Pro-arrhythmogenic effects of Trypanosoma cruzi conditioned medium proteins in a model of bovine chromaffin cells.

Asymptomatic sudden death is the principal cause of mortality in Chagas disease. There is little information about molecular mechanisms involved in the pathophysiology of malignant arrhythmias in Chagasic patients. Previous studies have involved Trypanosoma cruzi secretion proteins in the genesis of arrhythmias ex vivo, but the molecular mechanisms involved are still unresolved. Thus, the aim was to determine the effect of these secreted proteins on the cellular excitability throughout to test its effects on catecholamine secretion, sodium-, calcium-, and potassium-conductance and action potential (AP) firing. Conditioned medium was obtained from the co-culture of T. cruzi and Vero cells (African green monkey kidney cells) and ultra-filtered for concentrating immunogenic high molecular weight parasite proteins. Chromaffin cells were assessed with the parasite and Vero cells control medium. Parasite-secreted proteins induce catecholamine secretion in a dose-dependent manner. Additionally, T. cruzi conditioned medium induced depression of both calcium conductance and calcium and voltage-dependent potassium current. Interestingly, this fact was related to the abolishment of the hyperpolarization phase of the AP produced by the parasite medium. Taken together, these results suggest that T. cruzi proteins may be involved in the genesis of pro-arrhythmic conditions that could influence the appearance of malignant arrhythmias in Chagasic patients.

[Anticholinesterases in the treatment of Alzheimer's disease]. / Anticolinesterásicos en el tratamiento de la enfermedad de Alzheimer.

INTRODUCTION: Among the numerous pathophysiological theories that attempt to explain the development of Alzheimer's disease (AD) there are two facts that stand out above the rest: on the one hand, the formation of neurofibrillary tangles inside cells and, on the other, the extra-cellular deposition of beta-amyloid protein. These two mechanisms lead to neurodegeneration and the death of cells by means of a process called 'apoptosis' or 'programmed cell death'. In the early stages of this neurodegenerative process it is more pronounced in cholinergic-type brain centres. This led to the formulation of the so-called cholinergic theory of Alzheimer, which provides the rationale behind the use of the drugs that are currently available to treat this disease, namely, acetylcholine esterase (AChE) inhibitors (rivastigmine, donepezil and galanthamine). DEVELOPMENT AND CONCLUSIONS: We review the possible pharmacological approaches that could help to prevent or delay cell death, and which act on the mechanisms involved in the production of neurofibrillary tangles or the deposition of beta-amyloid protein. We also review the main characteristics of cholinergic neurotransmission, which will help us to understand the therapeutic approaches that have been applied in an attempt to enhance deficient cholinergic neurotransmission. One of the most notable of these is the amount of attention recently being paid to the enzyme AChE, which increases the bioavailability of the neurotransmitter in the cholinergic synapses by preventing the hydrolysis of acetylcholine; these are the only drugs currently available for the symptomatic treatment of this disease.

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells.

We established primary cultures of human pheochromocytoma chromaffin cells. We then tried to find what mechanism of their secretory apparatus could be altered to produce the massive release of catecholamines into the circulation and the subsequent hypertensive crisis observed in patients suffering this type of tumor. Their whole-cell Ca2+ channel currents could be pharmacologically separated into components similar to those found in normal human adrenal chromaffin cells: 20% L-type, 30% N-type, and 50% P/Q-type Ca2+ channels. However, modulation of the channels by exogenous or endogenous ATP and opioids, via a G-protein membrane-delimited pathway, was deeply altered; some cells having no modulation or very little modulation alternated with others having normal modulation. This may be the cause of the uncontrolled secretory response, measured amperometrically at the single-cell level. Some cells secreted for long time periods and were insensitive to nifedipine (L-type channel blocker) or to omega-conotoxin MVIIC (N/P/Q-type channel blocker), while others were highly sensitive to nifedipine and partially sensitive to omega-conotoxin MVIIC. Alteration of the autocrine/paracrine modulation of Ca2+ channels may lead to indiscriminate Ca2+ entry and exacerbate catecholamine release responses in human pheochromocytoma cells.

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.

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.

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells.

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.

Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions.

This study was carried out to characterize the set of voltage-dependent Ca2+ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM) Ba2+ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of -80 mV, averaged peak current in 10 mM Ba2+ was around 1 nA, and in 2 mM Ba2+ 0.36 nA. When using 2 mM Ba2+ as the charge carrier, nifedipine (3 microM) blocked IBa by 40-45%. omega-Conotoxin GVIA (1 microM) caused 26% inhibition, while omega-conotoxin MVIIC (3 microM) produced a 48% blockade. At low concentrations (20 nM), omega-agatoxin IVA caused 5-15% of current inhibition, while 2 microM gave rise to a 35-40% blockade. In 10 mM Ba2+, the blocking effects of nifedipine (40%) and omega-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba2+. In contrast, blockade by omega-conotoxin MVIIC was markedly reduced in 10 mM Ba2+ (20-25%) as compared to 10 mM Ba2+ (48%). The blocking actions of omega-agatoxin IVA (2 microM) were also slowed down in 10 mM Ba2+, though the final blockade was unaffected. In 2 mM Ba2+, IBa was quickly inhibited by over 94% with combined nifedipine + omega-conotoxin MVIIC + omega-conotoxin GVIA; in 10 mM Ba2+, IBa was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type (60%) high-threshold voltage-dependent Ca2+ channels. The current carried by non-L-type Ca2+ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also indicate that the fraction of current blocked by omega-conotoxin MVIIC and omega-agatoxin IVA might considerably change as a function of the Ba2+ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current is not expressed in mouse chromaffin cells.

Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes.

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (-) enantiomer R91154 on whole-cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of -80 mV, at 10 s intervals had an average magnitude of 1 nA. 2. Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration-dependent manner; their IC50s were 1.94 microM for lubeluzole and 2.54 microM for R91154. In a current-voltage protocol, lubeluzole (3 microM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I-V curve were detected. 3. After 10 min exposure to 3 microM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered. 4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at -110 mV and 87% at -50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at -110 mV, decreased at -80 mV and did not occur at -50 mV. 5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 microM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 microM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa. 6. The effects of lubeluzole (3 microM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At -80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole). 7. In mouse chromaffin cells approximately half of the whole-cell IBa was sensitive to 3 microM nifedipine (L-type Ca2+ channels) and the other half to 3 microM omega-conotoxin MVIIC (non-L-type Ca2+ channels). In omega-conotoxin MVIIC-treated cells, 3 microM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound. 8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca2+ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca2+ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca2+ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca2+ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.

Effects of omega-toxins on noradrenergic neurotransmission in beating guinea pig atria.

The effects of four omega-toxins, known to block various subtypes of neuronal voltage-activated Ca2+ channels, on the beating guinea pig left atrium have been analyzed. Atria were suspended in oxygenated Krebs-bicarbonate solution at 32 degrees C and driven with electrical pulses delivered by a stimulator at 1 Hz, 1 ms, 4 V. A 10-fold increase of voltage caused a potent and rapid enhancement of the size of contractions (about 3- to 4-fold above basal), which reflects the release of endogenous noradrenaline from sympathetic nerve terminals. omega-Conotoxin MVIIC, omega-conotoxin MVIIA and omega-conotoxin GVIA inhibited the inotropic responses to 10 x V stimulation with IC50 values of 191, 44 and 20.4 nM, respectively. omega-Agatoxin IVA did not affect the contractile responses. The inotropic responses to exogenous noradrenaline were unaffected by the toxins. The potent blocking effects of omega-conotoxin GVIA were present even in conditions in which the release of noradrenaline was strongly facilitated by presynaptic alpha 2-adrenoceptor blockade by phenoxybenzamine. These effects were not reversed upon repeated washing of the tissue with toxin-free medium. In contrast, the blockade induced by omega-conotoxin MVIIC and omega-conotoxin MVIIA were fully reversed, with t1/2 of 13.5 and 31.2 min, respectively. omega-Conotoxin MVIIC (1 microM) protected against the irreversibility of the blockade induced by omega-conotoxin GVIA (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-independent relaxation by 17-alpha-estradiol of pig coronary arteries.

We have studied the effects of 17-alpha-estradiol, a non-estrogenic steroid, on pig coronary arteries contracted by K+, Ca2+ or serotonin. Experiments were performed on helicoidal strips and rings of left circumflex coronary arteries from freshly slaughtered white pigs and on helicoidal strips of rat thoracic aorta. The strips and rings were suspended inside a water-jacketed muscle chamber in an oxygenated Krebs solution at 37 degrees C. From the initial K(+)-evoked contraction, 17-alpha-estradiol caused a relaxation with an IC50 (15 microM) which was in the range of the IC50s obtained for nitroglycerin (1.3 microM) and nicorandil (50 microM). Contractions evoked by Ca2+ were inhibited by 17-alpha-estradiol, but full blockade could not be achieved. Serotonin-evoked contractions were also blocked by 17-alpha-estradiol with an IC50 of 2.1 microM; 17-beta-estradiol also inhibited the serotonin-evoked contractions. In the presence of 100 microM of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester, the relaxing properties of 17-alpha-estradiol in pig coronary arteries and rat thoracic aorta were unaffected, suggesting that endothelial NO release was unrelated to these effects. 17-alpha-Estradiol also relaxed denuded pig coronary artery strips, suggesting that other endothelial-derived relaxing factors were not involved in its vascular effects. The results are compatible with the idea that 17-alpha-estradiol causes relaxation of coronary vessels by acting directly on the cell membrane of smooth muscle cells; these effects seem to be unrelated to the genomic physiological effects of estrogens.(ABSTRACT TRUNCATED AT 250 WORDS)

PCA 50941, a novel Ca2+ channel agonist.

PCA 50941 is a novel 1,4-dihydropyridine derivative. Its vasoconstricting effects prompted a systematic comparison with the prototypic Ca2+ channel activator, Bay K 8644. The two compounds exhibit marked analogies and differences in their cardiovascular profiles. PCA 50941 exhibits a pronounced vascular over cardiac selectivity while Bay K 8644 has both potent vasoconstrictor effects and strong cardiac positive inotropic actions. PCA 50941 exhibits either poor positive inotropic effects (isolated guinea-pig atria) or clear negative inotropic effects (isolated perfused rat heart). Both compounds reduced by 10-40% the coronary flow in the perfused rat heart. However, PCA 50941 had slight vasoconstrictor effects in pig coronary arteries, causing their relaxation at nanomolar/micromolar concentrations; this contrasts with the almost pure, marked vasoconstrictor effects of Bay K 8644 in coronary arteries. In the rat aorta PCA 50941 exhibited a biphasic pattern of vasoconstriction and vasorelaxation, and in portal vein it markedly reduced the Ca(2+)-evoked contractions; Bay K 8644 behaved as a pure vasoconstrictor in these two preparations. It is concluded that the racemic compound, PCA 50941, exhibits different degrees of Ca2+ agonism and Ca2+ antagonism by acting upon 1,4-dihydropyridine receptors of different cardiovascular tissues. Its tissue selectivity and its prolonged duration of action give PCA 50941 a cardiovascular profile more favourable than that of other 1,4-dihydropyridine Ca2+ agonist existing to date.

Membrane-mediated effects of the steroid 17-alpha-estradiol on adrenal catecholamine release.

The effects of 17-alpha-estradiol on the secretion of catecholamines from the perfused bovine and cat adrenal gland and bovine chromaffin cells in culture elicited by dimethylphenylpiperazinium (DMPP), methacholine and high potassium were studied. In perfused cat adrenal glands, secretion of catecholamines evoked by pulses of DMPP (1 microM for 30 sec) was decreased by 17-alpha-estradiol at concentrations of 1 and 10 microM by 50 and 80%, respectively. However, secretion evoked by pulses of methacholine (3 microM for 30 sec) was not affected by 1 microM of 17-alpha-estradiol and was affected to a variable extent by 10 microM 17-alpha-estradiol. Catecholamine secretion evoked by higher concentrations of methacholine (100 microM for 60 sec) was reduced by 50% by 10 microM 17-alpha-estradiol. 17-alpha-Estradiol decreased secretion evoked by pulses of 120 mM K+ for 10 sec to a similar extent in the perfused bovine and cat adrenal gland. The 45Ca++ uptake into bovine chromaffin cells in culture stimulated by DMPP (100 microM for 10 sec) or high K+ (59 mM for 10 sec) was almost inhibited completely by 100 microM 17-alpha-estradiol. The rapid action precludes a classical genomic mechanism and suggests effects at the cell membrane.

Different sensitivities to dihydropyridines of catecholamine release from cat and ox adrenals.

Catecholamine release evoked by quick pulses of Ca2+ (2.5 mM) given sequentially at 30 min intervals to cat and ox adrenal glands perfused continuously with Ca2+ free Krebs-Tris solutions containing 35 or 118 mM K+, was studied. In the feline, the secretory response was highly sensitive to various dihydropyridine (DHP) derivatives. For instance, secretion was completely blocked by nM concentrations of (+)isradipine (IC50 between 3 and 4 nM) and markedly potentiated by (-)Bay-K-8644. In contrast, the bovine secretory responses were resistant to blockade by nitrendipine or (+)isradipine, as well as to potentiation by Bay-K-8644, even at microM concentrations. From these experiments, it seems clear that distinct subtypes of Ca2+ channels might mediate a similar secretory response in ox and cat adrenal chromaffin cells, at least in the present experimental conditions.

Voltage-dependent inactivation of catecholamine secretion evoked by brief calcium pulses in the cat adrenal medulla.

1. Inactivation by voltage changes of 45Ca2+ uptake into and catecholamine release from cat adrenal glands perfused at a high rate (4 ml/min) at 37 degrees C with oxygenated Krebs-Tris solution has been studied. Experimental conditions were selected so that adrenal medullary chromaffin cells were depolarized for different time periods and with various K+ concentrations in the absence of Ca2+, prior to the application of 0.5 mM-Ca2+ for 10 s in the presence of 118 mM-K+ to test the rate of secretion (the 'Ca2+ pulse'). 2. Application of the Ca2+ pulse after perfusion with 5.9 mM-K+ led to a 100-fold increase of the basal rate of secretion. However, if the Ca2+ pulse was preceded by a 10 min period of perfusion with 118 mM-K+, the secretory response was decreased by over 80%. 3. Inactivation of secretion starts 10-30 s after high-K+ perfusion and is completed 2-5 min thereafter. Inactivation is readily reversed by perfusing the glands with normal K(+)-containing solution; the recovery phenomenon is also gradual and time-dependent, starting 30 s after repolarization and ending 300 s thereafter. 4. The rate of inactivation is much slower at 35 than at 118 mM-K+, suggesting that the process is strongly dependent on voltage. 5. Like catecholamine release, Ca2+ uptake into adrenal medullary chromaffin cells is inactivated in a voltage-dependent manner. This, together with the fact that Cd2+ blocked secretion completely and inactivation was seen equally using Ca2+ or Ba2+ as secretagogues, suggests that inactivation of a certain class of voltage-dependent Ca2+ channels is responsible for the blockade of secretion. Such channels must be slowly inactivated by voltage and highly sensitive to dihydropyridines, since (+)PN200-110 (an L-type Ca2+ channel blocker) enhanced the rate of inactivation and (+/-)Bay K 8644 (an L-type Ca2+ channel activator) prevented it, indicating that they might belong to L-subtype Ca2+ channels. 6. The effects of (+/-)Bay K 8644 (100 nM) were seen on both the voltage and time dependence of inactivation. At a moderate depolarization (35 mM-K+), the drug prevented inactivation and caused potentiation of secretion which developed gradually; at strong depolarizations (118 mM-K+), Bay K 8644 prevented the time-dependent development of inactivation. (+)PN200-110 (30 nM) did not suddenly decrease catecholamine release at the earlier times of depolarization; what the drug did was to accelerate the normal rate of inactivation induced by depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-dependence of nitrendipine provides direct evidence for dihydropyridine receptor coupling to calcium channels in intact cat adrenals.

Nitrendipine behaves as a powerful inhibitor of catecholamine release in perfused cat adrenal glands stimulated with pulses of Ca (0.5 mM) in high K (118 mM). Its blocking ability is considerably enhanced if, before Ca stimulation, the adrenomedullary tissue is impregnated with nitrendipine in depolarizing (IC50 = 0.99 nM) as opposed to hyperpolarizing (IC50 = 214 nM) conditions. 3H-nitrendipine binding to adrenomedullary tissue was considerably enhanced in depolarization; blockade of 45Ca uptake into and catecholamine secretion from the same glands was also enhanced in depolarized tissues. This study shows for the first time the a good correlation between these three parameters in the same intact preparation, providing direct evidence for a functional coupling between dihydropyridine receptors, Ca channels and secretion.