Effects of a Novel Antihypertensive Drug, Cilnidipine, on Catecholamine Secretion From Differentiated PC12 Cells

Abstract

Abstract-Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca 2ϩ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K ϩ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 Key Words: cilnidipine Ⅲ calcium channels Ⅲ calcium, cytoplasmic Ⅲ dopamine C ilnidipine (FRC-8653) is a newly synthesized DHP type of organic Ca 2ϩ channel blocker that has been developed as a slow-onset and long-lasting antihypertensive drug in Japan. 1,2 Recent electrophysiological data indicate that cilnidipine might be a dual-channel antagonist for peripheral neuronal N-type and vascular L-type Ca 2ϩ channels. [3][4][5] In humans and rodents, cilnidipine depressed the pressor response to acute cold stress but failed to induce tachycardia evoked by hypotensive baroreflexes. 6,7 In spontaneously hypertensive rats, vasoconstriction induced by electrical sympathetic nerve stimulation was also blocked by cilnidipine. 8 In in vitro experiments, cilnidipine also inhibited [ 3 H]norepinephrine release evoked by electrical stimulation in the rabbit mesenteric artery. 9 PC12 cells are derived from a rat pheochromocytoma cell line that is very popular for investigating neuronal differentiation. In response to externally applied NGF, PC12 cells acquire sympathetic neuronal characteristics such as neurite extension, increased CA synthesis, and expression of neuronal types of voltage-dependent N-type Ca 2ϩ channels. 10 -12 Therefore, the differentiated PC12 cells are widely used as model cells for studying the intracellular mechanisms of the stimulus-secretion coupling, including the activation of the N-type Ca 2ϩ channel. 13,14 With this model cell, it has been revealed that endogenous substances such as neuropeptide Y and proadrenomedullin N-terminal 20 peptide inhibit CA release by inhibiting Ca 2ϩ influx through N-type Ca 2ϩ channels. 15,16 In the present study, we examined the effects of cilnidipine on the cellular functions of the NGF-treated PC12 cells, including CA secretion and intracellular Ca 2ϩ mobilization triggered by membrane depolarizations. Consequently, in addition to its well-known L-type Ca 2ϩ channel blockade, 3 it was clarified that cilnidipine had a potent inhibitory effect for CA secretion from differentiated PC12 cells via the blockade of extracellular Ca 2ϩ influx through the N-type Ca 2ϩ channel. Methods Cell CulturePC12 cells were prepared as described previously. 17 In brief, cells were cultured in Dulbecco's modified Eagle's medium containing 7% fetal bovine serum (Gibco), 7% heat-ina...

Vasodilatory Effect of Cilnidipine, an L-type and N-type Calcium Channel Blocker, on Rat Kidney Glomerular Arterioles

“…[10][11][12]18) The N-type calcium channel is involved in sympathetic neurotransmission, [14][15][16] and it was shown that cilnidipine suppresses the release of norepinephrine from sympathetic nerve terminals in perfused SHR mesentery specimens. It was shown that cilnidipine suppresses sympathetic nerve activity of SHRs as well as normotensive rats.…”

“…[10][11][12] The L-type calcium channel is the usual target of DHP calcium blockers, thus it is DHP-sensitive, 13) and the Ntype calcium channel is DHP-insensitive. Several studies have shown that when norepinephrine (NE) is released from sympathetic nerve terminals there is an influx of extracellular calcium through calcium channels.…”

Neuronal Ca2+ Channel Blocking Action of an Antihypertensive Drug, Cilnidipine, in IMR-32 Human Neuroblastoma Cells

“…These results indicate that cilnidipine can suppress depolarization-induced elevation of intracellular Ca 2 concentrations through N-type Ca 2 channels in human neuronal cells as well as in rodent neuronal cells (10,11,22).…”

“…Using human neuronal cells from the cell line IMR-32, whose Ca 2 currents consist of both L-type and Ntype components (21), the N-type Ca 2 channel-blocking effect of cilnidipine was assessed in the presence of nifedipine to block L-type Ca 2 channels (22). In this model, cilnidipine as well as ω-conotoxin GVIA effectively suppressed the change in the intensities ratio induced by 40 mmol/l KCl, which finding was highly similar to that in our previous study that assessed the N-type Ca 2 channel-blocking effect of cilnidipine and ω-conotoxin GVIA using rat pheochromocytoma PC12 cells (22).…”

“…Using human neuronal cells from the cell line IMR-32, whose Ca 2 currents consist of both L-type and Ntype components (21), the N-type Ca 2 channel-blocking effect of cilnidipine was assessed in the presence of nifedipine to block L-type Ca 2 channels (22). In this model, cilnidipine as well as ω-conotoxin GVIA effectively suppressed the change in the intensities ratio induced by 40 mmol/l KCl, which finding was highly similar to that in our previous study that assessed the N-type Ca 2 channel-blocking effect of cilnidipine and ω-conotoxin GVIA using rat pheochromocytoma PC12 cells (22). Thus, it is expected that cilnidipine might be effective as a therapeutic tool to provide neuronal protection as well as its antihypertensive effects and antisympathetic actions, since blockade of N-type Ca 2 channels in the brain has been reported to reduce glutamate release and infarct volume in models of cerebral ischemia (23,24).…”