Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

Cyclic AMP and Ca2+ uptake by mastocytoma mitochondria

A thorough re-investigation was undertaken of a variety of factors that might explain the increased uptake of 45Ca2+ by mitochondria isolated from N6, O2'-dibutyryladenosine-3',5'-cyclic monophosphate (DB cyclic AMP)--treated PY815 cells. This showed that mitochondria isolated from DB cyclic AMP treated cells take up 45Ca2+ at a 30 per cent faster rate than mitochondria from untreated cells, although both mitochondria eventually reduce the total external Ca2+ to the same levels. 45Ca2+ precharged mitochondria from DB cyclic AMP-treated cells also leaked 45Ca2+ more slowly than those from untreated cells when they were recovered by filtration. Thus an apparently greater uptake of 45Ca2+ by mitochondria from DB cyclic AMP-treated cells was a consequence of the filtration procedure. In fact, mitochondria from DB cyclic AMP-treated cells contained less total Ca2+ than those from untreated cells, while DB cyclic AMP-treated cells also contained less total Ca2+ than untreated cells. The results suggest that mitochondria do not play an important role in controlling the growth of DB cyclic AMP-treated PY815 cells through effects on cytoplasmic Ca2+ availability.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic beta-cell

The cytoplasmic calcium concentration (Ca2+i) was measured in individual mouse pancreatic beta-cells loaded with fura-2 by recording the 340/380 nm fluorescence excitation ratio. An increase of the glucose concentration from 3 to 20 mM, caused initial lowering of Ca2+i followed by a rise with a peak preceding constant elevation at an intermediary level. However, at 11 mM glucose there were large Ca2+i oscillations with a frequency of 1 cycle per 2-6 min. The results indicate that both first and second phase secretion depend on elevated Ca2+i, and that many electrically coupled cells collectively determine the pace of rhythmic depolarization.     

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.     

Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

Calcium entry in nonexcitable cells occurs throughCa²⁺-selective channels activatedsecondarily to store depletion and/or through receptor- orsecond messenger-operated channels. In amphibian liver, hormones thatstimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in theplasma membrane, which allows a noncapacitative inflow ofCa²⁺. To characterize thisCa²⁺ channel, we studied theeffects of inhibitors of voltage-dependent Ca²⁺ channels and of nonselectivecation channels on 8-bromoadenosine 3',5'-cyclicmonophosphate (8-BrcAMP)-dependentCa²⁺ entry in single axolotlhepatocytes. Ca²⁺ entry provokedby 8-BrcAMP in the presence of physiologicalCa²⁺ followed first-order kinetics(apparent Michaelis constant = 43 µM at the cellsurface). Maximal values of cytosolicCa²⁺ (increment ~300%) werereached within 15 s, and the effect was transient (half time of 56 s).We report a strong inhibition of cAMP-dependentCa²⁺ entry by nifedipine[half-maximal inhibitory concentration(IC₅₀) = 0.8 µM], byverapamil (IC₅₀ = 22 µM), andby SK&F-96365 (IC₅₀ = 1.8 µM).Depolarizing concentrations of K⁺were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca²⁺ influx. This "profile"indicates a novel mechanism ofCa²⁺ entry in nonexcitable cells.

     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Cyclic AMP as a determinant for glucose induction of fast Ca2+ oscillations in isolated pancreatic beta-cells.

The effect of glucose on the cytoplasmic Ca2+ concentration ([Ca2+]i) of pancreatic beta-cells from ob/ob-mice was examined by dual wavelength recordings of the 340/380 nm fluorescence excitation ratio of fura-2. Single beta-cells responded to 11-20 mM glucose with an initial lowering of [Ca2+]i, followed by an increase usually manifested as large amplitude oscillations (300-500 nm) with a frequency of 0.2-0.5/min (a-type). Particularly in freshly isolated beta-cells, there were also superimposed fast oscillations with frequencies of 2-8/min amplitudes in the 70-250 nM range (b-type) and sometimes pronounced [Ca2+]i transients exceeding 250 nM with durations below 10 s (c-type). After addition of 1-100 nM glucagon or 1 mM of the dibutyryl or 8-bromo derivatives of cyclic AMP, glucose generated numerous b-type oscillations superimposed on those of the a-type or on an elevated steady-state level. The duration of the b-type oscillations increased slightly when glucose was raised from 11 to 16 mM. The c-type transients probably represent a separate reaction predominantly seen when raising cyclic AMP much above its normal concentration. It is concluded that glucose can induce fast oscillations of [Ca2+]i also in isolated beta-cells, especially when measures are taken to increase their cyclic AMP content.     

Nitric oxide induces apoptosis via Ca2+-dependent processes in the pancreatic beta-cell line MIN6

An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. In the present study, we show evidence that supports a tight link between NO, Ca2+, protease and apoptosis in beta-cells. Three different NO donors, SNAP, NOR3 and NOC7, induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP at 200 microM increased cytosolic Ca2+ concentration ([Ca2+]i) and induced apoptosis. The SNAP-induced apoptosis was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. In conclusion, an excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Glucose increases cytosolic Ca2+ activity in pancreatic islet cells

Isolated cells prepared from rat pancreatic islets were labelled with the tetraacetoxymethyl ester of the fluorescent Ca2+ indicator quin-2. An increase in the extracellular concentration of glucose provoked a rapid and sustained increase in the fluorescence of the labelled cells. This indicates that glucose increases cytosolic Ca2+ activity in pancreatic islet cells.     

Measurement of cytosolic free Ca2+ in individual pancreatic acini

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

EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism. Redistribution from intracellular stores and stimulated influx

The changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. The lack of Ca2+ rise seen in attached cells loaded with quin2 and treated with EGF in Ca2+-free medium was probably the result of a Ca2+ buffer artifact. Concomitantly to the Ca2+ signal, EGF induced phosphoinositide hydrolysis, with stimulated accumulation of inositol 1,3,4,trisphosphate and -1,3,4,5-tetrakisphosphate. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store (possibly mediated by generation of inositol trisphosphate) and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin (growth factors that cause much greater phosphoinositide hydrolysis and Ca2+ redistribution responses than EGF). It is therefore suggested that the Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism.     

Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells.

At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca(2+)-channel current (ICa) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on ICa amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of ICa and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.     

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.     

Somatostatin lowers the cytosolic free Ca2+ concentration in clonal rat pituitary cells (GH3 cells)

Changes in the cytosolic free Ca2+ concentration, [Ca2+]i, have been proposed to mediate the regulation of the secretion of pituitary hormones by hypothalamic peptides. Using an intracellularly trapped fluorescent Ca2+ probe, quin2, [Ca2+]i was monitored in GH3 cells. Somatostatin lowers [Ca2+]i in a dose dependent manner from a prestimulatory level of 120 +/- 4 nM (SEM, n = 13) to 78 +/- 9 nM (n = 5) at 10(-7)M; the effect is half maximal at 2 X 10(-9) M somatostatin. The decrease in [Ca2+]i occurs rapidly after somatostatin addition and a lowered steady state [Ca2+]i is maintained for several minutes. Somatostatin does not inhibit the rapid rise in [Ca2+]i elicited by thyrotropin releasing hormone (TRH) and can still cause a decrease in [Ca2+]i in the presence of TRH (10(-7)M). Concomitantly with its action on [Ca2+]i somatostatin causes hyperpolarization of GH3 cells assessed with the fluorescent probe bis-oxonol. The lowering of [Ca2+]i by somatostatin is however not only due to reduced Ca2+ influx through voltage dependent Ca2+ channels, since it persists in the presence of the channel blocker verapamil. These results suggest that somatostatin may exert its inhibitory action on pituitary hormone secretion by decreasing [Ca2+]i.     

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).