Tomoyuki Konda

The N- and L-Type Calcium Channel Blocker Cilnidipine Suppresses Renal Injury in Dahl Rats Fed a High-Sucrose Diet, an Experimental Model of Metabolic Syndrome

Background/Aims: The L/N-type calcium channel blocker (CCB) cilnidipine has been demonstrated to suppress progressive renal disease in a variety of experimental models, but the characteristic effects of N-type calcium channel blocking action on renal injury have not been examined in detail. Therefore, we investigated the beneficial effects of cilnidipine on renal injury in Dahl salt-sensitive (Dahl S) rats fed a high-sucrose diet (HSD), which mimics metabolic syndrome, and compared them with the effects of an L-type CCB, amlodipine. Methods: Male Dahl S rats were divided into groups with similar blood pressure at 8 weeks of age and fed an HSD. They received vehicle, cilnidipine or amlodipine for 27 weeks. At 35 weeks of age, urine and blood samples were collected for physiological analysis, and the kidneys were removed for histopathological evaluation. Results: Cilnidipine reduced albuminuria, glomerular hypertrophy, glomerular expression of ICAM-1, ED-1-positive cell infiltration and interstitial fibrosis compared with vehicle-treated rats. In contrast, amlodipine had no effect on these parameters. Urinary norepinephrine excretion, renal expression of renin mRNA and renal tissue levels of angiotensin II were increased only in the amlodipine-treated group. Conclusion: Cilnidipine provided superior protection against renal damage compared with amlodipine in Dahl S rats given an HSD. The different effects between these two drugs may be partly explained by their different actions on the renal sympathetic nerve activity and the renin-angiotensin system through the N-type calcium channel blocking action.

Selectivity of dihydropyridines for cardiac L-type and sympathetic N-type Ca2+ channels

The blocking effects of cilnidipine and other dihydropyridines on L-type cardiac Ca2+ channels (I(Ca,L)) and N-type sympathetic Ca2+ channel currents (I(Ca,N)) were studied using a whole-cell patch-clamp technique. At -80 mV, cilnidipine had little inhibitory effect below concentrations of 1 microM on I(Ca,L) (IC50 value; 17 microM). However, 1 microM cilnidipine strongly shifted the steady-state inactivation curve of I(Ca,L) toward negative potentials without changing the current-voltage relationship. Each action of cilnidipine was characterized by a high affinity for the inactivated channel in preference to the resting channel. The IC50 values of dihydropyridines for I(Ca,L) were in the range between 0.01 and 10 microM, and those for I(Ca,N) were between 3 and 30 microM. Cilnidipine had the strongest affinity for I(Ca,N) among the dihydropyridines tested. These results suggest that cilnidipine did not cause hypotension-evoked tachycardia deficiency by depression of cardiac L-type channels but by sympathetic N-type channels blockade.

Different Effects of L/N-Type and L-Type Calcium Channel Blockers on the Renin-Angiotensin-Aldosterone System in SHR/Izm

Background: The L/N-type calcium channel blocker (CCB) cilnidipine has been demonstrated to suppress sympathetic nerve activity. In the present study, we investigated the effects of cilnidipine on the renin-angiotensin-aldosterone system (RAAS) in SHR/Izm rats to confirm differences from the effects of L-type CCB. Methods: Male SHR/Izm rats received vehicle, cilnidipine (0.3, 3 mg/kg) or amlodipine (0.3, 3 mg/kg) by gavage for systolic blood pressure (SBP) measurement. For biochemical analyses, the experiments were performed under anesthesia. Results: Low doses of cilnidipine or amlodipine had no effect on SBP or RAAS parameters. A high dose of either drug produced similar effects on SBP levels. Although cilnidipine had no effect on plasma renin activity or the plasma angiotensin II level, amlodipine significantly increased these parameters as compared to levels in the vehicle group. The cilnidipine group had a significantly lower plasma aldosterone level and renal cortical tissue norepinephrine level than the vehicle group. Conclusions: Cilnidipine had effects different from those of L-type CCB on the RAAS in SHR/Izm rats. Our results indicate that suppression of RAAS hyperactivity by cilnidipine can be partly explained by its sympatholytic action.

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis

The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.

Effects of a dual L/N-type Ca2+ channel blocker cilnidipine on neurally mediated chronotropic response in anesthetized dogs

We investigated the effects of an L-type and N-type Ca(2+) channel blocker, cilnidipine, on neurally mediated chronotropic responses to clarify the anti-autonomic profile of cilnidipine in anesthetized dogs. Pretreatment with cilnidipine (0.3, 1.0 and 3.0 microg/kg, i.v.), which decreased mean blood pressure by 5 to 31 mm Hg, inhibited the changes in heart rate and plasma norepinephrine concentration induced by bilateral carotid artery occlusion, whereas it had no effect on vagal nerve stimulation-induced bradycardia. These results suggest that antihypertensive and antisympathetic doses of cilnidipine fail to influence chronotropic responses mediated by parasympathetic nerve activation in the in vivo canine heart.

L/N-type calcium channel blocker suppresses reflex aldosterone production induced by antihypertensive action

The L/N-type calcium channel blocker cilnidipine has been shown to suppress aldosterone production induced by angiotensin II (Ang II) in vitro. In addition, cilnidipine also suppresses the reflex tachycardia induced by its antihypertensive action in vivo. We investigated the effects of cilnidipine on the reflex aldosterone production induced by its antihypertensive action, to identify the differences in the effects of cilnidipine from those of the L-type calcium channel blocker nifedipine. Male SHR/Izm rats were anesthetized by intraperitoneal injection of pentobarbital sodium, and administered an intravenous infusion of saline supplemented or not with Ang II for 30 min. Blood pressure was monitored continuously in the femoral artery. Each of the calcium channel blockers under study was administered intravenously as a bolus through the femoral vein 1 min after the start of the Ang II infusion, and blood samples were collected 30 min after the start of the Ang II infusion. Following administration at nonhypotensive doses, all calcium channel blockers tended to decrease the plasma aldosterone. In particular, cilnidipine significantly suppressed the plasma aldosterone levels. On the other hand, under the condition of Ang II-induced hypertension, administration of a hypotensive dosage of cilnidipine showed no effect on the plasma aldosterone levels, whereas a hypotensive dosage of nifedipine significantly increased the plasma aldosterone levels. Our results suggest that the L/N-type calcium channel blocker cilnidipine reduces the plasma aldosterone level by suppressing the aldosterone production induced by reflex upregulation of the renin–angiotensin–aldosterone system associated with reduction of the blood pressure.

Effects of an N-type calcium antagonist on angiotensin II-renin feedback.

Background: Interrupting the renin-angiotensin system (RAS) with an angiotensin II receptor blocker (ARB) has been found to induce RAS overactivation. In this study, we investigated the effect of 2 calcium channel blockers (CCBs), cilnidipine (L-/N-type CCB) and amlodipine (L-type CCB), on the RAS activation induced by an ARB in a strain of spontaneously hypertensive rats (SHR/Izm, 10 weeks of age). Methods: Rats intravenously catheterized for blood collection were randomly divided into groups that were administered the vehicle, the ARB valsartan or valsartan combined with one of the 2 CCBs. Their blood and kidneys were collected 270 min after administration. Results: Valsartan increased the plasma angiotensin II (Ang II) level in a dose-dependent manner. Cilnidipine suppressed the increase in plasma renin activity and plasma Ang II levels induced by valsartan, but amlodipine did not. Combined administration of cilnidipine, but not amlodipine, and valsartan significantly reduced the noradrenaline content in the renal cortex. Conclusions: The results of this study suggest that the suppressive effect of cilnidipine on the valsartan-induced increase in RAS activity can be partly explained by its sympatholytic action mediated by N-type calcium channel blockade, and that combined administration of cilnidipine and valsartan might provide a synergistic therapeutic effect.

Regulation of adrenal aldosterone production by serine protease prostasin.

A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.