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Biomedical subjects

M G López

Publications and source records attributed to M G López.

At least 19 recordsLinked to original sources
PubMed2005

Depolarization evokes different patterns of calcium signals and exocytosis in bovine and mouse chromaffin cells: the role of mitochondria.

This study was planned on the assumptions that different high-voltage activated calcium channels and/or the ability of mitochondria to take up Ca(2+) could be responsible for different cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and catecholamine release responses in adrenal chromaffin cells of bovine and mouse species. Short K(+) pulses (2-5 s, 70 mM K(+)) increased [Ca(2+)](c) to a peak of about 1 microM; however, in bovine cells the decline was slower than in mouse cells. Secretory responses were faster in mouse but were otherwise quantitatively similar. Upon longer K(+) applications (1 min), elevations of [Ca(2+)](c) and secretion were prolonged in bovine cells; in contrast [Ca(2+)](c) in mouse cells declined three-fold faster and failed to sustain a continued secretion. Confocal [Ca(2+)](c) imaging following a 50-ms depolarizing pulse showed a similar Ca(2+) entry, but a rate of [Ca(2+)](c) increase and a maximum peak significantly higher in bovine cells; the rate of dissipation of the Ca(2+) wave was faster in the mouse. The mitochondrial protonophore CCCP (2 microm) halved the K(+)-evoked [Ca(2+)](c) and secretory signals in mouse cells, but had little affect on bovine responses. We conclude that the relative densities of L (15% in bovine and 50% in mouse) and P/Q Ca(2+) channels (50% in bovine and 15% in mouse) do not contribute to the observed differences; rather, the different intracellular distribution of Ca(2+), which is strongly influenced by mitochondria, is responsible for a more sustained secretory response in bovine, and for a faster and more transient secretory response in mouse chromaffin cells. It seems that mitochondria near the plasmalemma sequester Ca(2+) more rapidly and efficiently in the mouse than in the bovine chromaffin cell.

Aniline Compounds↗
PubMed2003

Combined nimodipine and citicoline reduce infarct size, attenuate apoptosis and increase bcl-2 expression after focal cerebral ischemia.

Cerebral ischemia triggers a multitude of pathophysiological and biochemical events that separately affect the evolution of focal ischemia and, therefore, stroke treatment should logically employ all known neuroprotective agents. We hypothesized that a treatment combining nimodipine and citicoline might have a potential neuroprotective effect. To assess this idea, Sprague-Dawley rats underwent transient bilateral common carotid artery ligation with simultaneous middle cerebral artery occlusion for 60 min. Four treatment groups were established. Animals received either: a) saline (control group); b) intracarotid nimodipine infusion during 30 min in the ischemia-reperfusion (nimodipine group); c) i.p. postischemic citicoline injections once daily for 7 days (citicoline group); or d) intracarotid nimodipine bolus during ischemia-reperfusion plus i.p. postichemic citicoline injections (combination group). They were killed after either 7 or 3 days after reperfusion. In the first case, the volume of the infarcted tissue was studied by a stereological procedure and in the second case, in situ end-labeling of nuclear DNA fragmentation (TUNEL) and Bcl-2 expression were employed to determine the level of apoptosis. The infarct volume was significantly reduced in both the nimodipine and the citicoline treatment groups after 7 days of reperfusion; combination of both drugs produced an additive effect. After 3 days of reperfusion, the number of Bcl-2-positive neurons was significantly increased while that of TUNEL-positive cells significantly decreased at the infarct border in the combined-treatment animals. Our findings demonstrate a neuroprotective effect from an acute single dose of nimodipine during ischemia-reperfusion and prolonged post-ischemic treatment with citicoline in a model of focal cerebral ischemia. These results suggest that a possible mechanism of neuroprotective action would be mediated by increased Bcl-2 expression and decreased apoptosis within the boundary zone of the infarct together with neutralization of the ischemia-reperfusion injury.

Animals↗
PubMed2001

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death.

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.

Adrenal Medulla↗
PubMed2001

Mechanisms of blockade by the novel migraine prophylactic agent, dotarizine, of various brain and peripheral vessel contractility.

The novel antimigraineur, dotarizine, inhibited 5-HT (5 hydroxytryptamine)-evoked contractions of rabbit vertebral, aorta, femoral and mesenteric arteries, with IC(50)s of 1.35, 1.40, 0.52 and 1.09 microM, respectively. Flunarizine had little effect on these contractions, while ketanserin was more potent (IC(50)s of 0.17 microM for vertebral, 0.22 microM for aorta, 0.05 microM for femoral and 0.03 microM for mesenteric arteries). At 10 microM, dotarizine caused 40% blockade of K(+)-evoked contractions of rabbit aorta, and 70% inhibition of 5-HT-evoked responses; these values were 30% and 20% for 10 microM flunarizine. Contractions of rabbit aorta elicited by noradrenaline, angiotensin II or prostaglandin F(2alpha) were not affected by 10 microM dotarizine or flunarizine. Ketanserin shifted to the right, in parallel, the concentration-response curves for 5-HT in rabbit aorta; however, dotarizine caused a non-competitive type of blockade, increasing the maximum 5-HT contraction at 30 nM and decreasing it at 3 and 30 microM. K(+)-evoked contractions of rabbit aorta were halved by 3 microM dotarizine in a voltage-independent manner; flunarizine caused a delayed-type, non-reversible post-drug blockade, and exhibited some voltage-dependence. Blockade by nifedipine was voltage-dependent and fully reversible. Ca(2+)-evoked contractions of depolarised bovine middle cerebral arteries were blocked by 1--3 microM dotarizine in a non-surmountable manner. Contraction of these vessels evoked by electrical stimulation was blocked 50% and 70% by 1 and 3 microM dotarizine, respectively. Dotarizine (1--3 microM) also inhibited to a similar extent the K(+)-evoked [(3)H]noradrenaline release from cultured rat sympathetic neurones. These data suggest that the mechanism of blockade by dotarizine of cerebral vessels contractility has three components: (i) presynaptic inhibition of noradrenaline release; (ii) blockade of postsynaptic vascular 5-HT receptors; (iii) blockade of Ca(2+)entry into the vascular smooth muscle cell cytosol. The compound does not affect the vascular receptors for noradrenaline, angiotensin II or prostaglandin F(2alpha).

Animals↗
PubMed2001

Effect of viable or dead Lactobacillus casei organisms administered orally to mice on resistance against Trichinella spiralis infection.

The capacity of viable, dead Lactobacillus casei and supernatant from L. casei culture, administered by oral route, to induce resistance in mice against Trichinella spiralis infection was evaluated. The percentage of adult worm reduction in the intestine five days after T. spiralis infection as compared with the worm burden in the control group fluctuated between between 53.1 and 58% in mice treated with viable L. casei, while reductions in animals treated with dead lactobacilli or supernatant from L. casei culture were of 44 and 32.5% respectively. The percentage of larvae per gram of muscle tissue reductions, as compared with controls, obtained 30 days after infection ranged from 48.4 to 70.7% in rodents which ingested viable L. casei compared with the percentage reductions of 65.9 and 24% obtained respectively in mice treated with dead lactobacilli or with L. casei supernatant. The protective response observed in the present study may be explained on the basis of 1) lactobacilli colonization of the intestine, 2) macrophage processing of dead lactobacilli in local immune tissues and presentation of L. casei antigens to Th1 cells which, in turn, produced IL-2 to activate B cells and other T cells.

Administration, Oral↗
PubMed2000

Ba(2+)-induced chromaffin cell death: cytoprotection by Ca(2+) channel antagonists.

Exposure of bovine adrenal medullary chromaffin cells to Ba(2+) ions (in the absence of Ca(2+) ions) caused their death, measured as lactate dehydrogenase (LDH) release. The concentration of Ba(2+) required to damage the cells by about 65% ranged between 1 and 10 mM (no Ca(2+) added); the required exposure time was rather brief (15 min-4 h). The simultaneous presence of Ca(2+), Mg(2+) or Zn(2+) together with Ba(2+) (2 mM, 4 h) afforded cyprotection (60-80%). Individual selective blockers of Ca(2+) channel subtypes afforded no protection. However, combined nifedipine (3 microM) plus omega-conotoxin MVIIC (3 microM) offered full protection. Substantial protection was also seen with the "wide-spectrum" Ca(2+) channel blockers penfluridol (0.3 microM), lubeluzole (3 microM), dotarizine (3 microM), flunarizine (3 microM), and mibefradil (3 microM). This protection was due to blockade of Ba(2+) entry through Ca(2+) channels because dotarizine (10 microM) inhibited the increase in cytosolic [Ba(2+)] seen in fura-2-loaded chromaffin cells. Once Ba(2+) accumulated in the cytosol, it was not extruded by the Na(+)/Ca(2+) exchanger, as shown by the prolonged and sustained elevation of the fura-2 signal. This contrasts with the fast dissipation of the fura-2 signal generated by [Ca(2+)](i) elevation. Thus, Ba(2+) overload can cause cell death by mechanisms similar to those reported for Ca(2+) overload and might be used as a novel and convenient tool to search for new cytoprotective compounds.

Animals↗
PubMed1999

Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells.

1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1.2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0.65 +/- 0.02 fmol cell-1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0.16 +/- 0.01 fmol cell-1. 3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 +/- 104 nM in hyperpolarized cells to 115 +/- 9 nM in depolarized cells. 4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1.2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 +/- 35 nA of catecholamines in hyperpolarized cells and 220 +/- 19 nA in depolarized cells. 5. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (2 microM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. 6. This was strengthened by two additional findings: (i) nifedipine (3 microM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 microM), an L-type Ca2+ channel 'activator', dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 microM nifedipine and dramatically enhanced by 3 microM FPL64176. 8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Adrenal Medulla↗
PubMed1999

Compartive study of volatile components and fatty acids of plants and in vitro cultures of parsley (Petroselinum crispum (Mill) nym ex hill).

Volatile compounds from plants, callus tissue cultures, and cell suspensions of parsley (Petroselinum crispum) were captured during the growth cycle using a dynamic headspace extraction and were identified by gas chromatography-mass spectrometry. Parsley plants were found to produce mainly monoterpenes, and the compound of major abundance was p-1,3,8-menthatriene, followed by beta-phellandrene and apiole. Callus cultures and cell suspensions produced aldehydes (nonanal and decanal) that were also detected in parsley plant. The former also produced limonene, acetophenone, and benzotiazol; these were not observed in the plants. The production of volatiles in plants, callus tissue, and cell suspensions was found to be time-dependent. Free and bound fatty acids were also monitored by an in situ method. Palmitic (C16:0) and stearic (C18:0) acids were the most abundant fatty acids in all materials; however, higher levels were found in plants. On the other hand, the unsaturated C16:1 and C16:3 were not detected in the in vitro cultures.

Aldehydes↗
PubMed1999

PF9404C, a new slow NO donor with beta receptor blocking properties.

1. PF9404C is the S-S diesteroisomer of a novel blocker of beta adrenergic receptors with vasodilatory properties. It causes a concentration-dependent relaxation of rat aorta helical strips pre-contracted with 10(-6) M noradrenaline (NA; IC(50) 33 nM). It was equipotent to nitroglycerin (NTG; IC(50) 49 nM), but much more potent than isosorbide dinitrate (ISD; IC(50) 15,000 nM). 2. Oxyhaemoglobin (10 microM) shifted to the right the concentration-response curve for the relaxation induced by PF9404C (IC(50) 530 nM) or NTG (IC(50) 61 nM). 3. Either methylene blue (MB) or ODQ (1 microM each) largely prevented the vasorelaxing responses to increasing concentrations of PF9404C or NTG. 4. In rat aorta smooth muscle cells, PF9404C increased the formation of cyclic GMP from 3 pmol mg(-1) protein in basal conditions, to 53 pmol mg(-1) protein in 10 microM PF9404C. Neither metoprolol nor carvedilol enhanced cyclic GMP. 5. In the electrically driven guinea-pig left atrium, PF9404C blocked the inotropic effects of isoprenaline in a concentration-dependent manner. Its IC(50) (30 nM) was similar to that for S-propranolol (22.4 nM) and lower than the IC(50)s for metoprolol (120 nM) and atenolol (192 nM). The beta-adrenergic ligand (-)-[(3)H]-CGP12177 (0.2 nM) was displaced from its binding to rat brain membranes with K(i) of 7 nM, 17 nM, 170 nM and 1.2 microM respectively for PF9404C, S-(-)propranolol, metoprolol, and atenolol. 6. The data are consistent with the idea that the S-S diesteroisomer PF9404C, is a potent vasorelaxing agent, as well as a blocker of cardiac beta adrenergic receptors. The mechanism of its vasorelaxing effects involves the slow generation of NO. This molecule can, therefore, exhibit antihypertensive and cardioprotective actions through a double mechanism, NO donation and beta blockade.

Adrenergic beta-Antagonists↗
PubMed1998

Unmasking the functions of the chromaffin cell alpha7 nicotinic receptor by using short pulses of acetylcholine and selective blockers.

Methyllycaconitine (MLA), alpha-conotoxin ImI, and alpha-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 microM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC50s of 100 nM for MLA and 300 nM for alpha-conotoxin ImI and alpha-bungarotoxin. MLA (100 nM), alpha-conotoxin ImI (1 microM), and alpha-bungarotoxin (1 microM) halved the entry of 45Ca2+ stimulated by 5-s pulses of 300 microM ACh applied to incubated cells. These supramaximal concentrations of alpha7 nicotinic receptor blockers depressed by 30% (MLA), 25% (alpha-bungarotoxin), and 50% (alpha-conotoxin ImI) the inward current generated by 1-s pulses of 100 microM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain alpha7 neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, alpha-conotoxin ImI, and alpha-bungarotoxin with IC50s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through alpha3 beta4 nAChR was unaffected by alpha-conotoxin ImI and alpha-bungarotoxin, and weakly blocked by MLA (IC50 = 1 microM). The functions of controlling the electrical activity, the entry of Ca2+, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to alpha3 beta4 nAChR; the present results constitute the first evidence to support a prominent role of alpha7 nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

Acetylcholine↗
PubMed1998

PCA50941, a new 1,4-dihydropyridine, reverses endothelin-induced cardiogenic shock in the anesthetized goat.

This study was designed to test the hypothesis that the properties of novel 1,4-dihydropyridine PCA50941 could favor the recovery of cardiogenic shock. Coronary blood flow (CBF), measured with an electromagnetic flow probe placed on the left circumflex coronary artery, systemic arterial pressure and heart rate were recorded in 24 anesthetized goats; left ventricular pressure and dP/dt were also recorded in 19 of these goats. Under control conditions, intracoronary injections in 5 goats of PCA50941 (10-120 microg) caused smaller reductions of CBF than those of Bay K 8644 (0.3-10 microg) (the reduction of CBF by 120 microg PCA50941 was 25% and that by 10 microg Bay K 8644 was 43%), and i.v. infusions in 4 goats of PCA50941 (10-300 microg/min) did not modify CBF nor the other hemodynamic variables recorded, whereas i.v infusion of Bay K 8644 (10-30 microg/min) reduced CBF by 20% and increased arterial pressure, left ventricular pressure and dP/dt. During control conditions and endothelin-induced cardiogenic shock, respectively, the values for 15 goats were: for CBF, 33+/-4 vs. 16+/-4 ml/min; for mean arterial pressure, 88+/-4 vs. 60+/-5 mm Hg; for left ventricular systolic pressure, 102+/-5 vs. 75+/-4 mm Hg; for dP/dt, 1453+/-147 vs. 925+/-101 mm Hg/s (all P<0.05), and for heart rate, 77+/-6 vs. 81+/-6 beats/min (P>0.05). Intravenous infusion of PCA50941 (100 microg/min) reversed the hemodynamic variables from the shock state to control values within 20 min in 5 of 6 animals, whereas i.v. administration of Bay K 8644 (10-30 microg/min) was not effective in 4 of 5 animals, and the vehicle (DMSO) was not effective in none of 4 animals in reversing the hemodynamic shock state. Therefore, it is suggested that PCA50941, a novel 1,4-dihydropyridine, has a cardiovascular profile that might be suitable for treating cardiogenic shock states.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1998

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine, barium, ouabain and 6-hydroxydopamine in chromaffin cells.

1. Incubation of bovine adrenal chromaffin cells with veratridine (10-100 microM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(-) enantiomer, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3-10 microM) decreased the veratridine-induced LDH release. 2. Penfluridol did not increase LDH release at concentrations 0.003-1 microM; 3-10 microM increased LDH release to 50-60%, after 24 h exposure. Penfluridol (0.03-0.3 microM) did not protect against the cytotoxic effects of veratridine; at 1 microM, 15% protection was produced. Higher concentrations (3-10 microM) enhanced the cytotoxic effects of veratridine. 3. Ba2+ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 microM lubeluzole and fully counteracted by 1 microM penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+. 4. Ouabain (10 microM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3-10 microM) and the lower concentrations of penfluridol (0.003-0.3 microM) prevented the ouabain cytotoxic effects. At higher concentrations (3 microM), penfluridol increased drastically the ouabain cytotoxic effects. 5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 microM. Lubeluzole (3-10 microM) or penfluridol (0.03-0.3 microM) had no cytoprotective effects against 6-OHDA. 6. Lubeluzole (3 microM), R91154 (3 microM) and penfluridol (1 microM) blocked the current through Na+ channels in voltage-clamped chromaffin cells (I(Na)) by around 20-30%. Ca2+ current through Ca2+ channels (I(Ca)) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversible. 7. Lubeluzole (3 microM) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura-2-loaded cells exposed to 30 or 100 microM veratridine. Penfluridol (1 microM) inhibited those oscillations in an irreversible manner. 8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as well as the [Ca2+]i oscillations. The Ba2+ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na+/Ca2+ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

Adrenal Glands↗
PubMed1997

Drastic facilitation by alpha-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i.

1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.

Acetylcholine↗
PubMed1997

'Wide-spectrum Ca2+ channel antagonists': lipophilicity, inhibition, and recovery of secretion in chromaffin cells.

Repetitive application of short depolarizing K+ pulses (70 mM K+, 2 mM Ca2+ Krebs-HEPES solution, for 10 s every 5 min) produced reproducible catecholamine secretory responses from superfused bovine chromaffin cells. At 10 microM for 15 min, the piperazine derivatives dotarizine, flunarizine and lidoflazine inhibited secretion by around 90%; cinnarizine halved the secretory response. Recovery of secretion after 30-min washout with Krebs-HEPES solution amounted to 75% in the case of dotarizine, 8% for flunarizine, 46% for lidoflazine and 21% for cinnarizine. The benzothiazol derivatives (10 microM) (+)-S-lubeluzole and R91154 (the (-)-R-enantiomer of lubeluzole) blocked the response by 75%; sabeluzole inhibited secretion by only 34% and R56865 (N-[1-(4-(4-fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiaz olamine) by 61%. Recoveries were around 70% in the case of these four benzothiazol derivatives. The diphenylbutyl-piperazine derivatives fluspirilene and penfluridol inhibited secretion by over 80%; no recovery was produced after 30-min washout. The inhibition of secretion was time dependent, as the recovery of the response was. Blockade of secretion by dotarizine and flunarizine occurred even in the absence of intermittent K+ stimulations of the cells. No obvious correlation was seen between the octanol/water partition coefficients of the ten compounds tested (that ranged between 6 and 4.61), the rate and extent of blockade of secretion, and the recovery of the secretory response upon washout. Rather than non-specific actions on ion channels (and secretion) due to their high lipophilicity, we believe that blockade of various Ca2+ channels relates to their binding properties to specific channel micro and macrodomains, as the case might be for 'narrow' (omega-conotoxin GVIA) and 'wide-spectrum' (omega-conotoxin MVIIC) peptide toxins.

Animals↗
PubMed1997

Distinct effects of omega-toxins and various groups of Ca(2+)-entry inhibitors on nicotinic acetylcholine receptor and Ca2+ channels of chromaffin cells.

The effects of omega-toxins and various Ca2+ antagonist subtypes on the 45Ca2+ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K+, have been compared. The conditions selected to stimulate the 45Ca2+ entry consisted of a 60-s period of exposure of cells to 100 microM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K+. The N-type voltage-dependent Ca2+ channel blockers omega-conotoxin GVIA and MVIIA (1 microM) inhibited 45Ca2+ entry stimulated by dimethylphenylpiperazinium or K+ by around 25-30%. The P-type Ca2+ channel blocker omega-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K+ responses; 1 microM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca2+ channel blocker omega-contoxin MVIIC (1 microM) inhibited the K+ evoked 45Ca2+ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P < 0.001). The L-type Ca2+ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 microM each) inhibited much more the dimethylphenylpiperazinium than the K+ response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K+ response by 38, 29, 22, and 10%. Combined omega-conotoxin MVIIC (1 microM) and furnidipine (3 microM) blocked 100% of the K+ evoked 45Ca2+ entry. However, combined omega-conotoxin GVIA (1 microM), and furnidipine left unblocked 50% of the K+ response. The "wide spectrum' Ca2+ channel antagonists flunarizine or dotarizine (3 microM each) blocked the dimethylphenylpiperazinium and the K+ responses to a similar extent (50%); cinnarizine (3 microM) inhibited more the dimethylphenylpiperazinium (82%) than the K+ response (21%). At 3 microM, the highly lipophilic beta-adrenoceptor antagonist (+/-)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K+ signal. Other high lipophilic beta-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K+ responses. The highly lipophilic agent penfluridol blocked the dimethylpiperazinium response by 30% and the K+ response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K+ responses. These data are compatible with the idea that the various omega-toxin peptides used to separate pharmacologically the different voltage-dependent Ca2+ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca2+ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca2+ channel or nicotinic acetylcholine receptor blockade.

Animals↗