Pedro Sánchez-García

A dopaminergic receptor modulates catecholamine release from the cat adrenal gland.

Nicotine evokes the release of catecholamines from perfused cat adrenal glands in a concentration‐dependent manner, the median effective concentration for nicotine being 5 microM. Two 2 min pulses of 5 microM‐nicotine, 40 min apart (S1 and S2) gave net catecholamine outputs of 7.64 and 3.55 micrograms/8 min, respectively. The ratio S2/S1 in control glands was 0.5. Increasing concentrations of apomorphine (1‐10 microM) markedly inhibited catecholamine release during the second nicotine pulse (S2). At 1 microM‐apomorphine, the release during S2 was significantly reduced to 16% of S1; with 10 microM‐apomorphine, the secretory response was reduced further to only 3% of S1, the ratio S2/S1 being 0.03. The presence of haloperidol, sulpiride or picobenzide (each 0.5 microM) during S2, completely reversed the inhibition of catecholamine release produced by apomorphine. Haloperidol itself increased the nicotinic secretory response during S2; so, while the ratio S2/S1 was 0.5 in control conditions, this ratio increased significantly to 0.95 if haloperidol (0.5 microM) was present during S2, suggesting that the presence of this dopaminergic antagonist removed a negative feed‐back mechanism that inhibits nicotine‐evoked catecholamine release. If present during S2, dopamine (1 microM) also markedly inhibited catecholamine release evoked by nicotine; this inhibition was again reversed by 0.5 microM‐haloperidol. Neither the opiate antagonist naloxone nor the alpha‐adrenoceptor blocking agent phentolamine (at concentrations of 0.5‐5 microM) affected the inhibition by apomorphine of the secretory response to nicotine. These data strongly suggest that the cat adrenal medulla chromaffin cell membrane contains a dopaminergic receptor which modulates the catecholamine secretory process triggered by stimulation of the nicotinic cholinoceptor. The fact that dopamine is released in measurable amounts, together with adrenaline and noradrenaline, from perfused cat adrenal glands in response to nicotinic stimulation (V. Ceña, unpublished results), favours a role for this dopaminergic receptor in modulating catecholamine release from the chromaffin cell.

Endothelium-independent relaxation by 17-α-estradiol of pig coronary arteries

We have studied the effects of 17-α-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°C. From the initial K+-evoked contraction, 17-α-estradiol caused a relaxation with an IC50 (15 μM) which was in the range of the IC50s obtained for nitroglycerin (1.3 μM) and nicorandil (50 μM). Contractions evoked by Ca2+ were inhibited by 17-α-estradiol, but full blockade could not be achieved. Serotonin-evoked contractions were also blocked by 17-α-estradiol with an IC50 of 2.1 μM; 17-β-estradiol also inhibited the serotonin-evoked contractions. In the presence of 100 μM of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester, the relaxing properties of 17-α-estradiol in pig coronary arteries and rat thoracic aorta were unaffected, suggesting that endothelial NO release was unrelated to these effects. 17-α-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-α-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. These acute vasorelaxant effects can best be explained by blockade of voltage-dependent Ca2+ channels and the ensuing restriction of extracellular Ca2+ availability to the contractile machinery. This is in line with the recent hypothesis that estrogens behave as ‘endogenous Ca2+ channel antagonists’.

Effects of collagenase on the release of [3H]‐noradrenaline from bovine cultured adrenal chromaffin cells

1 Bovine isolated adrenal chromaffin cells maintained in culture at 37°C for 1–7 days become polygonal and bipolar, with typical varicosity‐like extensions. Catecholamine levels and dopamine β‐hydroxylase activity decreased after 24–48 h of culture, but recovered to normal levels 3–7 days later. 2 Incubation of 1–7 day‐old cells in the presence of increasing concentrations of [3H]‐noradrenaline (3.91 to 125 nm) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day‐old cells took up and retained only one third of the radioactivity found in 2–7 day‐old cells. 3 The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. 4 Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mm K+ was very poor in 24 h‐old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2–7 days of culture. Bethanecol did not cause any secretory response. 5 When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatmen. 6 The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor‐mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage‐sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.

Pharmacological protection against the cytotoxicity induced by 6-hydroxydopamine and H2O2 in chromaffin cells

We present in this report the characteristics of the damage induced by 6-hydroxydopamine and H2O2 on bovine chromaffin cells in primary culture. Cytotoxicity was quantified using catecholamine cell contents, lactate dehydrogenase (LDH) release, trypan blue exclusion and morphological appearance. An excellent correlation between these four parameters was found. The cytotoxic effects of 6-hydroxydopamine were Ca(2+)-independent. In spite of this, the Ca2+ channel antagonists R56865 (N-[1-(4-(fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2-benzo-thiazo lamine) lidoflazine exhibited marked cytoprotective effects against both 6-hydroxydopamine and H2O2. The selective dopamine uptake blocker, bupropion, increased the viability of 6-hydroxydopamine and H2O2-treated cells from 20% to around 80%. Catalase drastically protected against the cytotoxic effects of 6-hydroxydopamine and H2O2. In contrast, desferrioxamine gave better protection against H2O2 cytotoxicity; glutathione and N-acetylcysteine only afforded substantial protection against 6-hydroxydopamine. Three main conclusions emerge from this study. (1st) 6-Hydroxydopamine causes chromaffin cell damage via a mechanism probably related to the production of free radicals, but unrelated to Ca2+ ions. Cytoprotection afforded by R56865 and lidoflazine must be unrelated to their Ca2+ antagonist properties. This suggests a novel component in the cytoprotective mechanism of action of these drugs. (2nd) The strong cytoprotective effects of bupropion seem to be unrelated to its ability to block the plasmalemmal dopamine carrier. (3rd) Bovine adrenal chromaffin cells in primary cultures are a suitable model for adult neurons to study the basic mechanism of cell damage, and to screen new drugs with putative neuroprotective properties.

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.

Presence and axonal transport of cholinoceptor, but not adrenoceptor sites on a cat noradrenergic neurone

1. Noradrenaline release and radioligand binding studies were carried out in the cat hypogastric nerve ligated in vito 2 cm distal to the inferior mesenteric ganglion for different time periods, and in different effector organs.

CORRELATION BETWEEN CATECHOLAMINE RELEASE AND SODIUM PUMP INHIBITION IN THE PERFUSED ADRENAL GLAND OF THE CAT

1 Ca2+ re introduction to retrogradely perfused and ouabain (10−4M)‐treated cat adrenal glands caused a catecholamine secretory response which was greater the longer the time of exposure to the cardiac glycoside. Such a response was proportional to the external Na+ concentration [Na+]0. 2 A qualitatively similar, yet smaller response was observed when glands were perfused with Krebs solution lacking K+ ions; thus, K+ deprivation mimicked the secretory effects of ouabain. Catecholamine secretion evoked by Ca2+ reintroduction in K+‐free solution (0‐K+) was also proportional to [Na+]0 and greater the longer the time of exposure of the gland to 0‐K+ solution. 3 The ionophore X537A also mimicked the ouabain effects, since Ca2+ reintroduction to glands treated with this agent (25 μm) caused a sharp secretory response. When added together with X537A, ouabain (10−4m) did not modify the response to the ionophore. 4 N‐ethylmaleimide (NEM), another Na+, K+‐ATPase inhibitor, did not evoke the release of catecholamines; on the contrary, NEM (10−4m) inhibited the catecholamine secretory response to high [K+]0, acetylcholine, Ca2+ reintroduction and ouabain. 5 Ouabain (10−4 m) inhibited the uptake of 86Rb into adreno‐medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30–40 min) to reach its peak. NEM (10−4 m) blocked 86Rb uptake in a similar manner. 6 The results are further evidence in favour of the presence of a Na+‐Ca2+ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca2+]i, and in the modulation of catecholamine secretion. This system is activated by increasing [Na+]i, either directly (ionophore X537A, increased [Na+]0) or indirectly (Na+ pump inhibition). However, the simple inhibition of Na+ pumping does not always lead to a catecholamine secretory response; such is the case for NEM.

Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca2

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca2+ channel antagonists, blockers of histamine H1 receptors, antimycotics, beta-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca2+]i oscillations elicited by veratridine. After 24-h exposure to 1-30 microM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 microM, 80% lactate dehydrogenase release was produced. At 1-3 microM flunarizine caused no cell damage; however 10 microM caused 20% lactate dehydrogenase release and 30 and 100 microM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 microM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10-30 microM) and flunarizine (3-10 microM) provided protection against veratridine-induced cell death; however, at 30 microM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 microM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca2+]i. Both compounds abolished the K+-induced increases of [Ca2+]i as well as the oscillations of [Ca2+]i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Contribution of SK and BK channels in the control of catecholamine release by electrical stimulation of the cat adrenal gland.

1. Transmural electrical stimulation (10 Hz, 1 ms, 40 V for 10 s) of cat adrenal glands perfused at room temperature with Krebs‐Hepes solution produced catecholamine secretory responses which were reproducible when stimulations were applied at 5 min intervals. Such responses were inhibited about 20% by atropine (1 microM) and 80% by hexamethonium (30 microM). Apamin (100 nM) increased the secretory response 2.5‐fold in the presence of atropine and 8‐fold in the presence of hexamethonium. 2. Potentiation by apamin of secretory responses evoked by 100‐pulse trains was similar at 5, 10 and 20 Hz (about 2‐fold). When glands were continuously stimulated at 3 Hz, apamin increased 4‐fold the initial secretion plateau. Continuous stimulation at a higher frequency (20 Hz) produced a sharp secretory peak followed by a small, sustained plateau; apamin did not alter this plateau. Apamin also enhanced the secretory responses obtained with sustained stimulation with acetylcholine (10 or 200 microM). 3. Secretion peaks induced by brief acetylcholine pulses (10 microM for 10 s) applied to isolated and superfused cat adrenal chromaffin cells were enhanced more than 3‐fold by 100 nM apamin. Charybdotoxin (10 nM) did not enhance these secretory peaks. 4. In perfused cat adrenal glands, charybdotoxin (10 nM) affected neither the secretion evoked by trains of electrical stimulation applied at different frequencies nor the secretion evoked by acetylcholine pulses. 5. In 0.5 mM [Ca2+]o, apamin enhanced 3‐fold the secretion evoked by electrical stimulation trains of 100 pulses (10 Hz, 10 s) and almost 6‐fold the acetylcholine (10 microM for 10 s)‐induced secretion. In 5 mM Ca2+, apamin enhanced the secretory responses to electrical stimulation and acetylcholine 2‐ and 10‐fold, respectively. Charybdotoxin enhanced 2.5‐fold the secretory response to electrical stimulation in 0.5 mM Ca2+, although this effect was not statistically significant. A synergistic interaction between the two toxins on catecholamine release induced by electrical stimulation was observed at low but not at high [Ca2+]o. 6. Simultaneous release of acetylcholine and catecholamines upon electrical stimulation was achieved in glands in which the endogenous acetylcholine stores in the splanchnic nerve terminals had been prelabelled by perfusion with [3H]choline. While apamin enhanced more than 2‐fold the postsynaptic release of catecholamines, the presynaptic release of acetylcholine remained unaffected. 7. The results are compatible with the hypothesis that, under physiological conditions, Ca(2+)‐activated SK channels present in chromaffin cells control the firing patterns of action potentials induced by the acetylcholine released from splanchnic nerves during stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of the dihydropyridine Bay K 8644 on the release of [3H]-noradrenaline from the rat isolated vas deferens.

1 The effects of Bay K 8644 on the release of [3H]‐noradrenaline evoked by potassium, electrical stimulation or tyramine from the rat isolated vas deferens labelled with [3H]‐noradrenaline were investigated. 2 Bay K 8644 (1 μM) by itself did not affect the spontaneous release of tritium from the rat isolated vas deferens. However, it increased the calcium‐dependent release of tritium elicited by both high potassium (59 mM) and electrical field stimulation. 3 The exposure of rat vas deferens to phentolamine (10 μM) increased the release of tritium induced by potassium (59 mM) and electrical field stimulation. Bay K 8644 (1 μM) failed to increase further the release of tritium elicited by both stimuli in preparations previously treated with phentolamine (10 μM). 4 The calcium‐independent release of [3H]‐noradrenaline evoked by tyramine (10 μM) was not affected by Bay K 8644 (1 μM). 5 The results of our study support the view that α2‐adrenoceptors modulate noradrenaline release by restricting calcium influx into sympathetic nerve terminals through voltage‐dependent channels.