Dihydropyridine Calcium Channel Agonist BAY Research Articles

Hyperosmotic regulation of voltage-gated calcium currents in rat anterior pituitary cells.

1. The sensitivity of voltage-gated calcium currents to hyperosmotic media containing mannitol or sucrose (373-723 mOsm) and to the dihydropyridine (DHP) calcium channel agonist Bay K 8644 was examined in enriched populations of rat anterior pituitary somatotrophs by using the whole cell mode of the patch-clamp technique. 2. Hyperosmotic media reduced the amplitude of voltage-gated calcium currents. With a 61.9% increase in extracellular medium osmolarity (523 mOsm), low voltage-activated (LVA) calcium currents were reduced to 67.9 +/- 17.8% of control size and high voltage-activated (HVA) calcium currents were reduced to 57.0 +/- 5.7% (mean +/- SD) of control size. The hyperosmotic suppression of HVA calcium currents was usually accompanied with a negative shift of 6.0 +/- 2.9 mV (mean +/- SD) in the activation curve of HVA currents. 3. The DHP calcium-channel agonist Bay K 8644 (10 microM), which stimulates hormone secretion from somatotrophs, increased the amplitude of HVA calcium currents to 212.6 +/- 67.2% of their control size, prolonged their tail currents, and negatively shifted the activation curve of HVA calcium currents by 6.2 +/- 2.8 mV. 4. Hyperosmotic media reduced the amplitude of DHP-sensitive HVA calcium currents and their associated prolonged tail currents, thus providing direct evidence for hyperosmotic suppression of DHP-sensitive currents. 5. Hence, exposure of pituitary cells to hyperosmotic media reduced voltage-sensitive calcium influx through LVA and DHP-sensitive HVA calcium channels. The inhibition of calcium influx through DHP-sensitive channels, which are implicated in regulation of hormone secretion in these cells, suggests that inhibitory hyperosmotic effects on hormone secretion from pituitary cells may stem from inhibition of calcium influx, before the exocytotic process. These results may also be relevant to effects of hypertonicity on neurosecretion in the nervous system.

Anticonvulsant properties of calcium channel blockers in mice: N-methyl-D-,L-aspartate- and Bay K 8644-induced convulsions are potently blocked by the dihydropyridines.

Ten calcium channel blockers were evaluated in mice after intraperitoneal (i.p.) administration for prevention of seizures induced by various convulsants. The dihydropyridines (class II calcium antagonists, i.e., nisoldipine, nitrendipine, nicardipine, nifedipine, and nimodipine) selectively prevented seizures elicited by administration of pentylenetetrazol (PTZ), N-methyl-D,L-aspartate (NMDLA) and the dihydropyridine calcium channel agonist BAY K 8644. With regard to prevention of NMDLA-induced seizures and the subsequent mortality, these compounds were similar in potency to the noncompetitive NMDA receptor antagonist MK801. Unlike MK801 (IC50 = 0.014 microM), the dihydropyridines did not inhibit in vitro binding of MK801 to synaptic membrane fractions prepared from rat cerebrohippocampal tissue. The dihydropyridines did not influence seizures elicited by maximal electroshock (MES). Flunarizine (diphenyl-alkylamine, class IV) was selectively active in the MES test, considerably less potent against NMDLA-induced convulsions/mortality, exhibited weak noncompetitive NMDA antagonism in vitro (IC50 = 28 microM), and was inactive in the PTZ and BAY K 8644 testing paradigms. Diltiazem, a class III benzothiazepine, possessed relatively weak broad spectra of activity against MES, PTZ, NMDLA, and BAY K 8644 test situations. It was inactive in vitro as a noncompetitive NMDA antagonist. The class I compound verapamil (phenylalkylamine) displayed only moderate inhibition of NMDLA-evoked seizures/mortality. Prenylamine (class V) was moderately active against convulsions produced by MES and NMDLA while retaining a degree (IC50 = 16 microM) of noncompetitive NMDA antagonism. Lidoflazine (class VI) was inactive in all tests. The Ca2+ channel blockers and MK801 were inconsistent in their ability to prevent bicuculline (BIC)-elicited convulsions.(ABSTRACT TRUNCATED AT 250 WORDS)

Single calcium channels in resistance-sized cerebral arteries from rats.

Unitary currents through single calcium channels were measured from cell-attached patches on smooth muscle cells isolated from resistance-sized branches of posterior cerebral arteries from Wistar-Kyoto normotensive rats. Barium (80 and 10 mM) was used as the charge carrier, with and without the dihydropyridine calcium channel agonist BAY R 5417. Unitary currents decreased on membrane depolarization, with a slope conductance of 19.4 pS (80 mM barium). Channel open-state probability (Po) was steeply voltage dependent. Peak Po during test pulses from -70 mV increased e-fold per 4.5-mV depolarization. Mean peak Po at potentials positive to +10 mV was 0.44. Po at steady membrane potentials was also steeply voltage dependent, changing e-fold per 4.5 mV in the absence of inactivation. Steady-state Po at positive potentials was substantially lower than peak Po elicited by test pulses, suggesting that steady-state inactivation can reduce Po by as much as 10-fold. Membrane depolarization decreased the longest mean closed time but had little effect on the mean open time of single calcium channels measured during steady-state recordings. Lowering the external barium concentration from 80 to 10 mM reduced the single channel conductance to 12.4 pS and shifted the relationship between steady-state Po and membrane potential by about -30 mV. BAY R 5417 also shifted this relationship by about -15 mV.

Bay K 8644 is a negative inotrope in the presence of arachidonic acid but not eicosapentaenoic acid.

The inotropic and radioligand binding characteristics of arachidonic acid (AA) and the fish oil derivative, eicosapentaenoic acid (EPA), were studied. AA alone was a concentration-dependent negative inotrope in isolated hamster papillary muscles. The negative inotropic effect of AA was enhanced 500-fold by the coadministration of indomethacin (cyclo-oxygenase inhibitor) and caffeic acid (lipoxygenase inhibitor). The dihydropyridine calcium channel agonist Bay K 8644 (BK) was a concentration-dependent positive inotrope. BK in the presence of AA was a concentration-dependent negative inotrope. The negative inotropic effect of BK in the presence of AA was blocked by caffeic acid but not indomethacin. EPA alone was a concentration-dependent negative inotrope. EPA had no effect on the positive inotropic effect of BK. Scatchard analyses of [3H]nitrendipine (dihydropyridine) binding to cardiac membranes in the presence of AA or EPA resulted in a decrease in the KD (-1/slope) with no effect on Bmax (x intercept). Kinetic studies further revealed that both AA and EPA enhanced the off rate of [3H]nitrendipine binding. These data suggest that both AA and EPA mediate similar direct negative inotropic effects through cardiac sarcolemmal calcium channels. A unique effect on E-C coupling by AA, not shared by EPA, is mediated through the lipoxygenase pathway (leukotrienes). AA and its eicosanoid products may play critically important roles in regulating myocardial contractility during acute and chronic myocardial ischemia. Differences between AA and EPA may contribute to the beneficial effects of dietary fish oils enriched in EPA.

Gonadotropin-releasing hormone-induced calcium signaling in clonal pituitary gonadotrophs.

In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.

Halothane Relaxes Preconstricted Small and Medium Isolated Porcine Coronary Artery Segments More Than Isoflurane

To compare the putative vasodilatory effects of isoflurane versus halothane on porcine coronary arteries, we studied the capacity of isoflurane and halothane to relax K(+)-constricted (30 mM) small (0.5-1.0 mm outside diameter [OD]) and medium (1.0-1.5 mm OD) porcine coronary arteries with use of in vitro tension recording. We also examined the effect of the dihydropyridine calcium channel agonist BAY K8644 on previously constricted epicardial porcine coronary artery segments in the presence of halothane or isoflurane. Our purpose was to determine (a) whether anesthetic effect on coronary arteries varied with arterial diameter, and (b) whether halothane and isoflurane inhibited BAY K8644-induced contraction of coronary vessels. Small and medium porcine coronary artery segments were constricted with K+ (30 mM) and the resulting contraction was allowed to stabilize. This was followed by exposure to 0.5%, 1.0%, 2.0%, and 3.0% isoflurane or halothane and the resultant tension was again measured. Potassium-induced contractions were significantly relaxed by halothane in small coronary artery segments at 0.5%, 1.0%, 2.0%, and 3.0% and in medium coronary artery segments at 1.0%, 2.0%, and 3.0%. Potassium-induced contractions were significantly reduced by isoflurane only at 3.0% in both small and medium coronary artery segments. Halothane caused significantly more relaxation of both small and medium porcine coronary arteries previously constricted with K+ (30 mM) than did isoflurane. There were no significant differences in coronary artery response to isoflurane or halothane with respect to coronary artery diameter. These experiments indicate that in porcine coronary arteries greater than 0.5 mm OD, studied in vitro after K(+)-induced contraction, isoflurane was not a potent coronary vasodilator.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic ethanol treatment alters ω-conotoxin and Bay K 8644 sensitive calcium channels in rat striatal synaptosomes

Two groups of adult Sprague-Dawley rats were maintained on a nutritionally complete liquid diet. In one group, 37% of the calories normally provided by dextrin were replaced with ethanol. Animals were maintained on this diet for eight weeks. The addition of ethanol (200 mM) in vitro significantly inhibited both calcium influx and dopamine release from control synaptosomes but did not alter calcium influx or dopamine release from synaptosomes isolated from ethanol-treated rats. The dihydropyridine calcium channel agonist Bay K 8644 (1 nM) significantly increased both calcium entry and dopamine release from control synaptosomes depolarized with 15 mM KCl. Bay K 8644 (1 nM) had no significant effect on either calcium entry or dopamine release in synaptosomes isolated from ethanol-treated animals. This loss of functional effect was accompanied by a slight (15%) but statistically insignificant increase in the binding of 3H-nitrendipine to striatal membranes from ethanol-treated rats as compared to control. The calcium-channel blocker, ω-conotoxin (500 nM) had no effect on voltage-dependent calcium uptake into synaptosomes prepared from control or ethanol-treated rats. Conotoxin (500 nM) inhibited the voltage-dependent release of endogenous dopamine from synaptosomes isolated from both groups by 36–44%. Ethanol (200 mM) added in vitro to control synaptosomes did not alter conotoxin's inhibition of dopamine release but completely abolished the ω-conotoxin-induced inhibition of dopamine release in synaptosomes isolated from ethanol-treated animals. These results suggest that DHP-sensitive and ω-conotoxin-sensitive calcium channels in rat brain respond differentially to chronic exposure to ethanol.

The effects of dihydropyridine calcium channel modulators on pentylenetetrazole convulsions

The effects of low doses of dihydropyridine (DHP) calcium channel antagonists nimodipine, nifedipine, (-)-R-202-791, and amlodipine, the DHP calcium channel agonist BAY K 8644 were investigated on clonic convulsions to pentylenetetrazole (PTZ) in mice. Nimodipine (2–20 mg/kg) produced a dose-dependent increase in the onset time for convulsions, but did not decrease the number of mice convulsing. Nifedipine, amlodipine (10 mg/kg) and BAY K 8644 (2 mg/kg) also produced an increase in the onset time for convulsions. (-)-R-202-791 (10 mg/kg) was without effect on clonic convulsions to PTZ. BAY K 8644 increased the number of mice dying from tonic-extension convulsions to PTZ. Nimodipine did not affect convulsions elicited by strychnine. Thus, low doses of DHP calcium antagonists possess anticonvulsant properties which are structurally dependent, while DHP calcium channel activators may act to promote convulsions. These observations suggest and support previous evidence that DHP receptors are important modulatory sites for the convulsive state.

Evidence for the mechanism of the inhibitory action of jatrophone in the isolated rat uterine muscle

1. 1. Jatrophone (JAT), a diterpene isolated from the plant Jatropha elliptica (1–300 μM), caused a concentration-dependent relaxation effect against acetylcholine (Ach)-oxytocin (Ot)- and KCl-induced uterine sustained contraction. The relative potency order was: Ach > Ot > KCl. 2. 2. The relaxant effect of JAT was not modified by phorbol ester, forskolin, MIX, TMB-8 and W-7. The increase concentration of calcium (0.2–2 mM) in the medium did not reverse the inhibitory effect caused by JAT. 3. 3. Pre-incubation of the preparations with JAT (16–32 μM) for 20 min, caused a concentration-dependent inhibition of KCl-induced contractile response. At 30 μM, JAT inhibited in an apparently non-competitive manner CaCl 2-induced contraction in K +-depolarized preparations. High concentrations of JAT (100 μM) also caused a time-dependent relaxation in CaCl 2-induced sustained uterine contraction ( T 1 2 = approx. 15 min ). 4. 4. JAT (30 μM) inhibited the dihydropyridine calcium channel agonist Bay K 8644-induced uterine contraction in an apparently non-competitive fashion, while verapamil (0.1 μM) caused in rightward displacement of Bay K 8644 contraction and marked inhibition of the maximal response.

Membrane receptors, involved in up-regulation of calcium channels in bovine adrenal chromaffin cells, chronically exposed to ethanol

Release of catecholamines, induced by carbachol, from bovine adrenal chromaffin cells, maintained in culture, is potently inhibited by the presence of ethanol (50 mM). This inhibition is prevented by the concomitant presence of bicuculline (1 μM) or by the inverse agonist at the benzodiazepine receptor, Ro 15 4513 (50 nM), arguing that the effect of ethanol is, at least, partly due to potentiation of endogenous GABA at the GABA A receptor sites on these cells. Exposing cells to medium containing ethanol (200 mM) for 4 days results in an approximately 100% increase in binding sites for [ 3H]dihydropyridine on membranes of adrenal chromaffin cells. This increase in binding sites was largely prevented by the presence in the culture medium of Ro 15 4513 (50 nM), the dihydropyridine calcium channel agonist BAY K 8644 (50 nM) or the calcium channel antagonist, nitrendipine (50 nM). The results strongly suggest that the increase in binding sites for [ 3H]dihydropyridine represents an adaptation of cells to overcome the inhibitory effect of ethanol on the excitability of cells (which is, at least partly, due to some action at the GABA A receptor). The protein containing the receptor site for dihydropyridine drugs is clearly also involved in controlling its up-regulation by ethanol, but this is probably not directly related to its channel function, since both activators and inhibitors of the dihydropyridine-sensitive channel prevented ethanol-induced up-regulation of binding sites for [ 3H]dihydropyridine.

Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K+ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [3H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or alpha 1-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [3H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1 microM atropine but not by 1 microM prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 microM atropine. The peptide calcium channel blocker omega-conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium and barium permeable channels fromAplysia nervous system reconstituted in lipid bilayers

Ion channels permeable to barium and calcium were reconstituted from the Aplysia nervous system into phospholipid bilayers formed on the tips of patch electrodes. With asymmetrical concentrations of barium or calcium on the two sides of the bilayer, the single-channel currents reversed at the calculated barium or calcium reversal potentials, indicating that the channels were cation selective. Channels with conductances of 10, 25 and 36 pS were routinely observed. Calcium and barium were equally effective as charge carriers for the 36-pS channel, whereas magnesium was at least fifteen-fold less effective. The gating of all three channels was independent of the voltage across the bilayer, but was affected by the dihydropyridine calcium channel agonist Bay K 8644 (Bay K). In the presence of Bay K but not in its absence, long discrete gating events were routinely observed, suggesting that the dihydropyridine increased the probability of long open states as it does for calcium channels in other systems. Bilayers invariably contained more than a single channel (or conductance state). This was observed even when the Aplysia nervous system membranes were prepared in the presence of cytoskeleton disrupting agents, or when the membrane proteins were diluted extensively with exogenous phospholipid. Furthermore, transitions between conductance levels were observed with high frequency. These findings, together with the fact that all of the conductance states share certain properties including voltage-independence and sensitivity to Bay K, suggest that the apparent multiple channel types may in fact represent subconductance states of a single ion channel.

Interaction of Dihydropyridine Calcium Channel Agonists and Antagonists with Adenosine Receptors

We have confirmed our previous (Fredholm et al. 1986a) finding that the dihydropyridine calcium channel agonist Bay K 8644 can displace [3H]-R-PIA from its binding site, the adenosine A1-receptor. Bay K 8644 had an apparent Ki of 5.2 X 10(-6) M. The effect was shared by the two dihydropyridine calcium channel antagonists nifedipine and felodipine (Ki 4.2 and 8.7 X 10(-6) M, respectively). By contrast, two non-dihydropyridine calcium channel antagonists, verapamil and diltiazem, did not affect binding. Bay K 8644 displaced [3H]-R-PIA from its binding sites in a solubilized preparation. [3H]-XAC, a novel, potent A1-receptor antagonist ligand, was also displaced by the dihydropyridine compounds with a similar or slightly higher potency as the displacement of R-PIA. This suggests a direct interaction with the adenosine receptor rather than an effect on regulatory GTP-binding proteins. However, at 1 mumol/l neither Bay K 8644 nor nifedipine significantly attenuated cyclic AMP accumulation in rat hippocampi or the R-PIA-mediated adenylate cyclase inhibition. The results show that dihydropyridine compounds that act as agonists or antagonists on L-type calcium channels can also affect adenosine receptors. The potency of the compounds for this effect is much lower than their potency as calcium channel agonists or antagonists. The results may therefore be of more experimental than clinical significance.

Selective Enhancement of Angiotensin II-and Potassium-Stimulated Aldosterone Secretion by the Calcium Channel Agonist BAY K 8644

Recent studies with dihydropyridine calcium channel antagonists have indicated that voltage-sensitive calcium channels (VSCC) play a major role in the control of aldosterone secretion. The modulation of VSCC by physiological regulators of zona glomerulosa function was further evaluated by analysis of the actions of the dihydropyridine calcium channel agonist BAY K 8644 (BK 8644) on basal and stimulated aldosterone production in isolated rat glomerulosa cells. In the presence of normal K+ concentrations (3.5-4.5 mM), only high concentrations of BK 8644 (greater than or equal to 100 nM) stimulated aldosterone secretion. However, addition of 10 nM BK 8644 markedly enhanced steroid production (70% over control) in cells stimulated by incubation in 7.5 mM K+ or 0.1 nM angiotensin II (AII). Greater enhancement was achieved with 1 microM BK 8644, with aldosterone secretion 150% and 300% above control levels for K+ and AII, respectively. In AII-stimulated cells, 30 nM BK 8644 enhanced aldosterone secretion at all peptide concentrations studied, including a 70% increase in the maximum steroid response, with no change in sensitivity to AII. In K+-stimulated cells, the effects of BK 8644 were dependent on the medium concentration of K+. At submaximally stimulating K+ concentrations (less than 9 mM), 30 nM BK 8644 increased the sensitivity of glomerulosa cells to K+ with no change in the maximal aldosterone response. However, at supramaximally stimulating concentrations of K+ (greater than 10 mM), BK 8644 reduced aldosterone production by 50%. In contrast to the effects of BK 8644 on cells stimulated with K+ or AII, the channel agonist had no effect on the action of ACTH. The ability of BK 8644 to enhance the maximum aldosterone response to AII suggests that AII, unlike K+, does not fully activate the Ca2+ influx pathway that leads to aldosterone secretion. Since BK 8644 is believed to facilitate Ca2+ influx primarily through previously activated channels, these results suggest that VSCC in the rat glomerulosa cell are partially operative under basal conditions, and that the same types of channels are further activated by AII and K+.

The effects of dihydropyridines of neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of ( 3H) norepinephrine and ( 3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of ( 3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.