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.

A preferential pole for exocytosis in cultured chromaffin cells revealed by confocal microscopy.

Histological studies suggest that adrenal medulla chromaffin cells in situ are polarised, but functional evidence is lacking. We present here the first demonstration for polarisation of exocytosis in isolated, spherical, bovine chromaffin cells. Cells were stimulated with 70 mM K(+) to cause a marked enhancement of catecholamine release, monitored amperometrically. FM1-43 and dopamine beta-hydroxylase antibodies provided fluorescence confocal pictures that were 2-3-fold more intense in the bottom of the cells, as compared to equatorial or apex planes. This suggests that the solid phase to which the cell attaches serves as a 'trophic' signal for the polarisation of its secretory apparatus.

Calcium channel subtypes and exocytosis in chromaffin cells: a different view from the intact rat adrenal.

In the intact rat adrenal gland perfused with an oxygenated Krebs-bicarbonate solution at 37 degrees C, the electrical field stimulation of splanchnic nerves (100 V, 0.5 ms duration, 10 Hz during 10 s) produced transient catecholamine release peaks that were reproduced in subsequent stimuli, applied at 8-min intervals. Omega-Conotoxin GVIA (0.3 microM) caused only a modest inhibition of the secretory response, suggesting that the N-subtype of voltage-dependent Ca2+ channels are scarcely involved in such a response. Both omega-conotoxin MVIIC (1 microM) and furnidipine (1 microM) halved the secretion, suggesting that the L- and P/Q-types of Ca2+ channels were involved. N-type Ca2+ channels appear to be involved in the maintenance of secretion in response to sustained stimulus since omega-conotoxin GVIA (0.3 microM) reduced the catecholamine output to 28%. When secretion was elicited by acetylcholine (10 microM), furnidipine reduced the catecholamine release by 50% and omega-conotoxin MVIIC by 40%, whereas omega-conotoxin GVIA did not modify the response. The K+-induced secretory responses (23.6 mM K+, 15 s) were reduced 75% by furnidipine and 45% by omega-conotoxin MVIIC, indicating that this type of stimulation preferentially recruited L-type channels. These data show that electrical stimulation recruits Ca2+ channel subtypes different from those recruited by direct depolarization of chromaffin cells.

Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin.

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

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.

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.

Different contributions of L- and Q-type Ca2+ channels to Ca2+ signals and secretion in chromaffin cell subtypes.

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.

The mechanism of calcium channel facilitation in bovine chromaffin cells.

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage-clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole-cell Ba2+ currents (IBa). 2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3.18 +/- 0.12 mM (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11.2 +/- 0.97 and 15.2 +/- 2 mM, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7-fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2+ channel facilitation, persisted in the presence of 3 microM nifedipine. 4. Intracellular dialysis of GDP-beta-S (0.5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml-1 for 18-24 h) prevented the reduction in peak current caused by a 1:100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 microM) or suramin (100 microM) and PPADS (100 microM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (< 10 kDa) considerably reduced the activity of SVL. 6. Stopping the flow of the external solution (10 mM Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow-stop conditions in the presence or absence of 3 microM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole-cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G-protein-mediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non-L-type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridine-sensitive Ca2+ channels are not tenable.

Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed.

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K+ glutamate-based solution containing 50 nM free Ca2+ and 2 mM adenosine 5'-triphosphate, dipotassium salt (K2ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca2+ concentrations ([Ca2+]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca2+] (3-100 microM), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200-400 vesicles per cell. Pretreatment of cells with tyramine (10 microM for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca2+] in the range of 0.1-3 microM, the activation rate of secretion increased from 2.3 to 35.3 nA.s-1, reached a plateau between 3-30 microM and rose again from 100 to 1000 microM [Ca2+] to a maximum of 91.9 nA.s-1. In contrast, total secretion first increased (0.1-1 microM Ca2+), then plateaud (1-100 microM Ca2+) and subsequently decreased (100-1000 microM Ca2+). At 30 and 1000 microM extracellular [Ca2+] or [Ca2+]o, the activation rates of secretion from intact cells depolarised with 70 mM K+ were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 microM Ca2+) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 microA.s at 1000 microM Ca2+) than in electroporated cells (42.4 microA.s at 1 microM Ca2+). Separation of the time-courses of changes in intracellular [Ca2+] or [Ca2+]i and secretion in intact chromaffin cells depolarised with 70 mM K+ was demonstrated at different [Ca2+]o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca2+]i. In contrast, the decline of secretion was faster than the decline of the peak [Ca2+]i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca2+ by the rates of opening and closing of voltage-dependent Ca2+ channels of the L- and Q-subtypes, which control the local [Ca2+]i near to exocytotic sites.

Effects of Ca2+ channel antagonist subtypes on mitochondrial Ca2+ transport.

This study was carried out to define the effects of various Ca2+ channel modulatory drugs on mitochondrial Ca2+ movements. Bovine adrenal medulla mitochondria took up Ca2+ at an initial rate of 6.8 nmol mg protein-1 5 s-1, with a Km of 15 microM and a Bmax of 30 nmol mg protein-1. At 30 microM, neither verapamil, diltiazem, nitrendipine nor Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] affected the initial rate of Ca2+ uptake. Ca(2+)-loaded mitochondria retained their Ca2+ contents in the presence of ruthenium red for at least 30 min. Cinnarizine and flunarizine, but not verapamil, diltiazem, isradipine, Bay K 8644 or nitrendipine, caused a fast and dramatic Na(+)-independent Ca2+ loss. Other Ca2+ channel antagonists assayed such as penfluridol, R56865 [N-[1-(4-(4-fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2- benzothiazolamine], lidoflazine, R87926 [(+)-(S)-4-(2-benzothiazolyl-methylamino)-alpha-[(3,4-difluorophenoxy ) methyl] 1 piperidine] and sabeluzole, also had a mitochondrial Ca2+ depleting effect which seemed to be directly related to their octanol/water partition coefficient. The Na(+)-dependent Ca2+ efflux from mitochondria was completely inhibited by diltiazem and greatly blocked by nitrendipine. Isradipine caused a moderate blockade and Bay K 8644 and verapamil had no effect. All these data open the possibility of developing novel Ca2+ channel antagonists having selective actions on plasmalemmal Ca2+ channels, and others with additional and different effects on mitochondrial Ca2+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat striatal astroglia induce morphological and neurochemical changes in adult bovine, adrenergic-enriched adrenal chromaffin cells in vitro.

Adult bovine chromaffin cells are generally resistant to plastic changes when exposed to various culture media. Here, we demonstrate that adrenergic-enriched bovine chromaffin cell populations show significant process extension when co-cultured with fetal rat brain glial fibrillary acid protein (GFAP)+, fibronectin astroglia. Basal medium was ineffective in promoting such growth in chromaffin cells. Initial process formation could be observed about 24 h after seeding, while well developed processes were seen after 3-4 days. Intimate contacts of cytoplasmic extensions with adrenergic chromaffin cells, or with astroglial cells, were frequently observed. Both co-culture with glial cells or exposure to glia-conditioned medium led to a significant increase in noradrenaline and adrenaline, but not in dopamine content. Since bovine chromaffin cells are widely used as models to study fundamental mechanisms of neurosecretion and phenotype transformation, their co-culture with astroglia may provide a useful strategy to study such phenomena.

Modulation by L-type Ca2+ channels and apamin-sensitive K+ channels of muscarinic responses in cat chromaffin cells.

In the perfused cat adrenal gland stimulated with the muscarinic agonist methacholine chloride (100 microM for 3 min), two components were detected in the catecholamine secretory response: 1) an early phasic component that peaked at 300 ng/5 s catecholamine release and 2) a tonic component whose peak was transient and declined to a plateau of about 140 ng/5 s. Apamin (0.1 microM) increased the phasic component to 1,200 ng/5 s and the tonic component to approximately 350 ng/5 s. In single fura 2-loaded cat adrenal chromaffin cells, the cytosolic Ca2+ concentration ([Ca2+]i) also followed a biphasic pattern after stimulation with methacholine. Depletion of extracellular Ca2+ reduced the phasic [Ca2+]i peak by > 50% and the phasic secretory peak by approximately 90%; both the tonic components of [Ca2+]i and secretion were abolished. Depletion of intracellular Ca2+ pools decreased the phasic and tonic components of [Ca2+]i and secretion with respect to control values; however, the phasic components diminished more than the tonic components of [Ca2+]i and secretion. Although 3 microM furnidipine (a dihydropyridine L-type Ca2+ channel blocker) inhibited the phasic component of [Ca2+]i and secretion, its effects were more pronounced on the tonic component. omega-Conotoxin GVIA (1 microM, an N-type Ca2+ channel blocker) did not affect the [Ca2+]i or the methacholine secretory responses. The secretion peak seems to depend on both extracellular free Ca2+ (Cao2+) entry through L-type Ca2+ channels as well as on the mobilization of Ca2+ from intracellular stores; the plateau depends only on Cao2+ entry through L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells.

The level of diadenosine 5',5"-P1-P4-tetraphosphate (diadenosine tetraphosphate or Ap4A), catecholamines, ATP and other nucleotides has been investigated in perchloric acid extracts of bovine adrenal medulla, chromaffin granules and cultured chromaffin cells. As a control, the amount of Ap4A and ATP has also been measured in human blood platelets. The following values (nmol/mg protein) were found in adrenal medulla: Ap4A, 0.019 +/- 0.004; ATP, 109 +/- 11; ADP, 23.8 +/- 5.8; AMP, 11.3 +/- 1.5; p4A, 0.18 +/- 0.08; catecholamines, 460 +/- 57. The level of Ap4A, catecholamines and ATP (nmol/mg protein) found in chromaffin granules and in chromaffin cells were, respectively: (0.15 +/- 0.07; 2175 +/- 99; 531 +/- 66) and (0.22 +/- 0.14; 1143 +/- 277; 222 +/- 53). In all the cases investigated, the ratio catecholamines/ATP and catecholamines/Ap4A were around 5 and in the order of 10(3), respectively. The amount of Ap4A found here, in bovine adrenal medulla, chromaffin granules and chromaffin cells, is two orders of magnitude lower than previously reported.

R56865 inhibits catecholamine release from bovine chromaffin cells by blocking calcium channels.

1. The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole-cell current through Ca2+ channels (IBa) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2. R56865 caused a time- and concentration-dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mM K+. After 5 min superfusion, a 3 microM concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85% after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 microM. The blocking effects of R56865 were reversible after 5-15 min wash out. In high Ca2+ solution (10 mM Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mM Ca2+. 3. In electroporated cells, R56865 (10 microM) did not modify the secretory response induced by the application of 10 microM free Ca2+. 4. R56865 blocked the peak IBa current in a concentration- and time-dependent manner; its IC50 was very similar to that obtained for secretion (3 microM). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 microM. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5. In fura-2-loaded single chromaffin cells the basal [Ca2+]i of around 100 nM was elevated to a peak of1.5 microM by the application of a 5 s pulse of 59 mM K+. R56865 (10 microM) did not affect the basal [Ca2+]but blocked by 90% the K+-evoked increase. This effect was fully reversible after 5-10 min of wash out.6. The results are compatible with the idea that R56865 blocks Ca2+ entry into K+-depolarized chromaffin cells by promoting the inactivation of voltage-dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.

Activation of adrenal medullary L-arginine: nitric oxide pathway by stimuli which induce the release of catecholamines.

The activation of the L-arginine: nitric oxide (NO) pathway in the cat adrenal medulla by different stimuli which induce the release of catecholamines was studied. Stimuli that evoke catecholamine release, such as electrical stimulation of splanchnic nerves (50 V, 5 Hz, 1 ms), methacholine (100 microM), dimethyl-4-phenylpiperazinium iodide (DMPP; 10 microM), high K+ (35 mM) and alamethicin (15 micrograms ml-1) also caused a rise in cyclic GMP in the perfused cat adrenal medulla. NG-nitro-L-arginine methyl ester (L-NAME; 1 mM) abolished the rise in cyclic GMP induced by these stimuli without affecting the catecholamine release. Bovine adrenal medulla cytosol contained an NO synthase which was L-arginine- and Ca(2+)-dependent. In conclusion cat and bovine adrenal medulla stimulated with a variety of secretagogues synthesize NO from L-arginine to activate the soluble guanylate cyclase. The present data do not rule out a role for cyclic GMP in the regulation of catecholamine secretion; however, it seems more plausible that cyclic GMP may play a role in controlling local blood flow and thus the access of the released catecholamines to the systemic circulation during stressful conflicts.

Separation between cytosolic calcium and secretion in chromaffin cells superfused with calcium ramps.

This paper describes experiments in which cytosolic Ca2+ concentrations ([Ca2+]i) and catecholamine release were measured in two populations of chromaffin cells stimulated with a solution enriched in K+ (100 mM). Once depolarized, external Ca2+ or Ba2+ ions were offered to cells either as a single 2.5 mM step or as a ramp that linearly increased the concentration from 0 to 2.5 mM over a 10-min period. A clear separation between the changes of the [Ca2+]i and the time course of secretion was observed. Specifically, secretion and [Ca2+]i rose in parallel when a Ca2+ step was used to reach a peak in a few seconds; however, while secretion declined to the basal level, [Ca2+]i remained elevated at a plateau of 400 nM. With a Ca2+ ramp, only a transient small peak of secretion was observed, yet the [Ca2+]i remained elevated throughout the 10-min stimulation period. The separation between secretion and [Ca2+]i was observed even when voltage-dependent Ca2+ channels were expected to remain open (mild depolarization in the presence of 1 microM Bay K 8644). By using Ba2+ steps or ramps, sustained noninactivating secretory responses were obtained. The results suggest that the rate and extent of secretion are not a simple function of the [Ca2+]i at a given time; they are compatible with the following conclusions: (i) A steep extracellular-to-cytosolic Ca2+ gradient is required to produce a sharp increase in the [Ca2+]i at exocytotic sites capable of evoking a fast but transient secretory response. (ii) As a result of Cai(2+)-dependent inactivation of Ca2+ channels, those high [Ca2+]i are possible only at early times after cell depolarization. (iii) The Cai(2+)-dependent supply of storage granules to the secretory machinery cooperates with the supply of Ca2+ through Ca2+ channels to regulate the rate and extent of secretion.

Muscarinic receptors in separate populations of noradrenaline- and adrenaline-containing chromaffin cells.

We have performed binding experiments of (a)[3H]quinuclidinyl benzilate to partially purified membranes from noradrenaline- and adrenaline-containing chromaffin cells and (b) [3H]N-methyl-quinuclidinyl benzilate to acutely isolated, or 48-h cultured, chromaffin cells subpopulations. Using this approach, we obtained enough evidence to conclude (1st) that muscarinic receptors are present in both noradrenaline- and adrenaline containing cells; (2nd) that noradrenaline cells contain in fact 2-3 fold higher density of those receptors; and (3rd) that those receptors undergo plastic changes upon chronic culturing of the cells.

(+)-isradipine but not (-)-Bay-K-8644 exhibits voltage-dependent effects on cat adrenal catecholamine release.

1. Catecholamine release from cat adrenal glands perfused at a high rate (4 ml min-1) at 37 degrees C with polarizing (1.2 or 5.9 mM K+) or depolarizing (17.7, 35, 59 or 118 mM K+) solutions, was triggered by 5 or 10 s pulses of Ca2+ (0.5 or 2.5 mM) in the presence of various concentrations of K+. 2. In polarized glands, secretion was greater the higher the K+ concentration present during the secretory K+/Ca2+ test pulse. Thus, in 17.7 mM K+, catecholamine released was 162 +/- 27 ng per pulse, while in 118 mM K+ secretion rose to 1839 +/- 98 ng per pulse. In depolarized glands, secretion reached a peak of around 1000 ng per pulse in 35-59 mM K+; in 118 mM K+, secretion did not increase further, suggesting that voltage changes are implicated in the control of the secretory process. 3. Blockade of secretion by increased concentrations of (+)-isradipine was much more manifest in polarized glands. The higher the degree of depolarization was (35, 59 or 118 mM K+), the lower the IC50 s were. So, the ratios between the IC50 s in polarized and depolarized glands rose from 3.92 in 35 mM K+ to 26.7 in 118 mM K+. 4. In contrast, the Ca2+ channel activator (-)-Bay K 8644 potentiated catecholamine release evoked by K+/Ca2+ pulses equally well in polarized or depolarized glands. The ratios between EC50 s in polarized or depolarized glands were, respectively, 0.30, 0.59 and 0.69 for 17.7, 35 and 118 mM K+. 5. In simultaneous experiments, the two enantiomers of Bay K 8644 exhibited opposite effects on secretion. (+)-Bay K 8644 (a Ca21 channel blocker) inhibited secretion better in depolarized than in polarized glands, whilst (-)-Bay K 8644 potentiated secretion in a voltage-independent manner. 6. Potentiation of secretion by (-)-Bay K 8644 was concentration-dependent from 10-8 to 10-6M. At 10- 5M, such potentiation largely disappeared in both polarized and depolarized glands. However, this dual effect of (-)Bay K 8644 was better seen in depolarizing conditions, suggesting that using the same enantiomer, the voltage-dependence is only seen when blockade of secretion dominates. 7. In the presence of increasing concentrations of (-)Bay K 8644 (3 x 10-9, 3 x 10-8 and 3 x 10-7M), the concentration-response curves for (+)isradipine to inhibit secretion were displaced to the right. However, a Schild plot of (dose ratio - 1) against (-)-Bay K 8644 concentrations gave a slope of 0.6, suggesting that the interactions between (+)-isradipine and (-)Bay K 8644 were non-competitive in nature. The pA2 for (-)-Bay K 8644 was 9.13. 8. Overall, the results suggest that potentiation of secretion by (-)Bay K 8644 (a voltage-independent phenomenon), and blockade by (+)-isradipine or (+-Bay K 8644 (a voltage-dependent phenomenon) might be exerted through binding of the dihydropyridines activators and blockers to separate sites on chromaffin cell L-type Ca2 + channels.

Mechanism of blockade by (+)isradipine of adrenal catecholamine release.

Cat adrenal glands were perfused at a high rate with various modified Krebs solutions containing different concentrations of K+ but no Ca2+. Catecholamine release was tested by applying brief Ca2+ pulses (10 s of a solution containing 120 mM K+ and 2.5 mM Ca2+). Under polarizing conditions (10 min perfusion with 1.4 mM K+ with no Ca2+), the total catecholamines released by the Ca2+ pulse amounted to 5 micrograms; in depolarizing conditions (10 min perfusion with a solution containing 70 mM K+ but no Ca2+), secretion was somewhat less (4-4.5 micrograms). (+)Isradipine, a 1,4-dihydropyridine Ca2+ channel blocker, did not affect the secretory response under polarizing conditions at 10(-8) M; at 10(-6) M, the secretory response was halved. When present under depolarizing conditions (70 mM K+ in 0 Ca2+), (+)isradipine (10(-8) M) blocked catecholamine release by 90%. In contrast, the inorganic Ca2+ channel blocker, Co2+, inhibited secretion equally well under polarizing or depolarizing conditions. Since 45Ca2+ uptake into adrenal medullary chromaffin cells was also inhibited by (+)isradipine (10(-8) M) in a voltage-dependent manner, it seems likely that blocking effects of the drug on catecholamine release are associated with inhibition of Ca2+ entry into cells through L-type Ca2+ channels. The association of (+)isradipine to its receptor is very rapid under polarizing conditions; dissociation is very slow in depolarized cells and very rapid upon polarization of such cells. Since chromaffin cells are being depolarized during stressful situations to secrete catecholamines into the circulation, (+)isradipine is likely to bind better to dihydropyridine receptors in this state; in this manner, the ensuing blockade of adrenal secretion could serve as a protective mechanism of cardiovascular tissues against massive increases in circulating catecholamines. If this suggestion is correct this mechanism could have additional therapeutic value in the treatment of hypertensive patients with (+)isradipine.

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.