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The present study was performed to evaluate the Ca v 1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Ca v 1.2 and Ca v 1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Ca v 1.2 and Ca v 1.3 subtypes, but not Ca v 1.1 or Ca v 1.4. Electrophysiological experiments were conducted to investigate the contribution of Ca v 1 channels to the exocytotic process and cell excitability. Ca v 1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3 μM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200 ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (−34 mV), which elicits 10.4 mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Ca v 1 channels in exocytosis and cell excitability.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on adrenal medullary (AM) cells as a neurotransmitter of the sympathetic preganglionic nerve. In guinea-pig AM cells, PACAP induces little catecholamine secretion, but enhances secretion evoked by stimulants, whereas in other animals, such as mouse, PACAP itself induces depolarization and/or catecholamine secretion. The present studies aim to explore the physiological implication of these species differences in PACAP actions, the ion channel mechanism for PACAP-induced depolarization, and the mechanism for facilitation of muscarinic receptor-mediated cation currents in mouse and guinea-pig AM cells. The perforated patch clamp technique was used to record the whole-cell current in isolated AM cells. The amplitudes of 3 nM PACAP-induced inward currents were significantly larger in mouse AM cells than guinea-pig, whereas 1 μM muscarine-induced currents were larger in guinea-pig AM cells than mouse. Exposure to PACAP consistently resulted in enhancement of muscarine-induced currents in guinea-pig AM cells and facilitation of cell membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarine in PC12 cells. The PACAP-induced current was inhibited by 30 μM 9-phenanthrol, a specific TRPM4 channel inhibitor, and abolished by replacement of external Na+ with N-methyl D-glucamine. TRPM4-like immunoreactivity was located at the cell periphery in AM cells. The present results indicate that PACAP and muscarinic receptors are major metabotropic receptors mediating generation of depolarizing inward currents in mouse and guinea-pig AM cells, respectively. We conclude that PACAP activates TRPM4-like channels and enhance the muscarinic current through facilitating the membrane insertion of TRPC1-TRPC4 channels in AM cells.

In the present study, the effect of chromaffin cell transplant in the spinal cord was evaluated on formalin-induced mechanical secondary allodynia in the rat. Chromaffin cells were transplanted into the lumbar subarachnoid space before or after formalin injection. Subcutaneous formalin injection (50 μl, 1%) produced long-lasting secondary allodynia in the ipsilateral and contralateral hind paws. Once secondary allodynia was established, treatment with chromaffin cells produced a significant reduction in the nociceptive behavior in both hind paws. The antiallodynic effect was time-dependent since it was observed 15 days after chromaffin cell transplants but not before. On the other hand, pre-treatment with chromaffin cells prevented the expression of secondary allodynia in both hind paws in the rat. Antiallodynic effect of chromaffin cells was reverted with the non-selective opioid receptor antagonist naltrexone and the non-selective α 2 -adrenoceptor antagonist rauwolscine. Clusters of viable chromaffin cells labeled with anti-tyrosine hydroxylase antibodies were observed in the retrieved transplants 15 days after transplant. These results establish the analgesic efficacy of intrathecal chromaffin cells on formalin-induced secondary allodynia. Our data suggest that chromaffin cells release neuroactive substances including opioid peptides and adrenergic amines that reduce secondary allodynia in rats through activation of their receptors.

Tobacco smokers have an increased risk of cardiovascular disease; this is likely associated to an enhanced catecholamine release by circulating nicotine. Here, we have explored how low concentrations of nicotine in the range of those found in the blood of tobacco smokers, might affect the release of catecholamines in bovine chromaffin cells. We have combined patch-clamp and Ca2+ imaging techniques to study cell excitability, cytosolic Ca2+ transients, vesicle movement, and secretory responses. We found that low concentrations of nicotine (1.5-3 οM) did not enhance catecholamine release by themselves. However, they drastically augmented the catecholamine release response triggered by a supramaximal K+ depolarising pulse. Furthermore, low nicotine concentrations caused slight depolarisation with superimposed action potentials, a transient elevation of [Ca2+]c and augmented Ca2+-dependent vesicle motion underneath the plasmalemma. We suggest that low nicotine concentrations overload the secretory machinery with secretory vesicles, which cause chromaffin cells to respond with an exaggerated adrenaline release into the circulation during stress. This might contribute to the higher cardiovascular risk of tobacco smokers. Copyright 2008 Elsevier B.V. All rights reserved., This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2006-03589 to AGG; and SAF2007-65181 to LG); Ministerio de Salud (Instituto de Salud Carlos III RETICS-RD06/0026); Comunidad Autónoma de Madrid (S-SAL-0275-2006), and Fundación Mutua Madrileña (to AGG and to LG).

Cardiac glycosides such as ouabain augment the release of neurotransmitters and hormones from various organs, tissues and cell systems. Here we have investigated novel aspects of the ouabain effects on fast-perifused bovine adrenal chromaffin cells subjected to repetitive stimulation during long-time periods with secretagogues that enhance Ca(2+) entry (i.e. 100 mM K(+)solution) or causing the release into the cytosol of the Ca(2+) stored in the endoplasmic reticulum (i.e. 20 mM caffeine or 100 microM histamine). After 1 h of intermittent stimulation, the amperometrically measured catecholamine release responses decayed to 50% with K(+) pulses, and to 10-15% with caffeine or histamine pulses. Ouabain (10 microM) augmented 2-fold the K(+) secretory responses and kept them high, along an hour. When given after the responses had decayed upon repetitive caffeine or histamine pulsing, ouabain gradually restored such responses to their initial control values. On the basis of these results, we raise the hypothesis that ouabain may facilitate the handling by the cell of the endoplasmic reticulum Ca(2+) fluxes that are known to be involved in secretory vesicle transport and the regulation of exocytosis. This may have physiological and pathological interest in the light of an endogenous ouabain steroid found in the adrenal cortex.

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.

Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC(50): 27 microM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na(+) (IC(50): 12 microM) and Ca(2+) (IC(50): 20 microM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca(2+) influx stimulated by high K(+). Binding of [3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na(+) concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na(+) influx through the channels and, consequently, attenuates both Ca(2+) influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.

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.

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 [mu]M) inhibited the early and late phases of the ionomycin (2 [mu]M)-evoked [Ca2+]i transients by 14-23%. [omega]-Agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50=50 nM). In contrast, 0.1 [mu]M [omega]-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (

( R )- N 2 -(diphenacetyl)- N -[(4-hydroxyphenyl)methyl]-argininamide (BIBP 3226) is a selective neuropeptide Y Y 1 receptor antagonist with structural similarity to the C-terminal tripeptide of neuropeptide Y. Based on this similarity we questioned whether BIBP 3226 could act as an agonist. Incubation of BIBP 3226 with bovine chromaffin cells in culture results in the inhibition of nicotinic receptor-stimulated catecholamine secretion (IC 50 =2.4 μM). The effect of BIBP 3226 is independent of neuropeptide Y action since the presence of neuropeptide Y in the culture medium does not alter the effect of BIBP 3226. BIBP 3226 decreased the efficacy of the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP), but did not change its potency suggesting non-competitive inhibition. BIBP 3226 has a similar effect on nicotinic receptor-stimulated 45 Ca 2+ influx. BIBP 3226 does not inhibit [ 3 H ]norepinephrine release induced by high K + and its effect is not pertussis toxin-sensitive. We conclude that not only can BIBP 3226 act as a neuropeptide Y receptor antagonist in bovine chromaffin cells but also act as an agonist and inhibit catecholamine secretion.

To investigate the relationship between the inhibitory effects of ginseng saponins (ginsenosides) on acetylcholine-evoked secretion of catecholamines and the structures of ginsenosides, we examined the effects of ginsenoside-Rg3 and -Rh2, which are panaxadiol saponins, 20(R)- and 20(S)-ginsenoside-Rg2, which are epimers involving the hydroxyl group at C-20 of sapogenin, and other plant saponins on the acetylcholine-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. The ginsenoside-Rg3 (1-100 microM) and -Rh2 (10-100 microM) greatly reduced the acetylcholine-evoked secretion in a concentration-dependent manner comparable to that of ginsenoside-Rg2, a panaxatriol saponin, which was the most potent inhibitor in our previous study. 20(R)- and 20(S)-ginsenoside-Rg2 (1-100 microM) similarly reduced the acetylcholine-evoked secretion. In contrast, saikosaponin-a, glycyrrhizin and the cardiac glycosides (100 nM-100 microM), digitoxin and digoxin, had no significant inhibitory effect on catecholamine secretion. Saikosaponin-c (10-100 microM), however, had an inhibitory effect, which was less than that of ginsenoside-Rg2 and -Rg3. These results strongly suggest that the inhibitory effects of ginsenosides on the acetylcholine-evoked secretion of catecholamines from bovine adrenal chromaffin cells are a unique property of ginseng. Further, the relationship between the inhibitory effects and the structures of ginsenosides is discussed.

The diterpene forskolin inhibits nicotine-evoked chromaffin cell Ca2+ influx, scinderin redistribution, F-actin disassembly and catecholamine secretion in a concentration-dependent (10–50 μM) fashion. On the other hand, forskolin showed weak inhibitory effects when the same responses were elicited by K+-induced depolarization. Similar concentrations of 1,9-dideoxy-forskolin, a forskolin analog which does not activate adenylate cyclase, blocked very effectively the responses evoked by either of the two stimuli. Patch-clamp (whole-cell configuration) studies demonstrated that both diterpenes blocked fast and reversibly peak and total chromaffin cell nicotinic acetylcholine receptor currents, effects not mediated through adenylate cyclase activation. Moreover, both forskolin and 1,9-dideoxy-for-skolin exhibited Ca2+ channel blocking properties. However, 1,9-dideoxy-forskolin was more potent than forskolin as a Ca2+ channel blocker. Furthermore, 1,9-dideoxy-forskolin was also more potent than forskolin as a nicotinic acetylcholine receptor and Ca2+ channel blocker and it was more potent as a nicotinic acetylcholine receptor blocker than Ca2+ channel blocker. The results showed powerful cAMP-independent effects of the diterpenes and suggest caution in interpretation of cAMP effects on chromaffin cells when its cellular levels are modified by forskolin.

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% (P0.001). 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.

Tacrine enhanced acetylcholine-induced catecholamine secretion with a concentration of up to 10 μM, but inhibited it at over 10 μM in perfused adrenal glands. Qualitatively the same result was obtaiend withphysostigmine. Both tacrine and physostigmine only inhibited the secretory responses to carbachol and/or nicotine in perfused glands and dispersed chromaffin cells. Acetylcholinesterase activity of adrenal homogenates was inhibited by tacrine and physostigmine in a concentration-dependent manner. In whole-cell patch-clamp experiments, tacrine and physostigmine caused reversible inhibition of nicotine-evoked inward currents with a dose range similar to that for the inhibitory action on the secretory response. These results suggest that the enhancing effect of tacrine and physostigmine on acetylcholine-induced catecholamine secretion results from the prevention of enzymatic hydrolysis of acetylcholine in adrenal glands and that the inhibitory effect is due to the inhibition of nicotinic receptor-mediated membrane currents in adrenal chromaffin cells.

The effects of ouabain, Li + and veratridine on the concentration of cytosolic free Ca 2+ ([Ca 2+ ] i ) were studied in single fura-2-loaded bovine adrenal chromaffin cells. Superfusion of cells with ouabain (10 μM for 60 min) caused only a delayed mild increase of the [Ca 2+ ] i , from around 0.1 μM to 0.2–0.3 μM; this increase was Na + o -dependent. Replacement of all NaCl of the Krebs-Hepes solution by LiCl (144 mM) produced a gradual increase of [Ca 2+ ] i , which remained elevated at a stable plateau of 0.4–0.5 μM for 40–50 min. When ouabain (in the presence of normal Na + o ) or Li + (in the absence of Na + o ) was given in Krebs-Hepes solution containing no Ca 2+ , the reintroduction of 2.5 mM Ca 2+ produced a fast elevation of the [Ca 2+ ] i . In the case of ouabain-treated cells, the [Ca 2+ ] i curve exhibited an initial phasic component which inactivated to a tonic component. ω-Conotoxin MVIIC (3 μM) and R56865 (10 μM) inhibited the phasic but not the tonic component. Veratridine (30 μM) induced large [Ca 2+ ] i oscillations. Both ouabain or Li + abolished such oscillations. These results are compatible with ouabain causing elevation of [Ca 2+ ] i in bovine chromaffin cells through a dual mechanism, i.e. cell depolarisation and slowing down of the Na + -Ca 2+ exchanger of their plasmalemma. Through its binding to the Na + site on the Na + -Ca 2+ exchanger, Li + ions generate powerful Ca 2+ i signals that might be relevant to its known effects on neurosecretory mechanisms.

Bovine chromaffin cells were separated by density gradient centrifugation into subfractions. After centrifugation on a self-generating Percoll gradient (42.75% isotonic Percoll, 30,000 x g for 22 min at 20 degrees C), the chromaffin cells were found in two clearly distinguishable peaks. The peak with the lower density contained most of the noradrenaline-producing cells (approximately 80%), whereas the adrenaline-producing cells were equally distributed between the two peaks. After collection of suitable fractions from the gradient, cell cultures were obtained, which were enriched with either90% adrenaline- or approximately 65% noradrenaline-producing cells. When stimulated by nicotine or carbachol, the dose-response curves of both cell fractions yielded similar EC50s for the release of adrenaline and noradrenaline. On the other hand, the cells of the less dense fraction released 30% more catecholamines (adrenaline as well as noradrenaline) than the cells of the more dense fraction. It is suggested that there are subpopulations among the adrenaline- and noradrenaline-producing cells with differences in receptor-effector coupling.

Long-term pretreatment (12–120 h) of cultured bovine adrenal chromaffin cells with cis-diamminedichloroplatinum (cisplatin, Pt(NH 3 ) 2 Cl 2 ) (33 μM), an antineoplastic drug, resulted in a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine. Acetylcholine-induced 45 Ca 2+ influx into the cells was also reduced in the cells pretreated with cisplatin for 48 h. The concentration-response curves (3–66 μM) for cisplatin inhibition of the secretion and 45 Ca 2+ influx were quite similar. Pretreatment of cells with 33 μM Pt 4+ or carboplatin, an analog of cisplatin, for 48 h also led to a decrease in acetylcholine-evoked secretion, but not with 33 μM Pt 2+ or other metals (Au + , Au 3+ , Ni 2+ , Os 3+ , Pd 2+ , Ir 3+ , and Ir 4+ ) that have properties similar to Pt 4+ . These results strongly suggest that in bovine adrenal chromaffin cells, cisplatin (3–66 μM) inhibits catecholamine secretion by the suppression of the Ca 2+ influx into the cells evoked by acetylcholine and that the inhibitory effect of cisplatin is attributable to the tetravalent platinum ion in its molecule.

Prolonged stimulation with nicotine (50 μM) enhanced the secretion of catecholamines from perfused cat adrenal glands. The profile of secretion consisted of a quick activation phase to peak of 7.68 μg/min followed by a second inactivation phase which exhibited a t 1 2 of 3.75 min. Sustained stimulation with a solution enriched in K+ (59 mM) also evoked a transient secretory response, with a peak release of 8.62 μg/2 min and a t 1 2 for inactivation of 4.8 min. Co2+ (10 mM) blocked the nicotinic response by 58% and the K+-evoked secretory response by over 96%. In the presence of Co2+; (5 mM), continuous perfusion with nicotine produced a transient but large initial secretory response; the gradual decrease of the extracellular Co2+ concentration, [Co2+]0, as a continuous ramp allowed the development of a second component of secretion which inactivated later on. When the glands were continuously stimulated with 59 mM K+ in the presence of Co2+, the first component of secretion was missing: the second component appeared as [Co2+]0 decreases as a ramp. In similar experiments performed in low-Na+ solution (10 mM Na+), only the first secretion component evoked by nicotine was observed. This finding suggests that the second component of secretion depends on Na+ entry through the nicotinic receptor, on the ensuing cell depolarization and on Ca2+ entry through voltage-dependent Ca2+ channels. These data are compatible with the idea that the nicotinic receptor-mediated catecholamine secretory response in the cat adrenal chromaffin cell has two components: the first seem to be triggered by Ca2+ entry through the nicotinic receptor channels, and the second by Ca2+ entry through voltage-dependent Ca2+ channels. The data also suggest that Co2+ ions protect against the Ca2+-dependent desensitization of nicotinic receptors.

How flunarizine, a class IV Ca2+ antagonist, affects the secretion of catecholamines in response to nicotinic receptor activation (10-s pulses with 100 microM dimethylphenylpiperazinium, DMPP) or direct depolarization of chromaffin cells (10-s pulses with 100 mM K+ and 2.5 mM Ca2+, 100 K+/2.5 Ca2+) was studied in bovine adrenal glands perfused with an oxygenated Krebs-Tris solution at 37 degrees C at a rate of 20 ml/min. Experimental protocols aimed to test voltage and time dependence of the flunarizine blocking effects on secretion are described. The DMPP pulses released an average of 217 micrograms catecholamines and the K+ pulses, an average of 117 micrograms. These responses were blocked by flunarizine concentration dependently; IC50s were 3.7 microM for DMPP and 1.1 microM for K+. Under polarizing conditions (60-s perfusion with a solution containing 5.9 mM K+ and nominally zero Ca2+), a 10-s pulse with 100 K+/2.5 Ca2+ released 117 +/- 26 micrograms of catecholamines (n = 12). Under depolarizing conditions (60-s perfusion with 118 K+/0 Ca2+ prior to the Ca2+ pulse), the pulse with 118 K+/2.5 Ca2+ released 307 +/- 36 micrograms of catecholamines (n = 14). Flunarizine blocked these secretory responses equally and concentration dependently with an IC50 of 3.4 microM under polarizing conditions and of 3.8 microM under depolarizing conditions. Thus, blockade by flunarizine of secretion was apparently not voltage-dependent. The blockade was, however, clearly dependent on the time of exposure of the adrenal medullary tissue to flunarizine.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used the whole cell patch clamp technique to investigate the action of pentobarbitone on the nicotinic channels of bovine chromaffin cells. Application of agonists induced an inward current associated with a large increase in current noise. The noise could be fitted by Lorentzian functions with time constants of 17 +/- 2 ms for 10 microM acetylcholine and 10 +/- 1 ms for 10 microM carbachol. The single channel conductance estimated from the current variance was about 25 pS in each case. Pentobarbitone decreased the time constants in a concentration-dependent fashion, but the unit conductances were unaffected. Single channel events were recorded in chromaffin cells held under voltage clamp. Pentobarbitone did not reduce the amplitude of channel openings or the probability of channel opening but reduced the mean channel open time. This reduction was sufficient to account for the decrease in inward current produced by pentobarbitone.

Marine sponges are becoming a rich source of potential new medicines. NP04634 is a synthetic derivative of 11,19 dideoxyfistularin, a natural product of the Mediterranean sponge Aplysina cavernicola. We report the cytoprotective effects of this new compound in isolated bovine chromaffin cells exposed to cytotoxic stimuli that have been related to neuronal cell death, i.e. Ca(2+) overload and mitochondrial dysfunction. Cell death was achieved by: (i) causing Ca(2+) overload through voltage-dependent calcium channels by exposing the cells to 30 mM K(+), 5 mM Ca(2+) plus 0.3 microM FPL64176 (an L-type Ca(2+)-channel activator); (ii) incubating the cells with veratridine, causing cytosolic Ca(2+) concentration ([Ca(2+)](c)) oscillations and mitochondrial disruption; and (iii) blocking mitochondrial complexes I and V using a combination of 30 microM rotenone and 10 microM oligomycin. At 10 microM, NP04634 caused significant protection against 30K(+)/5Ca(2+)/FPL-induced toxicity. NP04634 caused a concentration-dependent reduction in [Ca(2+)](c) induced by 70 mM K(+) in cells loaded with Fluo-4; maximum blockade was 67% at 30 microM. Veratridine caused continuous [Ca(2+)](c) oscillations that translated into 43.4+/-2% cell death. In this model, NP04634 caused 42% and 67% protection at 3 and 10 microM, respectively. NP04634 reduced [Ca(2+)](c) oscillations and mitochondrial depolarization caused by veratridine. NP04634 at 10 microM also protected against mitochondrial disruption caused by rotenone plus oligomycin. In conclusion, NP04634 is a novel compound of marine origin with cytoprotective properties that might have potential therapeutic implications under pathological circumstances involving Ca(2+) overload and mitochondrial disruption, such as in certain neurodegenerative diseases and/or stroke.

Transplants of adrenal medullary tissue or isolated chromaffin cells into the spinal cord subarachnoid space has been shown to reduce pain sensitivity in rats. This analgesia probably results from the release of neuroactive substances, particularly opioid peptides, from the transplanted cells since it is induced by nicotinic stimulation of chromaffin cell receptors, and can be blocked by naloxone. However, this analgesia is short-lived, most likely due to the rapid hydrolysis of opioid peptides. The purpose of this study was to determine whether protection of opioid peptide hydrolysis by the potent enkephalinase inhibitor kelatorphan could prolong this analgesia. Results indicated that the intrathecal injection of kelatorphan in animals with either adrenal medullary or chromaffin cell implants significantly prolonged nicotine-stimulated analgesia. Pretreatment with naloxone completely eliminated this analgesia. These results suggest that it may be possible to induce long-term reductions in pain sensitivity using enkephalinase inhibitors following the transplantation of opioid peptide-producing cells into the CNS.

In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.

The actions of maitotoxin were studied using cultured brainstem cells and adrenal chromaffin cells. Maitotoxin induced a profound increase in the Ca2+ influx into cultured brainstem cells after a brief lag period. The maitotoxin-induced Ca2+ influx was suppressed by various voltage-dependent Ca2+ channel blockers such as Co2+, Mn2+, verapamil and diltiazem. Maitotoxin-catecholamine release in brainstem cells initiated to increase after a lag period of about 1 min and the increase continued even at 4 min after treatment, while in the adrenal chromaffin cells the release started after an about 1-min lag period to attain a maximum within first 2-min and gradually decrease thereafter. These results suggest that maitotoxin acts on Ca2+ channels to increase the Ca2+ influx, accompanied by enhancement of catecholamine release in the brainstem cells with a different temporal profile from that in the adrenal chromaffin cells.

Nanomolar concentrations of atropine have been considered up to now to be selective for blockade of muscarinic receptors for acetylcholine. A collateral finding indicated to us that these low concentrations of atropine could also target the neuronal nicotinic receptors. We report here a detailed study on this novel property of atropine. Catecholamine release, measured on-line with amperometry in chromaffin cells stimulated with acetylcholine pulses was blocked by atropine in a competitive manner. To corroborate a direct action of atropine on nicotinic receptors, we have employed N,N-dimethyl-N'-phenyl-piperazinium (DMPP), a pure nicotinic receptor agonist; atropine blocked its secretory responses with an IC50 of 2.04 nM. Nicotinic currents, recorded with the whole cell configuration of the patch-clamp technique were blocked by atropine in a concentration-dependent manner (IC50 of 11 nM), also showing a competitive nature. Nicotinic receptor currents in oocytes expressing bovine alpha7 and alpha3beta4 nicotinic receptors were blocked by atropine with an IC50 of 11.2 and 46.8 nM, respectively. Atropine (30 nM) also decreased the increment of the cytosolic calcium concentrations after stimulation with 30 microM DMPP in bovine chromaffin cells. However, action potentials evoked by DMPP were not modified by atropine. Our results demonstrate that nicotinic currents and their downstream consequences (i.e. cytosolic calcium elevations and catecholamine release) were blocked by nanomolar concentrations of atropine; although the blockade was partial, it must be considered when using atropine to study cholinergic neurotransmission, particularly at synapses where both nicotinic and muscarinic receptors are present i.e., the adrenal medulla and autonomic ganglia.

Amphetamine, a psychostimulant, has been shown to act as a channel blocker of muscle nicotinic receptors and to induce a Ca 2+ -dependent secretion from adrenal chromaffin cells. In this study, the relationship between amphetamine and nicotinic receptors was studied using bovine adrenal chromaffin cells as a model system. Our results show that d -amphetamine sulfate alone induced an increase in the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) and [ 3 H]norepinephrine release in a dose-dependent and extracellular Ca 2+ -dependent manner. Two common nicotinic receptor antagonists, hexamethonium and mecamylamine, suppressed the d -amphetamine sulfate-induced [Ca 2+ ] c rise and [ 3 H]norepinephrine release. In addition, d -amphetamine sulfate inhibited the 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP)-induced [Ca 2+ ] c rise and [ 3 H]norepinephrine release, but not the high K + - or veratridine-induced [Ca 2+ ] c increase and [ 3 H]norepinephrine release. Antagonists, including α-bungarotoxin and choline, that are more specific for α7 nicotinic receptors were capable of inhibiting the d -amphetamine sulfate-induced [Ca 2+ ] c rise, while d -amphetamine sulfate was found to be capable of inhibiting the [Ca 2+ ] c rise induced by the α7-nicotinic receptor agonists, epibatidine and choline. Moreover, d -amphetamine sulfate dose-dependently suppressed [ 3 H]nicotine binding to chromaffin cells. We, therefore, conclude that d -amphetamine sulfate acts as a nicotinic receptor agonist to induce [Ca 2+ ] c increase and [ 3 H]norepinephrine release in bovine adrenal chromaffin cells.

Isolated adrenal chromaffin cells were used as a model for the release of catecholamines from adrenergic nerve endings. In this study we used an HPLC technique to determine the effects of acetylcholine and glycine on norepinephrine and epinephrine release. The amount of catecholamine released in response to glycine was 22% less than that released by acetylcholine. However, while the norepinephrine-to-epinephrine ratio was 1.6 after stimulation with acetylcholine, it was 0.6 after stimulation with glycine. This suggests that glycine preferentially affects epinephrine secretion as compared to acetylcholine, which preferentially releases norepinephrine. This differential effect could be of physiological importance considering our recent demonstration of a functional high-affinity chloride-gated glycine receptor on adrenal chromaffin cells.

The role of Ca(2+) influx in activating phospholipase C in bovine adrenal chromaffin cells has been investigated. Phospholipase C activity in response to K(+) depolarization (56 mM) was blocked by the L-type Ca(2+) channel antagonist nifedipine and partially inhibited by the omega-conotoxins GVIA and MVIIC. In contrast, phospholipase C activity in response to histamine receptor activation was unaffected by omega-conotoxin GVIA and partially inhibited by omega-conotoxin MVIIC or nifedipine. This response was however markedly inhibited by the non-selective Ca(2+) channel antagonists La(3+) or 1-[beta-[3-(4-Methoxyphenyl)propoxy]-4-methoyphenethyl]-H-imidazol e (SKF-96365). Despite this Ca(2+) dependence phospholipase C activity was not increased during periods of "capacitative" Ca(2+) inflow generated by histamine-, caffeine- or thapsigargin-mediated depletion of internal Ca(2+) stores. Thus, while Ca(2+) influx in response to K(+) depolarization or G-protein receptor activation can increase phospholipase C activity in these cells, in the latter case it appears to be ineffective unless there is concurrent agonist occupation of the receptor.

Two alpha-conotoxins PnIA and PnIB (previously reported as being "mollusc specific") which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 microM, with IC(50) = 0.7 microM, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC(50) = 2.0 microM) completely inhibits catecholamine release at 10 microM whereas [N11S]PnIA shows little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB.

The novel K + channel blocker 6,10-diaza-3(1,3)8,(1,4)-dibenzena-1,5(1,4)-diquinolinacyclodecaphane (UCL 1684) has been tested for its ability to inhibit Ca 2+ -activated K + currents in cultured rat chromaffin cells. Low nanomolar concentrations of UCL 1684 produced a rapid and reversible inhibition of the slow, apamin-sensitive, tail current activated by a depolarizing voltage command. This compound also inhibited the muscarine activated outward current with an IC 50 of 6 nM. These results confirm UCL 1684 to be the most potent non-peptidic blocker of the apamin-sensitive Ca 2+ -activated K + channel so far described.

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.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

The effects of cytosolic ATP on Ca 2+ -dependent K + (K Ca ) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated K Ca channels in a dose-dependent manner at 30 μ M to 10 mM. The K Ca channels also were activated by 3 mM of adenosine 5′- O -(3′-thiotriphosphate) (ATP γ S), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μ M to 3 mM). Furthermore, other nucleotides also activated K Ca channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg 2+ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 ( N -[2-( p -bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated K Ca channel activation. Following complete removal of Mg 2+ , a higher concentration of ATP (10 mM) and other nucleotides was required to activate K Ca channels; however, Mg 2+ was ineffective in altering the activation of K Ca channels by itself. It is concluded that intracellular ATP and other nucleotides activate K Ca channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.

Bovine chromaffin cells were separated by density gradient centrifugation into subfractions enriched with either >90% adrenaline- or 70–80% noradrenaline-producing cells. Concentrations of epibatidine (an alkaloid with nicotinic receptor activity) as low as 10 nM released adrenaline and noradrenaline from both fractions of cells maintained as monolayer cultures. The maximal effect was evoked by 30 nM epibatidine and was comparable to that evoked by 10 μM nicotine. The catecholamine release from the noradrenaline fraction was 30–40% higher than from the adrenaline fraction. Initial exposure to 50 nM epibatidine reduced release induced by a second exposure to the drug. There was cross-desensitization between epibatidine and nicotine. Substance P inhibited the epibatidine-evoked catecholamine release from both fractions by up to 85% (IC 50 =3–5 μM). The release of noradrenaline was inhibited more than that of adrenaline. In addition, substance P protected the chromaffin cells against desensitization of the nicotinic receptor by epibatidine. The C-terminal heptapeptide sequence of substance P was 10× less active, two N-terminal sequences did not modulate the catecholamine release.

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.

The effects of ibogaine, a putative anti-addictive drug, on stimulated catecholamine release were examined in cultured chromaffin cells to clarify its mechanism(s) of action. Low concentrations of ibogaine (1-10 microM) had a selective inhibitory action on nicotinic receptor-mediated catecholamine release, while higher concentrations (100 microM) inhibited additional modes of stimulated catecholamine release. These results suggest a selective inhibitory action of ibogaine at the nicotinic acetylcholine receptor, possibly at the receptor ion channel site.

The effects of the protein kinase C inhibitor CGP 41251 (31-benzoyl-staurosporine) on nicotinic responses of cultured bovine adrenal chromaffin cells have been investigated. CGP 41251 inhibited tyrosine hydroxylase activation by phorbol 12,13-dibutyrate, with an IC50 of0.3 microM and complete inhibition at 1 microM. In contrast, it had little effect on nicotine-stimulated tyrosine hydroxylase activity up to 1 microM, and did not fully inhibit it even at 10 microM. From 1 to 10 microM, CGP 41251 caused a similar concentration-dependent inhibition of tyrosine hydroxylase activity stimulated by nicotine, K+, forskolin and 8-Br-cyclic AMP. CGP 42700 (19,31-dibenzoyl-staurosporine), a structural analogue of CGP 41251 that lacks activity as a protein kinase C inhibitor, had no effect on tyrosine hydroxylase activity stimulated by any of the agonists. CGP 41251 had no effect on catecholamine secretion induced by nicotine. The results suggest phorbol ester-sensitive protein kinase C isozymes do not play a major role in nicotinic stimulation of tyrosine hydroxylase activity or catecholamine secretion in chromaffin cells.