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UBC Theses and Dissertations

Endothelium and smooth muscle function in rat mesentric vasculature He, Yi


The mesenteric arterial bed (MAB) comprises medium and small arteries as well as arterioles. They are important in generating and controlling peripheral resistance, thereby regulating blood flow and maintaining blood pressure. This hemodynamic function is mainly determined by the smooth muscle tone and contractility of mesenteric arteries and arterioles. Endothelial cells lining blood vessels help smooth muscle in this function by releasing various vasoactive substances. Abnormal vascular reactivity and impaired endothelium function has been found in several forms of hypertension. The purpose of the research in this dissertation was to study some cellular mechanisms involved in regulating smooth muscle reactivity and endothelium vasodilator function in rat mesenteric vasculature, and their abnormalities in hypertensive states. Cl⁻ currents represent a depolarizing mechanism in vascular smooth muscle cells, thus in the first part of the study the contribution of Cl⁻ channels to α₁-adrenoceptor-mediated vasoconstriction was studied in mesenteric arteries in vitro and in vivo from sham normotensive and two-kidney, one-clip (2K1C) hypertensive rats. Blockade of Cl⁻ channels with niflumic acid (NFA) significantly inhibited cirazoline-induced vasoconstriction in isolated MAB from both groups of rats. Cirazoline-evoked vasoconstriction was also significantly inhibited following removal of Cl⁻ from the perfusion buffer. Removal of Cl⁻ resulted in a significantly greater inhibition of cirazoline-mediated vasoconstriction in MAB from sham rats as compared with 2K1C rats. In vivo, intravenous infusion of cirazoline caused a dose-dependent decrease in superior mesenteric vascular conductance. Pretreatment with NFA significantly attenuated the cirazoline-mediated decrease in vascular conductance. To further investigate how the Cl⁻ channel blockade impaired α₁-adrenoceptor-mediated vasoconstriction, the inhibitory effect of NFA on cirazoline-induced vasoconstriction in isolated MAB was compared with that produced by the voltage-operated calcium channel (VOC) blocker nifedipine (NFDP). The extent to which the contractions to cirazoline were reduced by nifedipine compared to NFA plus NFDP was similar. Thus, effects of NFA and NFDP were not additive. In addition, in the absence of extracellular Ca²⁺, the transient phasic contraction to cirazoline was not affected by NFA , or by NFDP. NFA also had no effect on contraction induced by the depolarizing agent KCl. These observations suggest that Cl⁻ channels play an important role in α₁-adrenoceptor-induced vasoconstriction in mesenteric blood vessels. They may act by producing membrane depolarization, thereby indirectly inhibiting activation of VOCs. The contribution of Cl⁻ channels in α₁-adrenoceptor- mediated vasoconstriction in mesenteric blood vessels from 2K1C hypertensive rats appears to be reduced. This effect may reflect an adaptive change due to increased vascular resistance in hypertension. In the second part of the dissertation, the role of Cl⁻ channels in endothelium-dependent relaxation to acetylcholine (ACh) in superior mesenteric artery and the factors that mediate the endothelium-dependent relaxation were investigated. The aorta was also studied as a comparison. ACh concentration-dependently relaxed phenylepinephrine (PE)-induced tone in rat endothelium-intact mesenteric arteries and aorta. Inhibition of Cl⁻ channels with NFA had no effect on the dilator responses to ACh in either mesenteric arteries or aorta. The BKCa antagonist, TEA, decreased the potency (pD₂) to ACh without affecting the maximum response (Rmax) in mesenteric arteries, whereas it had no effect in aorta. In the presence of NFA plus TEA, there was no further inhibition seen in mesenteric arteries as compared to TEA alone. In contrast, in the aorta, the pD₂ to ACh was significantly inhibited by NFA plus TEA although without changing the Rmax. In addition, neither NFA nor TEA alone, nor TEA plus NFA had any effect on relaxation to the Ca²⁺ ionophore A23187 in aorta. These data suggest that besides BKCa, Cl⁻ channels play a functional role in ACh-induced endothelium-dependent relaxation in aorta, possibly by preventing the depolarization-mediated inactivation of receptor-operated Ca²⁺ channels (ROC), thereby resulting in a sustained Ca²⁺ influx and NO synthesis. By contrast, in mesenteric arteries, K⁺ channels, but not Cl⁻ channels, mediated the relaxation. In addition, we found that indomethacin has no effect on, while L-NMMA only slightly impaired, the relaxation to ACh, suggesting that the effect of PGI₂ is negligible, while the contribution of NO is small in mesenteric arteries. Furthermore, the L-NMMA/ indomethacin-insensitive component of the relaxation response of mesenteric artery to ACh was greatly inhibited in the presence of SKCa and BKCa antagonists. High K⁺ (30 mM) further decreased the maximum relaxation to ACh, but did not abolish it. Thus, the observations suggest that EDHF contributes to a large part of the ACh-induced vasorelaxation in rat superior mesenteric arteries. Another relaxing factor or (possibly more than one) that is distinct from EDHF, such as NO and PGI₂, may also play a role. In the third part of the dissertation, the contribution of endogenous EDRF (NO) and endothelium-derived contraction factors (prostaglandins) to reactivity to NE in MAB from hypertensive Zucker obese rats with hyperinsulinemia and insulin resistance was studied. The influence of insulin on the NE response was examined. There was no major difference in pressor responses to NE in MAB between hypertensive Zucker obese and hormotensive Zucker lean rats, except for a small decrease in responsiveness to the highest concentration o f NE (90 nmol) tested. Inhibition of NO synthesis with L-NMMA enhanced the vasoconstriction to NE, while blockade of prostanoid production by indomethacin decreased the NE response. A pathophysiological concentration of insulin (200 mU/l) potentiated responses to the two lowest concentrations of NE (0.3 and 0.9 nmol) used in MAB from Zucker obese rats, but not lean rats. The potentiating effect of insulin was further enhanced after blockade of NO synthesis, while it was prevented by inhibition of prostanoid production. These data suggested that NE-induced vasoconstrictor responses are normally modulated by concurrent release of NO and vasoconstrictor cyclooxygenase product(s) in MAB from both obese and lean Zucker rats. Insulin increases the release of contracting cyclooxygenase product(s) and enhances reactivity to NE in MAB from obese rats. This altered action of insulin may play a role in hypertension in this hyperinsulinemic/insulin resistant model.

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