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Abstract-The level of renal blood flow (RBF) is controlled by opposing vasoconstrictor and vasodilator influences. In a recent investigation in normotensive dogs, we found that combined blockade of endothelin type A (ET A ) receptors and angiotensin II formation induces marked increases in RBF that were much larger than the effects of blocking either system alone. The aim of the present study was to determine the contribution of nitric oxide (NO) to this vasodilator response. Experiments were made in 6 conscious, chronically instrumented dogs subjected to 5 different experimental treatments on separate days. Blockade of ET A receptors alone by the selective antagonist LU 135252 had only minor effects on RBF compared with time-control experiments. Additional blockade of angiotensin II formation by angiotensin-converting enzyme inhibition with trandolaprilat caused a substantial increase of RBF by Ϸ50%. This vasodilation was entirely suppressed when NO formation was prevented by inhibition of NO synthase with N G -nitro-L-arginine methyl ester HCl. However, when during NO synthase inhibition renal vascular NO concentrations were clamped at control levels by infusing the NO donor S-nitroso-N-acetyl-D,L-penicillamine, the vasodilator response to combined blockade of ET A receptors and angiotensin II formation was completely restored (⌬RBF Ϸ60%). These results indicate that the vasodilation after combined ET A receptor blockade and angiotensin-converting enzyme inhibition is not mediated by an increase in NO release but results from the unmasking of the tonic influence that is normally exerted by constitutively released NO. Accordingly, the tonic activity of endothelial NO synthase appears to be of major importance in the physiological regulation of renal vascular resistance by determining the vasomotor responses to endothelin and angiotensin II. (Hypertension. 1999;34:1254-1258.) Key Words: endothelin Ⅲ receptors, endothelin Ⅲ nitric oxide Ⅲ renal circulation Ⅲ renin-angiotensin system U nder physiological conditions, renal blood flow (RBF) is maintained at its normal level by a balance of opposing vasoconstrictor and vasodilator influences. 1 Besides the renal nerves, angiotensin II (Ang II) and endothelin-1 (ET-1) are the most potent renal vasoconstrictors. The renal circulation is particularly sensitive to small changes in Ang II plasma concentrations even within the physiological range, 2,3 and chronic infusions of low doses of Ang II cause sustained reductions of RBF and increases in systemic blood pressure in dogs. 4,5 The physiological function of ET-1 in the regulation of renal hemodynamics is less clear; under pathophysiological conditions, it apparently can assume a major vasoconstrictor role and contribute to end-organ damage. 6 -10 In a recent investigation in normotensive dogs, we have observed that combining angiotensin-converting enzyme (ACE) inhibition and endothelin type A (ET A ) receptor blockade caused a pronounced renal vasodilation, whereas blockade of either system alone had only minor effec...

cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GADUB and GAD 67 ) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD 95 and GAD 67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD 65 and GAD 67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD 65 and GAD 67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed.Type 1 (insulin-dependent) diabetes mellitus (EDDM) results from selective destruction of the insulin-producing y9-cells of the pancreas mediated by a chronic, clinically silent, autoimmune process (1, 2). The detection of circulating islet cell specific autoantibodies in non-diabetic individuals indicates a risk for IDDM and has lead to a search for the /?-cell specific components that may be the targets of this autoimmune response (3,4). Autoantibodies directed to a 64 kDa protein seem to be the earliest detectable /?-cell antibodies, being found up to 8 years before the clinical onset of IDDM in 80-90% of patients (5-12). The 64 kDa islet antigen representing one of the major target antigens in IDDM has been identified as glutamic acid decarboxylase (GAD). GAD is also expressed in brain tissue (13). Recent studies of GAD expression and purification have demonstrated at least two isoforms of GAD, with apparent molecular masses of 65 kDa (GAD 66 ) and 67 kDa (GAD 67 ), which are encoded by two genes located on 1 This work was supported by grants from the Steinbeis Stiftung, Baden-WUrttemberg, the Deutsche Forschungsgemeinschaft Sche 225/6-2, Sche 225/6-2, the Juvenile Diabetes Foundation (to W.A.S.), and the Deutsche Diabetes Stiftung (to J.S.). Since this manuscript was submitted, Karlsen et al. (34) have demonstrated that only the 65 kDa GAD isoform can be detected in human islets, whereas both the 65 kDa and 67 kDa GAD isoforms were expressed in rat islet cells as well as in human and rat brain. 2 To.whom correspondence should be addressed. Abbreviations: AET, 2-aminoethylisothiouronium bromide; GAD e5 and GAD» 7 , 65 kDa and 67 kDa glutamic acid decarboxylases; IDDM, insulin-dependent diabetes mellitus; IPTG, isopropylthiogalactoside; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PLP, pyridoxal-5-phosphate; PMSF, phenylmethylsulfonyl fluoride. two different chromosomes (14,15). Both GAD...

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