Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

GABAA receptor-mediated increase of cytosolic Ca2+ in isolated bovine adrenal chromaffin cells

We have studied the effects of GABA on cytosolic free Ca2+ concentration ([Ca2+]i) as a means of investigating the role of GABA in adrenal catecholamine (CA) secretion. It was demonstrated that GABA caused an elevation of [Ca2+]i via the GABAA receptor in a concentration-dependent manner, which was well correlated with an increase of 45Ca uptake, an increase of CA release and a depolarization of chromaffin cells assessed with bis-oxonol fluorescence. Since the GABA-induced rise of [Ca2+]i was absolutely dependent on the presence of extracellular Ca2+ and partly sensitive to nifedipine, at least one entry route for Ca2+ facilitated by GABA via a voltage-sensitive Ca2+ channel was suggested. When extracellular Cl- was lowered, GABA-induced CA release, depolarization, and rise of [Ca2+]i were all markedly enhanced. It is possible that GABA plays a modulatory role in the regulation of adrenal CA secretion as a facilitatory modulator.     

Spatial localization of the stimulus-induced rise in cytosolic Ca2+ in bovine adrenal chromaffin cells. Distinct nicotinic and muscarinic patterns

The spatial distribution of the intracellular free Ca2+ (Ca2+i) rise elicited by different stimuli in bovine adrenal chromaffin cells was examined in single fura-2-loaded cells. In response to the potent secretagogues nicotine and high K+, Ca2+i was initially localized exclusively to the entire subplasmalemmal area of the cell. In response to the ineffective secretagogues, methacholine and muscarine, the rise in Ca2+i originated only in one pole of the cell and even at the peak of the response Ca2+ was still generally restricted to this same area of the cell. These results suggest that the triggering of exocytosis from these cells requires a specific spatial distribution of Ca2+i.     

Histamine evoked sustained elevations of cytosolic Ca2+ in bovine adrenal chromaffin cells independently of Ca2+ entry

Whole-cell patch-clamp experiments and optical measurements with the Ca2+ fluorescent dye fura-2 were performed to examine histamine induced cytosolic Ca2+ changes in bovine adrenal chromaffin cells. The purpose of this study was to find out whether the sustained plateau phase, which followed the rapid transient increase, was due to Ca2+ influx. The extracellular Ca2+ dependence appeared to be minor, because substitution of Ca2+ with EGTA or BAPTA did not cause obvious changes in the biphasic Ca2+ response. Application of histamine in a Mn2+ containing external solution did not quench the fura-2 signal. It was neither possible to detect a histamine induced depolarisation, nor a Ca2+ permeable current. Changing the driving force for Ca2+ during the plateau phase did not result in a correlating fura-2 signal. Metal ions like Cd2+, La3+ and Co2+ which are known to block Ca2+ influx were unable to abolish the typical histamine induced Ca2+ response. These results suggest that primarily intracellular Ca2+ was responsible for generating the characteristic biphasic Ca2+ response due to histamine in bovine adrenal chromaffin cells.     

Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.     

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.     

Regulation of free cytosolic Ca2+ in the isolated guinea pig gastric chief cells

Stimulation with COOH-terminal octapeptide of cholecystokinin (CCK8) or carbachol resulted in a rapid increase in Quin-2 fluorescence of isolated guinea pig gastric chief cells, whereas histamine, vasoactive intestinal peptide, secretin or forskolin had no effect. The minimum effective dose of CCK8 or carbachol to elicit the rise in Quin-2 fluorescence was almost similar to that for pepsinogen secretion. Removal of Ca2+ from extracellular medium or Ca2+ channel blockers did not affect CCK8- or carbachol-induced increase in Quin-2 fluorescence. Moreover, following addition of CCK8, carbachol was unable to stimulate a second increase in Quin-2 fluorescence. These results suggest that CCK8 and carbachol share common Ca2+ pools and an increase in free cytosolic Ca2+ concentration may mediate CCK8- or carbachol-induced pepsinogen secretion from gastric chief cells.     

Ca2+ depletion and inositol 1,4,5-trisphosphate-evoked activation of Ca2+ entry in single guinea pig hepatocytes

Store-operated Ca(2+) entry was investigated by monitoring the Ca(2+)-dependent K(+) permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca(2+) stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca(2+)-Mg(2+)-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca(2+) store depletion on Ca(2+) influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP(3)) release from caged InsP(3) after treatment of the cells with 2, 5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca(2+)-free solutions. We show that the photolytic release of 1-d-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP(3), and Ca(2+) store depletion have additive effects to activate a high level of Ca(2+) entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP(3) receptors and Ca(2+) channels in the plasma membrane, although the nature of these Ca(2+) channels in hepatocytes is unclear.     

Homogeneous Ca2+ stores in rat adrenal chromaffin cells

The localization and function of Ca(2+) stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP(3)), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP(3), and the majority of BODIPY-Ry-binding area was bound by fluorescent IP(3). Under Ca(2+)-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca(2+) increases, as observed with fluo-3, lasted for a few tens of seconds under Ca(2+)-free conditions, whereas a caffeine-induced Ca(2+) transient diminished rapidly with a half decay time of 3s and this spike-like Ca(2+) transient was then followed by a sustained increase with a low level. These results indicate that IP(3) receptors and Ry receptors (RyRs) are present in common ER Ca(2+) storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.     

Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ～30 s, whereas contraction ceased within ～3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.     

PAC1 receptor activation by PACAP-38 mediates Ca2+ release from a cAMP-dependent pool in human fetal adrenal gland chromaffin cells

Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.     

Na(+)-dependent Ca2+ influx in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells were loaded with Na+ via either acetylcholine receptor-associated ion channels or voltage-sensitive Na+ channels. There were increases in [Ca2+]i, 45Ca2+ uptake and catecholamine secretion in both types of Na(+)-loaded cells relative to control cells in which Na+ loading had been prevented by hexamethonium and tetrodotoxin, respectively. These results show the presence of Na(+)-dependent Ca2+ influx activity in chromaffin cells which is probably mediated by the reverse mode of a Na+/Ca2+ exchanger.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminal Ca2+ controls the activation of the inositol 1,4,5-trisphosphate receptor by cytosolic Ca2+.

Luminal Ca2+ controls the sensitivity of the intracellular Ca2+ stores to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3-induced Ca2+ release is also controlled by cytosolic Ca2+; low concentrations of Ca2+ stimulate the release. The aim of this work was to investigate whether luminal Ca2+ would affect the stimulation of the Ins(1,4,5)P3 receptor by cytosolic Ca2+ in permeabilized A7r5 smooth muscle cells. We also report that the Ins(1,4,5)P3 receptor in A7r5 cells is activated by low concentrations of cytosolic Ca2+. Cytoplasmic Ca2+ increases the Ins(1,4,5)P3 sensitivity without affecting the cooperativity. The increase in Ins(1,4,5)P3 sensitivity becomes relatively more pronounced when the Ca2+ content of the stores decreases. This modulatory effect of luminal Ca2+ on the responsiveness to cytosolic Ca2+ is an intrinsic property of the Ins(1,4,5)P3 receptor.     

InsP3 receptor type 2 and oscillatory and monophasic Ca2+ transients in rat adrenal chromaffin cells

Muscarinic receptor stimulation induced oscillatory and monophasic Ca(2+) transients in rat adrenal chromaffin cells in the absence of external Ca(2+). As this Ca(2+) mobilization may be mediated by InsP(3), we first explored types of InsP(3) receptors and their intracellular distribution in chromaffin cells. The InsP(3) receptor type 1 was not immunodetected in precipitates of adrenal medulla homogenates and in dissociated adrenal chromaffin cells, whereas an anti-type 3 mAb recognized a faint band with about 250 kDa, but no significant immunoreaction was visible in chromaffin cells. The anti-type 2 mAb strongly detected a band with about 220 kDa and the immunoreaction was observed perinuclearly and at the cell periphery. These results indicate that InsP(3) receptor type 2 is predominant in chromaffin cells. The oscillatory and monophasic Ca(2+) transients were reproduced in simulation based on a three-state kinetic model (shut, open, and inactivated states). Ca(2+) ions were found experimentally and theoretically to turn over rapidly between stores and the cytosol during stimulation. The results suggest that InsP(3) receptor type 2 is responsible for both oscillatory and monophasic Ca(2+) transients and that change in mode of Ca(2+) responses may be accounted for by the kinetic property of the type 2 receptor.