calcium in the perfused submandibular gland of the cat

1. In the perfused cat submandibular gland efflux and influx of (45)Ca, and concentrations of K, (40)Ca and Mg in the effluent from the gland were measured under different experimental conditions.2. When the standard perfusion fluid was shifted to a high Mg (5 mM) or a low Ca (0.25 mM) solution the efflux of (45)Ca from the pre-labelled gland declined. The magnitude and the duration of the effect of the high Mg concentration was more marked at a low external Ca concentration and was abolished by Mersalyl (1 mM). When the standard perfusion fluid was shifted to a Mg-free solution the efflux of (45)Ca from the pre-labelled gland increased.3. After shift of (45)Ca containing perfusion fluid from normal to a high Mg (5 mM) solution the influx of (45)Ca to the gland increased rapidly.4. Both acetylcholine (ACh) and adrenaline caused a marked increase in the efflux of (45)Ca from the pre-labelled gland. This increase in efflux was also seen under conditions where the gland was unable to secrete, i.e. during perfusion with Ca-free and Na-free tetraethylammonium Locke solutions.5. Stimulation with ACh failed to reveal any rapidly occurring increase in influx of (45)Ca.6. Stimulation with ACh evoked a small temporary increase in the concentration of (40)Ca. and Mg in the effluent.7. It is suggested that Ca uptake by intracellular Ca-accumulating systems of the submandibular gland depends on the external Mg concentration and that ACh and adrenaline cause a release of Ca bound intracellularly.     

Characterization of platelet cytoplasmic Ca2+ mobilization in patients with congenital cyclo-oxygenase deficiency and with defective platelet aggregation to A23187

Agonist-induced platelet cytoplasmic Ca2+ concentrations ([Ca2+]i) in patients with congenital cyclo-oxygenase deficiency (A) and with impaired aggregation to A23187 (B) were measured with aequorin in the presence or absence of extracellular Ca2+. The influence of TMB-8 or ONO3708 on agonist-induced [Ca2+]i in those platelets was also investigated. In Patient 1, there was a single aequorin luminescence peak in response to arachidonate, which was a thromboxane A2(TXA2) independent Ca2+ influx. The luminescence peak due to the formation of TXA2 was not detectable. The A23187-induced [Ca2+] i was decreased in the presence of extracellular Ca2+, but was within normal limits in the absence of extracellular Ca2+. A thrombin or STA2-induced elevation of [Ca2+] i was always within normal limits under any conditions. These results suggest that cyclo-oxygenase activity (CO activity) contributes to the A23187-induced Ca2+ influx, but does not contribute to the Ca2+ release from intracellular stores, and that the thrombin or STA2-induced Ca2+ influx and release do not depend on the CO activity. In Patient 2, the time lag from the addition of A23187 to the aequorin luminescence peak was found both in the presence and absence of extracellular Ca2+, which was more obvious in the latter. This A23187-induced elevation of [Ca2+] i disappeared after treatment of the platelets with TMB-8 in the absence of extracellular Ca2+, which is rarely seen in normal platelets. The most striking finding was that the thrombin-induced rise in [Ca2+] i in the absence of extracellular Ca2+ was not detectable. These findings might be closely related to abnormal platelet function in this patient.     

Effects of CO2, acetylcholine and caerulein on45Ca efflux from isolated mouse pancreatic fragments

Mouse pancreatic fragments were loaded with⁴⁵Ca and placed in a flow cell. The concentration of⁴⁵Ca in the effluent was measured. The effects of changing the tension of carbon dioxide on⁴⁵Ca efflux were observed and compared with effects of pancreatic secretagogues.The normal control solution was equilibrated with 5% CO₂, 95% O₂. Shift to solutions equilibrated with 10, 20, 50 or 100% CO₂ evoked a dose-dependent increase in fractional⁴⁵Ca efflux, with a just detectable effect at 10% and a maximal one at 50%.The CO₂-evoked Ca release was not due to anoxia, since a short period of exposure to a 100% N₂-equilibrated solution had no effect. A decrease in extracellular pH (tris buffering) had only a very modest effect on⁴⁵Ca efflux.CO₂-evoked Ca release under conditions avoiding extracellular pH changes (20% CO₂, 100 mM NaHCO₃). This CO₂-evoked enhanced⁴⁵Ca efflux was sustained during a 30 min stimulation period, but was abruptly terminated on return to the control solution (5% CO₂, 25 mM NaHCO₃). NH₃ (10 mM) added to the 20% CO₂-equilibrated solution for a brief interval in the middle of a period of CO₂-evoked enhanced⁴⁵Ca efflux evoked a rapid return of the fractional Ca efflux towards the resting level. This effect was rapidly reversible.While the CO₂-evoked Ca release was largely sustained, the ACh-evoked increase in⁴⁵Ca fractional efflux was entirely transient. The CO₂-evoked Ca release was not inhibited by a background of sustained ACh stimulation. ACh-evoked Ca release, however, was markedly inhibited in the presence of sustained CO₂ stimulation.2,4 Dinitrophenol (1 mM) in combination with iodoacetate (2 mM), while markedly reducing⁴⁵Ca uptake into the fragments during the loading period had little or no effect on the ACh-evoked increase in⁴⁵Ca fractional efflux. The CO₂-evoked Ca release, however, was markedly reduced by these metabolic inhibitors.The local anaesthetic procaine (1 mM) virtually abolished ACh- or caerulein-evoked Ca release without having any influence on the CO₂ effect.It is concluded that CO₂ releases Ca from pancreatic acinar cells by means of intracellular acidification. This effect may in part be due to H⁺ displacement of Ca²⁺ from intracellular membrane binding sites and partly due to release of Ca from compartments (organelles) into which Ca has been actively accumulated.     

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.     

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.     

Interaction of islet hormones with cholecystokinin octapeptide-evoked secretory responses in the isolated pancreas of normal and diabetic rats

This study investigates the effects of the islet hormones, insulin (Ins), glucagon (Glu) and somatostatin (Som) with cholecystokinin octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i and their pattern of distribution in the isolated pancreas of normal and diabetic rats. Ins and Glu evoked small increases in amylase output from pancreatic segments compared with a much enhanced effect of CCK-8. In contrast, Som induced a biphasic response comprising an initial decrease followed by a secondary increase and this biphasic response may be dependent upon the concentration. Combining the islet hormones with CCK-8 resulted in marked potentiation in amylase output compared with either CCK-8 alone or the individual hormone. Genistein and tyrphostin A25, the tyrosine kinase inhibitors, evoked a small decrease in amylase output from pancreatic segments. They had no effect on the CCK-8-evoked secretory response but markedly inhibited the potentiation of the islet hormones with CCK-8. In pancreatic acini and acinar cells Ins, Glu and Som individually evoked small increases in amylase output compared with a much larger response with CCK-8. When the islet hormones were combined with CCK-8 there was no potentiation of amylase output. Similarly, when rats were rendered diabetic by prior treatment with streptozotocin Ins, Glu and Som failed to potentiate the secretory response of CCK-8. In fura-2-loaded pancreatic acinar cells Ins or Glu evoked small increases in [Ca2+]i compared with a much larger elevation with CCK-8. Ins, Glu and Som each enhanced the CCK-8-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of the islet hormones. It also decreased the elevation in [Ca2+]i resulting from the combined presence of CCK-8 with either Ins or Glu but it had no effect on CCK-8 in combination with Som. In pancreatic acinar cells from diabetic rat Ins, Glu and Som had no detectable effect on CCK-8-evoked elevation in [Ca2+]i compared with the response obtained with CCK-8 alone. CCK-8-immunopositive cells were distributed around the walls of blood vessels, numerous Ins-positive cells in the central and peripheral parts of the islets of Langerhans, Glu-immunoreactive cells in the periphery of islets and Som-positive cells in the outer part of the islets. During diabetes, the number of CCK-immunopositive cells remained unchanged whereas the number of Ins-positive cells decreased coupled with an increase in the number of Glu-positive cells. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with CCK-8. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with CCK-8.