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The present study evaluated the possible changes in the autonomic control of heart rate in the hypertensive model induced by the inhibition of nitric oxide synthase. Rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME group) in the drinking water during 7 days, whereas control groups were treated with tap water (control group) or with the N(G)-nitro-D-arginine methyl ester (D-NAME group), an inactive isomer of the L-NAME molecule. The L-NAME group developed hypertension and tachycardia. The sequential blockade of the autonomic influences with propranolol and methylatropine indicated that the intrinsic heart rate did not differ among groups and revealed a sympathetic overactivity in the control of heart rate in the L-NAME group. The spectral density power of heart rate, calculated using fast-Fourier transformation, indicated a reduced variability in the low-frequency band (0.20-0.60 Hz) for the L-NAME group. The baroreflex sensitivity was also attenuated in these animals when compared with the normotensive control or D-NAME group. Overall, these data indicate cardiac sympathetic overactivity associated with a decreased baroreflex sensitivity in L-NAME hypertensive rats.

. Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme. Am J Physiol Heart Circ Physiol 285: H775-H783, 2003. First published April 24, 2003 10.1152/ajpheart.00818. 2002We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-AlaAla-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro 11 ,D-Ala 12 ]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature. endothelium; chymostatin SEVERAL STUDIES HAVE POSTULATED the existence of alternative pathways to angiotensin (ANG)-converting enzyme (ACE) for ANG II generation in tissues of different species based on evidence derived from experiments carried out with a combination of protease inhibitors and ANG II receptor antagonists. In a pioneering study (9) in this area, it was demonstrated that the vasoconstrictor response induced by ANG I in blood vessels of the hamster cheek pouch was blocked only partially by ACE inhibitors but completely abolished by ANG II receptor antagonists, leading to the conclusion that this vascular bed converts significant amounts of ANG I to ANG II by a route that does not involve ACE. In other investigations, the partial or total blockade of ANG II formation by different combinations of ACE inhibitors with chymostatin or other protease inhibitors has provided clues as to the nature of the enzymes involved in ANG I conversion in a particular tissue or pharmacological preparation (23,45). Since the advent of [Pro 11 ,D-Ala 12 ]-ANG I, a biologically inactive precursor that selectively yields ANG II on incubation with chymases but not with ACE or carboxypeptidases (22), several reports (18,22,26,38,41,43,61,63) have described the relative contribution of chymases in ANG II formation in isolated preparations derived from various species.The vascular endothelium actively participates in the control of vascular tone through the synthesis and metabolism of seve...

An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursor [Pro 11 -D-Ala 12]-Ang I was converted into Ang II by the rat MAB elastase-2 with catalytic efficiency of 8.6 min-1 microM-1, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1 microM-1 and 7.6 min-1 microM-1, respectively. The non-cleavable peptide inhibitor CH-5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50 = 49 microM) and N-succinly-Ala-Ala-Pro-Phe-p-nitroanilide (IC 50 = 4.8 microM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone, an effective active site-directed inhibitor of pancreatic elastase-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC 50 = 4.5 microM). Altogether, the data presented here confirm and extend the enzymological similarities between pancreatic elastase-2 and its rat MAB counterpart. Moreover, the thus far unrealized interaction of elastase-2 with [Pro 11-D-Ala 12]-Ang I and CH-5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways.

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