In our previous studies, we reported that spontaneously hypertensive rats (SHRs) had high plasma norepinephrine (NE) levels  , similar to hypertensive patients  , and that young SHRs had increased NE overflow from nerve endings in the mesenteric arteries  . 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  and P/Q-type  calcium channels existed in nerve endings and initiated the neurotransmitter release. α1A-adrenoceptor links L-type calcium channels of vascular beds and α1B- adrenoceptor linked phospholipase C, which stimulate calcium mobilization from intracellular calcium store. Calcium antagonists are also major anti-hyper- tensive 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 α1A adrenoceptor-linked L-type calcium channel and the extent of calcium mobilization from the smooth-surfaced endoplasmic reticulum, an intracellular calcium store.
2.1. Animals and Protocols
Six-week-old SHR/Izm and Wistar-Kyoto rats (WKY/Izm) were used. Male SHR/Izm strain rats were obtained from the Disease Model Cooperative Research Association and were housed under a 12-hour light/dark cycle. Animals were handled according to the institutional guidelines for animal research of Wakayama Medical University. An isolated mesenteric arterial preparation was made as previously reported  . Briefly, polyethylene tubes were inserted into the superior mesenteric artery and the mesenteric artery-intestine loop preparation was perfused with Krebs-Henseleit solution. The superior mesenteric arterial preparation was electrically stimulated (10 Hz, 1 ms duration, supramaximal voltage) before and after treatment with ω-conotoxinGVIa   (N-type calcium channel blocker [CgTX]; 5 × 10−9 M) and ω-agatoxinIVa (P/Q-type calcium channel blocker [AgaTX  ]; 5 × 10−11 M). Pressor response to NE was measured before and after treatment with the α1A blocker WB-4101  (WB; 10−9 M) and the α1B blocker chloroethylclonidine  (CEC; 5 × 10−6 M). The dosages used were based on previous work      and preliminary studies. To determine the intracellular calcium store size, the effects of ryanodine  (1 × 10−5 M) on pressor response to NE and caffeine  (20 mM)- induced vascular contraction were also tested. Perfusion pressure was measured by a pressure transducer (TR-200, Gulton Industries Inc.). NE overflow was measured by HPLC using an electrochemical detector.
2.2. 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.
The blood pressure levels of SHR/Izm and WKY/Izm rats were 167 ± 15 mmHg and 139 ± 8 mmHg, respectively. Pressor response and NE overflow evoked by electrical stimulation were increased in SHR/Izm (n = 6) compared with WKY/ Izm rats (n = 6) (pressor response: SHR/Izm, 35.9 ± 3.2 mmHg vs. WKY/Izm, 14.1 ± 2.4 mmHg; NE overflow: SHR/Izm, 1.56 ± 0.4 ng/g wet.wt vs. WKY/Izm, 0.65 ± 0.1 ng/g wet.wt) (Figure 1).
CgTX significantly suppressed pressor response and NE overflow evoked by electrical stimulation in SHR/Izm (n = 6) compared with WKY/Izm rats (n = 6). The percent decrease in pressor response to electrical stimulation was higher in SHR/Izm (−82.1% ± 1.7%) than in WKY/Izm rats (−65.4% ± 0.8%, p < 0.05) and the percent decrease in NE overflow showed a similar trend in SHR/Izm (−79.9% ± 1.5%) and WKY/Izm rats (−68.4% ± 0.7%, p < 0.05).
In contrast, AgaTX more markedly suppressed the pressor response and NE overflow evoked by electrical stimulation in WKY/Izm rats (n = 6) compared with SHR/Izm (n = 6). The percent decrease in pressor response to electrical stimulation was lower in SHR/Izm (−20.3% ± 1.0%) than in WKY/Izm rats (−53.1% ± 0.8%, p < 0.05) and the percent decrease in NE overflow was −5.5% ± 1.0% in SHR/Izm and −50.4% ± 0.7% in WKY/Izm rats (p < 0.05) (Figure 2).
Pressor response to NE was increased in SHR/Izm (n = 6) (NE 0.5 micro g, 39.0 ± 0.9 mmHg; NE 1.0 micro g, 60.7 ± 7.5 mmHg) compared with WKY/Izm rats (n = 6) (NE 0.5 micro g, 13.1 ± 3.1 mmHg; NE 1.0 micro g, 23.0 ± 3.1 mmHg, p < 0.01). WB-4101 suppressed pressor response to NE in SHR/Izm (n =
Figure 1. Pressor response and NE overflow evoked by electrical stimulation were increased in SHR/Izm compared with WKY/Izm rats (*p < 0.05).
Figure 2. Percent decrease in NE overflow with ω-conotoxin (N-type calcium channel blocker, CgTX) and ω-agatoxin (P/Q-type calcium channel blocker, AgaTX) in SHR/Izm and WKY/Izm rats. The effect of CgTX on NE overflow was significantly greater in SHR/Izm than in WKY/Izm rats (left panel, *p < 0.05). AgaTX suppressed NE overflow to a lesser extent in SHR/Izm than in WKY/Izm rats (*p < 0.05).
6) (NE 0.5 micro g, −35% ± 2%; NE 1.0 micro g, −53% ± 2%) but not in WKY/ Izm rats (n = 6) (NE 0.5 micro g, −78% ± 8%; NE 1.0 micro g, −78% ± 12%, p < 0.01). CEC showed no effects on pressor response to NE in both strains of rats (SHR [n = 6]: NE 0.5 micro g, −37% ± 4%; NE 1.0 micro g, −41% ± 1%; WKY [n = 6]: NE 0.5 micro g, −45% ± 5%; NE 1.0 micro g, −41% ± 6%).
Caffeine (20 mM)-induced contraction was 7.7 ± 0.9 mmHg in SHR/Izm (n = 6) and 9.1 ± 0.8 mmHg in WKY/Izm rats (n = 6) (p < 0.05). High potassium (128 mM)-induced maximal contraction was 45.5 ± 6.3 mmHg in SHR/Izm and 25.4 ± 1.8 mmHg in WKY/Izm rats (p < 0.01). The ratio of caffeine-induced contraction to maximal contraction was lower in SHR/Izm (0.17 ± 0.01) than in WKY/Izm rats (0.36 ± 0.02) (p < 0.001) (Figure 3).
Ryanodine significantly reduced NE-induced pressor response in both SHR/ Izm (n = 6) (from 47.9 ± 3.5 mmHg to 37.5 + 4.0 mmHg) and WKY/Izm rats (n = 6) (from 34.1 ± 2.0 mmHg to 22.3 ± 5.7 mmHg) (p < 0.05). In addition, the percent reduction in pressure response was −13.8% ± 2.7% in SHR/Izm and −22.8% ± 4.9% in WKY/Izm rats. The magnitude of the reduction in the pressure response was blunted in SHR/Izm compared with WKY/Izm rats (p < 0.05).
Our previous data show that isolated mesenteric nerve endings had increased NE release in young SHR/Izm  and that their hyperactive condition was suppressed more by calcium antagonist compared with normal rats  . 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,
Figure 3. Ratio of caffeine-induced contraction to KCl-induced maximal contraction. The ratio was significantly smaller in SHR/Izm than in WKY/Izm rats (*p < 0.001).
suppressed NE overflow more in WKY/Izm rats than in SHR/Izm. These results indicate that N-type calcium channel contribute more to hyperactive nerve terminals of SHR/Izm and that P/Q-type calcium channel contribute less to NE release in nerve ending of hypertension.
NE promotes contraction of the vascular bed through α1A-linked L-type calcium channels and α1B-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.
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.
This study was supported by a Grant-in-Aid for Scientific Research (C) (#26460915) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan.