Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice

We cloned a rat vascular chymase (RVCH) from smooth muscle cells (SMCs) that converts angiotensin I to II and is up-regulated in SMC from spontaneously hypertensive vs. normotensive rats. To determine whether increased activity of RVCH is sufficient to cause hypertension, transgenic mice were generated with targeted conditional expression of RVCH to SMC, with the use of the tetracycline-controlled transactivator (tTA). We confirmed conditional expression of RVCH by mRNA, protein, and chymase activity in the absence, but not in the presence, of dietary doxycycline. The systolic blood pressure (mmHg), measured by carotid artery cannulation at 10–12 weeks of age, was higher in tTA+/RVCH+ mice than in nonbinary transgenic littermates (136 ± 4 vs. 109 ± 3) (P < 0.05), as were the diastolic and mean pressures. Hypertension was completely reversed by doxycycline, suggesting a causal link with chymase expression. Medial thickening of mesenteric arteries from tTA+/RVCH+ mice vs. littermates (0.82 ± 0.1 vs. 0.42 ± 0.02) (P < 0.05) was associated with increased SMC proliferation, as judged by positive immunoreactivity, with the use of an antibody to the proliferating cell nuclear antigen. These structural changes were prevented by doxycycline. Perfusion myography of mesenteric arteries from tTA+/RVCH+ mice also revealed increased vasoconstriction in response to phenylephrine and impaired metacholine-induced vasodilatation when compared with littermate controls or with the doxycyline-treated group. Our studies suggest that up-regulation of this vascular chymase is sufficient to cause a hypertensive arteriopathy, and that RVCH may be a candidate gene and a therapeutic target in patients with high blood pressure.

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.