Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.     

Mobilization of calcium from intracellular stores as one of the mechanisms underlying the antiopioid effect of cholecystokinin octapeptide.

In enzymatically dissociated brain cells prepared from neonatal rats, KCl produced a significant increase in [Ca2+]i and this increase could be prevented by verapamil or nifedipine, known to block voltage-sensitive calcium channels. The opioid receptor agonists ohmefentanyl (OMF, mu agonist), [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist), and 66A-078 (kappa agonist) produced a marked suppression of the Ca2+ influx induced by high K+ depolarization. The suppressive effect of OMF, DPDPE, and 66A-078 on the high K(+)-induced increase in [Ca2+]i was markedly reversed by their respective antagonists beta-funaltrexamine (beta-FNA), ICI174864, and nor-binaltorphimine (nor-BNI). Cholecystokinin octapeptide (CCK-8), at concentrations of 0.3, 3.0, and 30 nM, dose-dependently mobilized Ca2+ from intracellular stores. While CCK-8 30 nM did not affect significantly the increase of [Ca2+]i following high K+, it did reverse the suppression of the high K(+)-induced increase in [Ca2+]i by the mu agonist OMF and the kappa agonist 66A-078, but not that by the delta agonist DPDPE. The results suggested that while opioid ligands suppress [Ca2+]i by blocking voltage-operated Ca2+ influx, the antiopioid effect of CCK-8 seems to be operated via mobilization of Ca2+ from intracellular stores.     

Release of intracellular Ca2+ and elevation of inositol trisphosphate by secretagogues in parietal and chief cells isolated from rabbit gastric mucosa

The fluorescent intracellular Ca2+ indicator, fura2/AM, was used to determine the effects of carbachol, cholecystokinin octapeptide (CCK-8), gastrin and histamine on intracellular Ca2+ ([Ca2+]i) in parietal cells from rabbit gastric mucosa enriched to more than 95% purity by a new Nycodenz gradient/centrifugal elutriation technique. Changes in [Ca2+]i in response to the same agonists were also measured in enriched chief cells. Carbachol, histamine, gastrin and CCK-8 increased parietal cell [Ca2+]i with the response to carbachol greater than CCK -8 = histamine = gastrin. Prestimulation with msximal doses of carbachol blocked histamine-induced increases in [Ca2+]i. In chief cells, carbachol increased [Ca2+]i but to a lesser degree than CCK-8, while histamine had no significant effect on [Ca2+]i. Neither removal of extracellular Ca2+ coupled with acute addition of 1 mM EGTA nor addition of the Ca2+-channel blocker nicardipine prevented agonist-induced changes in [Ca2+]i in either cell type. In the presence and absence of 10 mM LiCl2, carbachol and CCK-8 were found to increase inositol trisphosphate (IP3) content in both parietal and chief cells while histamine had no significant effect on this phosphoinositide hydrolysis product. From these results and previous observations with gastric glands (Chew, C.S. (1986) Am. J. Physiol. 13, G814-G823) we conclude that: carbachol, CCK-8, gastrin and histamine increase parietal cell [Ca2+]i initially by release of Ca2+ from the same intracellular store(s); the release of [Ca2+]i in response to carbachol and CCK-8 in both chief and parietal cells appear to be mediated by IP3; however, other mechanisms may be involved in histamine-induced release of parietal cell Ca2+.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.     

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.     

Dose-dependency in spatial dynamics of [Ca2+]c in pancreatic acinar cells

Spatial dynamics of cytosolic concentration of Ca2+, [Ca2+]c, in stimulus-secretion coupling of rat pancreatic acinar cell was monitored by a digital image analysing technique using Fura-2. When freshly isolated acini were stimulated with lower concentrations of CCK-8 (5-30 pM), [Ca2+]c increase began at the region beneath the basolateral membrane and the [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCK-8 at higher concentrations (100 pM and 1 nM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. Low concentrations of G-protein activator, NaF (10 mM or lower), caused [Ca2+]o-dependent increase in [Ca2+]c, whereas higher concentrations of NaF (15 mM or higher) increased [Ca2+]c in the absence of [Ca2+]o. These results are compatible with the view that G-protein activated by a physiological concentration of secretagogue accelerates Ca2+ entry. This process is in contrast to the process of Ca2+ release from intracellular stores, which can be predominant when pharmacological or toxic concentration of the secretagogue was applied.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10-30 min of ischemia followed by 5-30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(-)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon beta-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.

The role of intracellular Ca2+ pools in oscillations of the cytosolic Ca2+ concentration ([Ca2+]c) triggered by Ca2+ influx was investigated in mouse pancreatic B-cells. [Ca2+]c oscillations occurring spontaneously during glucose stimulation or repetitively induced by pulses of high K+ (in the presence of diazoxide) were characterized by a descending phase in two components. A rapid decrease in [Ca2+]c coincided with closure of voltage-dependent Ca2+ channels and was followed by a slower phase independent of Ca2+ influx. Blocking the SERCA pump with thapsigargin or cyclopiazonic acid accelerated the rising phase of [Ca2+]c oscillations and increased their amplitude, which suggests that the endoplasmic reticulum (ER) rapidly takes up Ca2+. It also suppressed the slow [Ca2+]c recovery phase, which indicates that this phase corresponds to the slow release of Ca2+ that was taken up by the ER during the upstroke of the [Ca2+]c transient. Glucose promoted the buffering capacity of the ER and amplified the slow [Ca2+]c recovery phase. The slow phase induced by high K+ pulses was not affected by modulators of Ca2+- or inositol 1,4,5-trisphosphate-induced Ca2+ release, did not involve a depolarization-induced Ca2+ release, and was also observed at the end of a rapid rise in [Ca2+]c triggered from caged Ca2+. It is attributed to passive leakage of Ca2+ from the ER. We suggest that the ER displays oscillations of the Ca2+ concentration ([Ca2+]ER) concomitant and parallel to [Ca2+]c. The observation that thapsigargin depolarizes the membrane of B-cells supports the proposal that the degree of Ca2+ filling of the ER modulates the membrane potential. Therefore, [Ca2+]ER oscillations occurring during glucose stimulation are likely to influence the bursting behavior of B-cells and eventually [Ca2+]c oscillations.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.     

Evidence for cAMP-dependent protein kinase in mediating the parathyroid hormone-stimulated rise in cytosolic free calcium in rabbit connecting tubules

We investigated cellular mechanisms mediating the parathyroid hormone (PTH)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated perfused rabbit connecting tubules. Prior and/or concomitant exposure to 0.5 mM of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8), a cyclic nucleotide-dependent protein kinase inhibitor, abolished the rise in [Ca2+]i produced by 0.1 nM PTH in five connecting tubules and suppressed it by approximately 50% in another five. In the latter, there was a delayed onset in the rise of [Ca2+]i. Such responses contrasted to the prompt increase in [Ca2+]i in PTH-stimulated control tubules. However, when H-8 was withdrawn, [Ca2+]i rose within minutes to reach a plateau value similar to the uninhibited response to PTH in controls, indicating rapidly reversible inhibition by H-8. In an otherwise identical protocol, 0.5 mM H-8 also reversibly suppressed the rise in [Ca2+]i induced by 0.175 mM 8-Br-cAMP. In contrast to the stimulatory effect of 8-Br-cAMP on [Ca2+]i, 1 mM 8-Br-cGMP caused no increase. At a concentration of 0.4 mM, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), a well-characterized cAMP-dependent protein kinase inhibitor, totally abolished the rise in [Ca2+]i caused by 0.1 nM PTH. We conclude that a cAMP-dependent protein kinase plays an important role in the PTH-stimulated rise in [Ca2+]i in the rabbit connecting tubule. Since the increase in [Ca2+]i was shown previously to depend on extracellular Ca2+, we propose that cAMP-dependent protein phosphorylation is important in mediating PTH-stimulated Ca2+ fluxes across plasma membranes of connecting tubule cells.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated for 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By contrast, glutathione had no effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.     

Growth hormone promotes Ca(2+)-induced Ca2+ release in insulin-secreting cells by ryanodine receptor tyrosine phosphorylation

Elevation in cytoplasmic free Ca2+ concentration ([Ca2+]i) is a common mechanism in signaling events. An increased [Ca2+]i induced by GH, has been observed in relation to different cellular events. Little is known about the mechanism underlying the GH effect on Ca2+ handling. We have studied the molecular mechanisms underlying GH-induced rise in [Ca2+]i in BRIN-BD11 insulin-secreting cells. GH (500 ng/ml, 22 nm) induced a sustained increase in [Ca2+]i. The effect of GH on [Ca2+]i was prevented in the absence of extracellular Ca2+ and was inhibited by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+)-channel inhibitor nifedipine. However, GH failed to induce any changes in Ca2+ current and membrane potential, evaluated by patch-clamp recordings and by using voltage-sensitive dyes. When the intracellular Ca2+ pools had been depleted using the Ca(2+)-ATPase inhibitor thapsigargin, the effect of GH was inhibited. In addition, GH-stimulated rise in [Ca2+]i was completely abolished by ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and caffeine. GH induced tyrosine phosphorylation of ryanodine receptors. The effect of GH on [Ca2+]i was completely blocked by the tyrosine kinase inhibitors genistein and lavendustin A. Interestingly, treatment of the cells with GH significantly enhanced K(+)-induced rise in [Ca2+]i. Hence, GH-stimulated rise in [Ca2+]i is dependent on extracellular Ca2+ and is mediated by Ca(2+)-induced Ca2+ release. This process is mediated by tyrosine phosphorylation of ryanodine receptors and may play a crucial role in physiological Ca2+ handling in insulin-secreting cells.     

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic beta-cells

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.