Cd and Co at micromolar concentrations stimulate catecholamine secretion by increasing the cytosolic free Ca concentration in cat adrenal chromaffin cells

Cd²⁺ and Co²⁺ at micromolar concentrations increased the cytosolic free Ca²⁺ concentration, which was measured by fura-2 microfluorometry, in cat adrenal chromaffin cells. Simultaneously, these divalent cations stimulated catecholamine secretion from the perfused adrenal. The present findings suggest that these cations increase the Ca²⁺ influx by depolarizing the cell membrane and consequently stimulate catecholamine secretion.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Cd and Co at micromolar concentrations mobilize intracellular Ca via the generation of inositol 1,4,5-triphosphate in bovine chromaffin cells

To understand the mechanisms underlying the Cd²⁺- and Co²⁺-induced intracellular Ca²⁺ mobilization, we measured the levels of inositol phosphates using bovine chromaffin cells. Studies using HPLC indicated that Cd²⁺, Co²⁺ and methacholine significantly increased the generation of 1,4,5-IP₃. The results suggest that Cd²⁺ and Co²⁺ mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores, possibly through the presumptive Cd²⁺ receptor.     

A muscarinic receptor agonist mobilizes Ca from caffeine and inositol-1,4,5-trisphosphate-sensitive Ca stores in cat adrenal chromaffin cells

To gain some understanding of the characteristics of intracellular Ca²⁺ stores of cat adrenal chromaffin cells, we investigated the effects of ryanodine, a blocker of Ca²⁺-induced Ca²⁺ release channels in muscle, on both cytosolic Ca²⁺ concentration and catecholamine secretion induced by caffeine or methacholine. The results suggest that Ca²⁺ stores consist of at least two compartments, one which is sensitive to both caffeine and inositol-1,4,5-trisphosphate (IP₃), and the other which is sensitive to IP₃ alone.     

Possible occurrence of Na-dependent Ca influx mechanism in isolated bovine chromaffin cells

Chromaffin cells were isolated from bovine adrenals and the effects of experimental manipulations which alter the level of internal Na⁺ on the release of catecholamines and⁴⁵Ca uptake by these cells were investigated. In response to Na⁺ deprivation both parameters were increased or decreased when internal Na⁺ was raised or reduced, respectively. The results suggest the existence of Na⁺-dependent Ca²⁺ influx mechanism in these cells.     

Pharmacology of nicotine-induced increase in cytosolic Ca concentrations in chick embryo ciliary ganglion cells

Chick embryo ciliary ganglion cells were acutely isolated, and the mechanism(s) underlying the increase in the cytosolic Ca²⁺ concentration ([Ca]_in) induced by high concentrations of nicotine examined using fura-2 microfluorometry. The order of potencies of nicotinic receptor agonists in increasing [Ca]_in was ACh > nicotine = dimethylphenylpiperazinium > cytisine. The nicotine-induced increase in [Ca]_in was inhibited not only by nicotinic antagonists but also by muscarinic antagonists, while the muscarine-induced [Ca]_in increase was little affected by nicotinic antagonists. The nicotine-induced [Ca]_in increase was inhibited by both L- and N-type Ca²⁺ channel blockers and potentiated by an L-type Ca²⁺ channel agonists, Bay-K-8644. Nicotine also increased the cytosolic Na⁺ concentration ([Na]_in) as measured by sodium binding benzofuranisophthalate microfluorometry, and this [Na]_in increase was inhibited by various agents which reportedly affected nicotinic receptor channels resulting in chromaffin cells. These results suggest that nicotine increased Na⁺ influx nicotinic receptor channels resulting in membrane depolarization, which in turn increased Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, nicotine still increased influxes of Ca²⁺ and Mn²⁺ in the absence of external Na⁺, suggesting that nicotinic receptor channels in these cells are permeable not only to monovalent cations but also to Ca²⁺ and Mn²⁺.     

Effects of ginsenosides on Ca channels and membrane capacitance in rat adrenal chromaffin cells

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca²⁺ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca²⁺ currents (I_Ca) was elicited by depolarization and the change in cell membrane capacitance (ΔC_m) was monitored. The application of GTS (100 μg/ml) induced rapid and reversible inhibition of the Ca²⁺ current by 38.8 ± 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca²⁺ current inhibition, the effects of five ginsenosides (ginsenoside Rb₁, Rc, Re, Rf, and Rg₁) on the Ca²⁺ current were examined. The inhibitions to the Ca²⁺ current by Rb₁, Rc, Re, Rf, and Rg₁ were 15.3 ± 2.2% (n = 5); 36.9 ± 2.4% (n = 7); 28.1 ± 1.9% (n = 12); 19.0 ± 2.5% (n = 10); and 16.3 ± 1.6% (n = 15), respectively. The order of inhibitory potency (100 μM) was Rc > Re > Rf > Rg₁ > Rb₁. A software based phase detector technique was used to monitor membrane capacitance change (ΔC_m). The application of GTS (100 μg/ml) induced inhibitory effects on ΔC_m by 60.8 ± 9.7% (n = 10). The inhibitions of membrane capacitance by Rb₁, Rc, Re, Rf, and Rg₁ were 35.3 ± 5.5% (n = 7); 41.8 ± 7.0% (n = 8); 40.5 ± 5.9% (n = 9); 51.2 ± 7.6% (n = 9); and 35.9 ± 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on ΔC_m were Rf > Rc > Re > Rg₁ > Rb₁. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca²⁺ currents and ΔC_m in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.     

Inhibition by neuroprotective drug NS-7 of nicotine-induced Na influx, Ca influx and catecholamine secretion in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀=15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na⁺,K⁺-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of ²²Na⁺ without altering the EC₅₀ value of nicotine. Also, NS-7 diminished nicotine-induced ⁴⁵Ca²⁺ influx via nicotinic receptors and voltage-dependent Ca²⁺ channels (IC₅₀=14.1 μM) and catecholamine secretion (IC₅₀=19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.     

Activation of ATP receptor increases the cytosolic Ca(2+) concentration in ventral tegmental area neurons of rat brain

ATP increased the cytosolic Ca(2+) concentration ([Ca](i)) in neurons of ventral tegmental area acutely dissociated from rat brain. The ATP response was dependent on external Ca(2+) and Na(+), and was blocked by voltage-dependent Ca(2+) channel blockers. The results suggest that the ATP-induced depolarization increases Ca(2+) influx via voltage-gated Ca(2+) channels resulting in the increase in [Ca](i).     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Evidence for paracrine modulation of voltage-dependent calcium channels by amperometric analysis in cultured porcine adrenal chromaffin cells

We investigated the endogenous control through vesicular contents of voltage-dependent Ca2+ channels (VDCCs) in cultured porcine adrenal chromaffin cells. To examine paracrine regulation of VDCCs, catecholamine release was monitored amperometrically together with patch-clamp recording under culture conditions at different cell densities. A depolarizing pulse evoked Ca(2+)- (ICa) and Ba(2+)-currents (IBa) in Ca(2+)- and Ba(2+)-containing solutions, respectively. In cells cultured at high density, stop-flow of the external solution decreased the I(Ba) concomitant with a sustained increase of amperometric current (Iamp), but not in cells at low density, suggesting the endogenous modulation of VDCCs in a paracine fashion. The degree of the prepulse facilitation was similar regardless of the flow condition. Application of noradrenaline (NA), ATP, methionine-enkephalin (ENK) or protons decreased IBa. The extent of the prepulse facilitation of the endogenous VDCC inhibition was similar to those induced by NA and ATP. GDPbetaS, pertussis toxin (PTX), blockers for alpha-adrenoceptors and P2-purinoceptors significantly reduced the endogenous VDCC inhibition. These results suggest that VDCCs are regulated by vesicular substances in a paracrine fashion, at least by noradrenaline and ATP, through activation of alpha-adrenoceptors and P2-purinoceptors, respectively, in porcine adrenal chromaffin cells.     

Activation of ATP receptor increases the cytosolic Ca concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca²⁺ concentration ([Ca]_i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca²⁺ and Na⁺, and was blocked by voltage-dependent Ca²⁺ channel blockers. The results suggest that the ATP-induced depolarization increases Ca²⁺ influx resulting in the increase in [Ca]_i.     

Angiotensin II increases intracellular Ca concentration in folliculo-stellate cells of the rat anterior pituitary in primary culture

It has been reported that pituitary gonadotrophs and lactotrophs contain angiotensin II (Ang II) and suggested that Ang II modulates hormone secretion from endocrine cells of the anterior pituitary through paracrine mechanism among the endocrine cells. However, there has been no report on the effect of Ang II on the folliculo-stellate cells (FSC) which are thought to play a regulatory role in the release of hormones from pituitary endocrine cells. We, therefore, examined the effect of Ang II on FCS in primary culture by Ca²⁺ imaging technique. Certain proportion (42%) of FSC responded to 100 nM Ang II by increasing [Ca²⁺]_i. In addition, Ang II elicited the Ca²⁺ response in about 50% of the pituitary endocrine cells. The results indicate that Ang II functions as a paracrine factor among pituitary cells including FSC.     

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca-ATPase inhibitor in adrenal chromaffin cells

Long-term (≥12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), caused a time (t_1/2=16.3 h)- and concentration (IC₅₀=37.8 nM)-dependent decrease of cell surface ¹²⁵I-insulin binding by 35%, but did not change the K_d value. TG caused a sustained increase of cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_c) in a biphasic manner, and the effect of TG on ¹²⁵I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) β-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR β-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca²⁺]_c down-regulates cell surface IR by retarding externalization of IR from the TGN.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Possible occurrence of Na+-dependent Ca2+ influx mechanism in isolated bovine chromaffin cells

Chromaffin cells were isolated from bovine adrenals and the effects of experimental manipulations which alter the level of internal Na+ on the release of catecholamines and 45Ca uptake by these cells were investigated. In response to NA+ deprivation both parameters were increased or decreased when internal NA+ was raised or reduced, respectively. The results suggest the existence of Na+-dependent Ca2+ influx mechanism in these cells.     

The contribution of voltage-gated Ca currents to K channel activation during ovine adrenal chromaffin cell development

Prior to the development of adrenal innervation, the adrenal medulla is capable of responding to low blood oxygen directly. However, this response is lost once adrenal innervation is established. Previous work by our group has outlined mechanisms involved in this direct hypoxic response and the means by which innervation causes the loss of the direct hypoxic response in the ovine adrenal. The current study further investigates mechanisms which may underlie the developmental loss of the direct hypoxic response by concentrating on two aspects of cell function which regulate catecholamine secretion: the contribution of different types of Ca²⁺ channels to the total Ca²⁺ current and the contribution of each Ca²⁺ channel type to K⁺ channel activation. We identified that Ca²⁺ current size at −40 to −10 mV is increased in amplitude in fetal chromaffin cells. This is not due to the increased prevalence or size of T-type Ca²⁺ currents present at these voltages. The relative contribution of L-, N- or P/Q-type Ca²⁺ channels to total Ca²⁺ current and to activation of the K⁺ current is unchanged during chromaffin cell development, however K⁺ current density increases with age. Our results indicate that there is a developmental shift in relative expression of T-type, but not L-, N- or P/Q-type, Ca²⁺ channels in ovine chromaffin cells. The increased K⁺ current density in adult cells may result in an altered response to an equal stimulus, while larger Ca²⁺ current at negative voltages in fetal cells may facilitate Ca²⁺ entry and catecholamine secretion in response to small depolarisations such as those induced by hypoxia.     

Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells

Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress Ca(2+) channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2 suggesting the involvement of P(2Y) purinergic receptors. Prepulses to positive potentials that relieve the Ca(2+) channel block largely relieve the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca(2+) influx there is no apparent change in the relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes secretion by inhibiting Ca(2+) influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca(2+) channels, may be an important regulator of catecholamine release in chromaffin cells.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Phosphorylation promotes the desensitization of the opioid-induced Ca increase in NG108-15 cells

Using the fluorescent Ca²⁺ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca²⁺]_i increase, while exposure of desensitized cell to 50 mM K⁺ restores the response EK-induced, suggesting a role of intracellular Ca²⁺ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl- -tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4,5-trisphosphate (IP₃) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP₃ generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca²⁺ influx by either verapamil or removal of extracellular Ca²⁺ or the depletion of Ca²⁺ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca²⁺ transporters involved in Ca²⁺ influx and Ca²⁺ release. Our results imply that emptying of intracellular Ca²⁺ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.