Altered Calcium Handling in Peripheral Nerve Terminals and Blood Vessels in Spontaneously Hypertensive Rats

The present study was designed to clarify the roles of N-type and P/Q-type calcium channels in the increased sympathetic activity of spontaneously hypertensive rats (SHR/Izm). We also tested in SHR/Izm the contribution to increased vascular tone of α1A adrenoceptor-linked L-type calcium channels and α1B receptor-mediated calcium mobilization from the sarcoplasmic reticulum. Methods: Six-week-old SHR/Izm and Wistar-Kyoto rats (WKY/Izm) were used. A superior mesenteric arterial preparation was electrically stimulated before and after treatment with ω-conotoxin GVIa (N-type calcium channel blocker [CgTX]) and ω-agatoxinIVa (P/Q-type calcium channel blocker [AgaTX]). Pressor response to norepinephrine was measured before and after treatment with the α1A blocker WB-4101 and the α1B blocker chloroethyl clonidine (CEC). To determine the intracellular calcium store size, the effects of ryanodine on pressor response and caffeine-induced vascular contraction were also tested. Results: Norepinephrine overflow evoked by electrical stimulation was increased in SHR/Izm. CgTX but not AgaTX suppressed the increased NE overflow in SHR/Izm. WB-4101 suppressed the pressor response to norepinephrine in SHR/Izm but not WKY/Izm rats. CEC had no effects on pressor response to norepinephrine in both types of rats. Caffeine-induced contraction to a high potassium-induced maximal contraction ratio was reduced in SHR/Izm. The effect of ryanodine on pressor response was reduced in SHR/Izm. Conclusion: N-type calcium channels but not P/Q-type calcium channels play an important role in the increased sympathetic tone in SHR/ Izm. Although α1A adrenoceptor-linked L-type calcium channels contribute to the increased vascular tone, the intracellular calcium store size was reduced in SHR/Izm.


Introduction
In our previous studies, we reported that spontaneously hypertensive rats (SHRs) had high plasma norepinephrine (NE) levels [1], similar to hypertensive patients [2], and that young SHRs had increased NE overflow from nerve endings in the mesenteric arteries [3].In a series of publication, mechanisms of hypertension of SHR were similar to essential hypertension and WKY strain was commonly used as normotensive controls.Calcium ions play a role in NE release from nerve terminals and vascular contraction.N-type [4] and P/Q-type [5] calcium channels existed in nerve endings and initiated the neurotransmitter release.α 1A -adrenoceptor links L-type calcium channels of vascular beds and α 1Badrenoceptor linked phospholipase C, which stimulate calcium mobilization from intracellular calcium store.Calcium antagonists are also major anti-hypertensive drugs and their magnitude of depressor effect is higher in hypertensive patients than in normal individuals.However, the precise role of calcium in hypertension is unclear.
The present study was conducted to clarify the roles of N-type and P/Q-type calcium channels in the increased sympathetic activity of SHRs.In addition, we tested in SHRs the contribution to the increased vascular tone of α 1A adrenoceptor-linked L-type calcium channel and the extent of calcium mobilization from the smooth-surfaced endoplasmic reticulum, an intracellular calcium store.

Statistical Analysis
Group differences in pressure response and NE overflow and their percent changes in both strains of rats were examined using independent t-tests.Differences between dose-dependent pressor responses and the effects of chemicals on pressor response were analyzed by two-way ANOVA.Differences were considered significant at p < 0.05.

Discussion
Our previous data show that isolated mesenteric nerve endings had increased NE release in young SHR/Izm [3] and that their hyperactive condition was suppressed more by calcium antagonist compared with normal rats [2].In peripheral nerve endings, N-type calcium channels play an important role in NE release, although P/Q-type calcium channels also participate.The magnitude of the relative contributions of N-type and P/Q-type channels in nerve terminals is unclear.In the present study, CgTX, N-type calcium antagonists, suppressed NE overflow more in SHR/Izm.In contrast, AgaTX, P/Q-type calcium antagonists, NE promotes contraction of the vascular bed through α 1A -linked L-type calcium channels and α 1B -linked phospholipase-mediated calcium mobilization from the sarcoplasmic reticulum, an intracellular calcium store.Calcium ion influx from the extracellular fluid and mobilized from an intracellular calcium store induces vascular contraction.It is unclear which mechanism contributes more to the hyper-reactive vascular bed in SHR/Izm.In the present study, WB-4101 but not CEC suppressed vascular contraction in SHR/Izm.This finding means that L-type calcium channels predominantly act on the blood vessels of SHR/Izm compared with phospholipase-mediated calcium mobilization from intracellular calcium stores.In addition, our data using caffeine and ryanodine showed that the calcium store in the blood vessels was smaller in SHR/Izm than in WKY/Izm rats.

Conclusion
In conclusion, N-type calcium channels but not P/Q-type calcium channels play an important role in the increased sympathetic tone in SHRs.In addition, although α1A-linked L-type calcium channels mainly contribute to the increased vascular tone, the calcium store size was reduced in SHRs.