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There are conflicting data regarding the effects of transplantation of bone marrow-derived cells (BMDCs) on the severity of diabetes. We therefore inquired whether the competence of BMDCs is preserved on adoptive transfer into diabetic (db/db) mice and how the adoptive transfer of BMDCs affects vascular and metabolic abnormalities in these mice. Recipient db/dbmice received infusions of BMDCs prepared from either db/dbor non-diabetic heterozygout mice (db/m) mice and effects on endothelium-dependent relaxation, insulin sensitivity, and renal function were evaluated. Recipients of BMDCs from db/m, but not db/dbdonors showed better glucose control, exhibited striking improvement in endothelium-dependent relaxation in response to acetylcholine, and had partially restored renal function. Improved glucose control was due to enhanced insulin sensitivity, most likely secondary to improved vascular function. Enhanced apoptosis of endothelial progenitor cells under oxidative stress, as well as decreased endothelial progenitor cell numbers were responsible for the apparent functional incompetence of BMDCs from db/dbdonors. Treatment of db/dbmice with Ebselen restored the resistance of both BMDCs and endothelial progenitor cells to oxidative stress, improved acetylcholine-induced vasorelaxation, and reduced proteinuria in db/dbrecipients of BMDC transplantation. In conclusion, infusion of BMDCs obtained from db/mdonors to db/dbrecipient mice benefited macrovascular function, insulin sensitivity, and nephropathy. BMDCs obtained from db/dbmice were functionally incompetent secondary to the increased proportion of apoptotic cells on oxidative stress challenge; their competence was restored by Ebselen therapy.

Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of NG-monomethyl-l-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward “metabolic hypoxia” through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 μM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A-derived arachidonic acid metabolite, is a potent vasoconstrictor and a modulator of vascular reactivity. We have shown that CYP4A1 and CYP4A2 are the major CYP4A isoforms expressed in the rat renal microcirculation. In the present study, we constructed two bicistronic vectors, pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2, and examined their functional efficacy in COS-1 and vascular smooth muscle (A7r5) cells and in microdissected rat interlobar arteries. Immunocytochemistry coupled with fluorescence microscopy of pIRES2-EGFP-4A1- or pIRES2-EGFP-4A2-transfected COS-1 and A7r5 cells indicated that both enhanced green fluorescence protein (EGFP) and CYP4A1/4A2 were expressed in 80 to 90% of the cells. Western blot analysis showed a 3- to 5-fold increase of CYP4A1 and CYP4A2 proteins in pIRES2-EGFP-4A1- and pIRES2-EGFP-4A2-transfected cells as compared with control pIRES2-transfected cells. Cells transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 catalyzed arachidonic acid omega-hydroxylation to 20-HETE at rates of 0.85 +/- 0.29 and 0.27 +/- 0.04 nmol/10(7) cells/h, respectively. Transfection of interlobar arteries with either plasmid yielded EGFP immunofluorescence that was localized to the intima, media, and adventitia. Arteries transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 showed increased vasoreactivity displaying EC50 to phenylephrine of 0.24 +/- 0.07 and 0.11 +/- 0.03 microM, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 +/- 0.21 microM; n=6, p <0.05). The increased vasoreactivity to phenylephrine was inhibited by N-methylsulfonyl-12,12-dibromododec-11-enamide, an inhibitor of CYP4A-catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. Removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1 cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin.

Heme oxygenase (HO)-derived carbon monoxide (CO) attenuates vascular reactivity to constrictor stimuli. ANG II produces vasoconstriction and induces HO-1 isoform expression. However, direct evidence that ANG II promotes HO product generation is lacking. Therefore, we examined the effects of ANG II on CO release and HO isoform expression in isolated rat kidneys. Kidneys were perfused with oxygenated Krebs buffer. ANG II (1 μmol/l) increased (P< 0.05) perfusion pressure from 97 ± 9 to 150 ± 14 mmHg; it also increased (P< 0.05) the concentration of CO in the venous effluent (from 27.1 ± 11.9 to 45.6 ± 11.7, 62.5 ± 16.7, 94.8 ± 20.7, and 101.9 ± 13.1 nmol/l after 30, 60, 90, and 120 min, respectively). The pressor effect of ANG II was blunted (P< 0.05) in kidneys perfused with buffer containing losartan (10 μmol/l) or PKC inhibitors staurosporine (0.1 μmol/l) or calphostin C (1 μmol/l). Kidneys perfused with buffer containing ANG II for 120 min also displayed increased (P< 0.05) HO-1 expression. Stannous mesoporphyrin (30 μmol/l) decreased CO release (P< 0.05) in preparations perfused with and without ANG II; the HO inhibitor also increased (P< 0.05) perfusion pressure, more so in kidneys perfused with that without ANG II. We conclude that ANG II stimulates CO production and release in isolated, perfused rat kidneys. This action of ANG II is linked to the activation of AT1receptors and involves PKC activation and upregulation of renal HO-1 but not of HO-2 protein expression. The study suggests upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction.

20-Hydroxyeicosatetraenoic acid (20-HETE), which promotes renal vasoconstriction, is formed in the rat kidney primarily by cytochrome P-450 (CYP) 4A isoforms (4A1, 4A2, 4A3, 4A8). Nitric oxide (NO) has been shown to bind to the heme moiety of the CYP4A2 protein and to inhibit 20-HETE synthesis in renal arterioles of male rats. However, it is not known whether NO interacts with and affects the activity of CYP4A1 and CYP4A3, the major renal CYP4A isoforms in female rats. Incubation of recombinant CYP4A1 and 4A3 proteins with sodium nitroprusside (SNP) shifted the absorbance at 440 nm, indicating the formation of a ferric-nitrosyl-CYP4A complex. The absorbance for CYP4A3 was about twofold higher than that of CYP4A1. Incubation of SNP or peroxynitrite (PN; 0.01–1 mM) with CYP4A recombinant membranes caused a concentration-dependent inhibition of 20-HETE synthesis, with both chemicals having a greater inhibitory effect on CYP4A3-catalyzed activity. Moreover, incubation of CYP4A1 and 4A3 proteins with PN (1 mM) resulted in nitration of tyrosine residues in both proteins. In addition, PN and SNP inhibited 20-HETE synthesis in renal microvessels from female rats by 65 and 59%, respectively. We previously showed that microvessel CYP4A1/CYP4A3 expression and 20-HETE synthesis are decreased in late pregnancy. Therefore, we investigated whether such a decrease is dependent on NO, the synthesis of which has been shown to increase in late pregnancy. Administration of NG-nitro-l-arginine methyl ester (l-NAME) to pregnant rats for 6 days (days 15-20of pregnancy) caused a significant increase in systolic blood pressure, which was prevented by concurrent treatment with the CYP4A inhibitor 1-aminobenzotriazole (ABT). Urinary NO2/NO3excretion decreased by 40 and 52% in l-NAME- and l-NAME + ABT-treated groups, respectively. Interestingly, renal microvessel 20-HETE synthesis showed a marked increase following l-NAME treatment, and this increase was diminished with coadministration of ABT. These results demonstrate that NO interacts with CYP4A proteins in a distinct manner and it interferes with renal microvessel 20-HETE synthesis, which may play an important role in the regulation of blood pressure and renal function during pregnancy.

We studied the effects of the heme oxygenase (HO) inhibitor stannous mesoporphyrin (SnMP; 40 μmol/kg iv) on renal hemodynamics in anesthetized rats with and without 48-h pretreatment withNG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide (NO) synthesis. SnMP decreased renal blood flow (RBF) and increased renal vascular resistance (RVR) in both groups. The SnMP-induced reduction of RBF inl-NAME-pretreated rats was more prominent than in rats without pretreatment (43 ± 7 vs. 13 ± 3%) as was the SnMP-induced elevation of RVR (87 ± 31 vs. 14 ± 5%). The renal vasoconstrictor effect of SnMP is linked, in part, to amplification of prevailing neurohormonal constrictor mechanisms, since in l-NAME-pretreated rats it was prevented by concurrent administration of prazosin or losartan. However, SnMP (15 μmol/l) also elicits vasoconstriction in isolated, pressurized renal interlobular arteries and the response is more intense in vessels obtained from l-NAME-pretreated rats than from rats without pretreatment. These data indicate that the status of NO synthesis conditions the vascular response to HO inhibition in the rat kidney.

20-HETE, a cytochromeP-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca2+-activated K+channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-Kmarachidonic acid ω-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50from 0.37 ± 0.04 μM in plasmid-transfected arteries to 0.07 ± 0.01 μM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated byN-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, with the release of iron and carbon monoxide (CO). HO not only controls the availability of heme for the synthesis of heme proteins but also is responsible for the generation of CO, which binds to the heme moiety of heme proteins thus affecting their enzymatic activity. Cyclooxygenase (COX) is a heme protein that catalyzes the conversion of arachidonic acid to prostaglandin H(2), the precursor of prostanoids that participate in the regulation of vascular function. The goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells. Endothelial cells stably transfected with the human HO-1 gene exhibited a severalfold increase in human HO-1 mRNA levels, which was accompanied by an increase in HO activity and a marked decrease in prostaglandin (PG) E(2) and 6-keto-PGF(1alpha) levels. Exposure of cells to CoCl(2), an inducer of HO-1 gene expression, resulted in increases in HO-1 protein levels and HO activity. The increase in HO activity was associated with a subsequent decrease in COX activity, which returned to normal levels following normalization of HO activity. The addition of heme resulted in an increase in COX activity with an increase in PGE(2) and 6-keto-PGF(1alpha) levels. The degree of HO-1 expression and, consequently, the level of cellular heme, were directly related to COX activity. These results demonstrate that the heme-HO system can function as a cellular regulator of the expression of vascular COX, thus influencing the generation of prostanoids, PGE(2) and PGI(2), known to play a role in vascular homeostasis.

Our previous study found that angiotensin-converting enzyme (ACE) inhibitors and amlodipine induce NO release from coronary microvessels through a kinin-dependent mechanism. The goal of this study was to determine whether amlodipine could potentiate NO formation during ACE inhibition. Coronary microvessels were isolated from 16 mongrel dogs. Nitrite, the hydration product of NO, from coronary microvessels was quantified by using the Griess reaction. Bradykinin and kallikrein all significantly increased nitrite release from coronary microvessels in a concentration-dependent manner. The ACE inhibitor, ramiprilat, potentiated these effects. Amlodipine also markedly potentiated nitrite production by ramiprilat. For instance, amlodipine (10−10M) enhanced nitrite release induced by ramiprilat (10−7M) from 122 ± 9 to 168 ± 14 pmol/mg (p < 0.05 vs. ramiprilat). Nitrite release potentiated by ramiprilat and amlodipine was entirely blocked by Nω-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase), HOE 140 (Icatibant, a specific B2-kinin receptor antagonist), and dichloroisocoumarin (DCIC, a serine protease inhibitor that blocks local kinin formation). These results clearly show that there is a synergistic effect on NO formation when amlodipine is combined with ACE inhibition. Our data suggest that kinin-mediated coronary NO production may contribute importantly to the beneficial therapeutic action of ACE inhibitors, especially in combination with amlodipine in the treatment of heart disease.

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt−1⋅ day−1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

Increasing evidence suggests that angiotensin-converting enzyme (ACE) inhibitors can increase vascular nitric oxide (NO) production. Recent studies have found that combined inhibition of ACE and neutral endopeptidase (NEP) may have a greater beneficial effect in the treatment of heart failure than inhibition of ACE alone. Amlodipine, a calcium channel antagonist, has also been reported to have a favorable effect in the treatment of patients with cardiac dysfunction. The purpose of this study was to determine whether and the extent to which all of these agents used in the treatment of heart failure stimulate vascular NO production. Heart failure was induced by rapid ventricular pacing in conscious dogs. Coronary microvessels were isolated from normal and failing dog hearts. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. ACE and NEP inhibitors and amlodipine significantly increased nitrite production from coronary microvessels in both normal and failing dog hearts. However, nitrite release was reduced after heart failure. For instance, the highest concentration of enalaprilat, thiorphan, and amlodipine increased nitrite release from 85 +/- 4 to 156 +/- 9, 82 +/- 7 to 139 +/- 8, and 74 +/- 4 to 134 +/-10 pmol/mg (all *p <.01 versus control), respectively, in normal dog hearts. Nitrite release in response to the highest concentration of these two inhibitors and amlodipine was reduced by 41% and 31% and 32% (all #p <.01 versus normal), respectively, in microvessels after heart failure. The increase in nitrite induced by either ACE or NEP inhibitors or amlodipine was entirely abolished by Nw-nitro-L-arginine methyl ester, HOE 140 (a B2-kinin receptor antagonist), and dichloroisocoumarin (a serine protease inhibitor) in both groups. Our results indicate that: 1) there is an impaired endothelial NO production after pacing-induced heart failure; 2) both ACE and NEP are largely responsible for the metabolism of kinins and modulate canine coronary NO production in normal and failing heart; and 3) amlodipine releases NO even after heart failure and this may be partly responsible for the favorable effect of amlodipine in the treatment of heart failure. Thus, the restoration of reduced coronary vascular NO production may contribute to the beneficial effects of these agents in the treatment of heart failure.

Rings of thoracic aortae taken from rats made hypertensive by aortic coarctation express a calcium-dependent basal tone. We investigated whether this basal tone is mediated by prostanoids. To this end, we contrasted the effects of indomethacin, an inhibitor of cyclooxygenase, and of ifetroban, an antagonist of thromboxane A2/prostaglandin endoperoxide H2 receptors, on basal tone in aortic rings taken from normotensive and hypertensive rats. Rings with endothelium from normotensive rats were unaffected by indomethacin and ifetroban. However, in endothelium-intact rings from hypertensive rats, the basal tone was reduced 65 to 75% by indomethacin and ifetroban, but not by CGS13080, an inhibitor of thromboxane synthase. The reductions in tone elicited by indomethacin and ifetroban in rings from hypertensive rats were eliminated upon removal of the endothelium and were attenuated when the rings were pretreated with an inhibitor of nitric oxide synthase (N omega-nitro-L-arginine methyl ester or N omega-nitro-L-arginine) or an inhibitor of soluble guanylate cyclase. Neither indomethacin nor ifetroban affected tissue cGMP levels or nitrite release in aortic rings taken from hypertensive rats. However, sodium nitroprusside offset the inhibitory effects of N omega-nitro-L-arginine methyl ester, on the relaxant responses to indomethacin and ifetroban. These data suggest that a constrictor prostanoid other than thromboxane A2, presumably prostaglandin endoperoxide H2 contributes to the implementation of the basal tone in rings from hypertensive rats and that part of the relaxant response to indomethacin and ifetroban is linked to nitric oxide.

1. Renal arterial injection of bolus doses of angiotensin II or noradrenaline, (0.06, 0.12 and 0.25 µg) caused renal vasoconstriction and decreased blood flow to the kidney in a dose-related manner in dogs anaesthetized by sodium pentobarbital. 2. The effect of angiotensin II and noradrenaline in lowering renal blood flow was reduced during renal arterial infusion of either bradykinin (10 ng min-1 kg-1) or prostaglandin E2 (4 ng min-1 kg-1). 3. Pretreatment of the dogs with an inhibitor of prostaglandin synthesis, sodium meclofenamate (5 mg/kg), blunted the inhibitory action of bradykinin, but not that of prostaglandin E2, on renal vascular reactivity to angiotensin II and noradrenaline. 4. These results indicate that bradykinin reduces the renal vasoconstriction induced by angiotensin II and noradrenaline in the dog by a mechanism dependent upon synthesis of prostaglandins.

1. To test the hypothesis that endogenous prostaglandins contribute to the regulation of blood pressure, we studied the effect of an inhibitor of prostaglandin synthesis, indomethacin, on mean aortic blood pressure in the normotensive, unanaesthetized rabbit. 2. Daily administration of indomethacin at 42 μmol/kg subcutaneously, but not of vehicle only, for 14 consecutive days, elevated the average mean arterial pressure in seven rabbits from 88 ± 3 mmHg on the last day of the control period to 105 + 3 mmHg (P < 0·01) and 107 ± 2 mmHg (P < 0·01) on days 6 and 14 of indomethacin treatment respectively, and reduced the urinary excretion of prostaglandin-like substance from 1·06 ± 0·26 to 0·17 ± 0·05 nmol of prostaglandin E2 equivalents/ day (P < 0·05; n = 5). Neither indomethacin nor the vehicle affected the intake of water, the 24 h urine volume, the cumulative difference between sodium intake and urinary sodium excretion, or the plasma volume. 3. The results of the study are compatible with the hypothesis that one or more prostaglandins contribute to maintain normotension in the rabbit and that reduction in prostaglandin biosynthesis may cause blood pressure to rise.

Bradykinin is a substrate for both neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). Our previous studies showed that ACE inhibitors can stimulate nitric oxide production in coronary microvessels, which is mediated by local kinins. Whether inhibition of NEP also can affect local vascular NO production has not been established. To determine the role of NEP in the control of NO production, coronary microvessels were isolated from seven mongrel dogs. Two NEP inhibitors, phosphoramidon and thiorphan, and an ACE inhibitor, ramiprilat, were used. Nitrite, the metabolite of NO in aqueous solution, was measured by using the Griess reaction. Phosphoramidon and thiorphan (10−6M) increased nitrite production from 80 ± 6 to 136 ± 6 and 144 ± 7 pmol/mg, respectively. Ramiprilat (10−8M) increased nitrite production from 78 ± 6 to 155 ± 7 pmol/mg wet weight. The effect of these agents on nitrite release was blocked by L-NAME, which inhibits NO synthase, HOE-140, which blocks bradykinin B2-receptor, and dichloroisocoumarin, which blocks kinin-forming enzymes. These results clearly indicate that inhibition of kinin metabolism by using neutral endopeptidase inhibitors increases NO production from coronary microvessels. Thus neutral endopeptidase plays an important role in local kinin-modulated NO production in the coronary microcirculation and NEP inhibitors may be useful clinical tools in treatment of cardiovascular disease.

This study was designed to assess the contribution of thromboxane (Tx) A2 to the pathogenesis of renal dysfunction in rats with angiotensin II (Ang ID-salt hypertension. Hypertension was induced in rats drinking 0.15mol/l NaCI by infusion of Ang II (125ng/min, intraperitoneaily) for 12 days. Relative to values in water- and saline-drinking rats without Ang II infusion, rats with Angll-salt hypertension exhibited increased renal vascular resistance, decreased renal blood flow, and increased renal excretion and glomerular synthesis of TxB2. Treatment with an inhibitor of TxA2 synthesis, UK 38,485, had no effect on renal function in normotensive and hypertensive rats. Similarly, the TxA2 and prostaglandin endoperoxide antagonist SQ 29,548 did not affect renal function in normotensive rats. In contrast, in rats with Angll-salt hypertension of 12 days' duration, SQ 29,548 caused a reduction in renal vascular resistance, allowing for maintenance of renal blood flow in the face of an accompanying reduction in blood pressure. A comparable reduction in renal perfusion pressure, produced by constriction of the abdominal aorta above the renal arteries, was not accompanied by a reduction in renal vascular resistance in Ang ll-salt hypertensive rats. Therefore, the SQ 29,548-induced lowering of renal vascular resistance is attributable not to renal blood flow autoregulation, but to blockade of the renal vasoconstrictor actions of TxA2 and/or prostaglandin endoperoxides. This interpretation implies that pressor eicosanoids contribute to increase renal vascular resistance in rats with severe Angll-salt hypertension.

Plasma levels of norepinephrine, epinephrine, and dopamine were measured in conscious, male rabbits. Twenty-minute infusions of 5, 50, and 500 ng/kg/min angiotensin II elevated mean arterial blood pressure by 8 (P< 0.05), 23 (P< 0.01), and 33 mm Hg (P< 0.01), respectively. Plasma catecholamines were unchanged at all levels of angiotensin II infusion except epinephrine which rose from 15 ± 3 to 52 ± 17 pg/ml at 500 ng/kg/min. These data suggest an action of the peptide at the adrenal medulla but offer no evidence of a generalized stimulation of sympathetic discharge by antiotensin II.

This study was designed to examine the influence of angiotensin II on the renal hemodynamic response to blockade of thromboxane A2 and prostaglandin H2 receptors with SQ29548 (2 mg/kg, i.v. bolus, plus 2 mg kg-1 hr-1 infusion) in anesthetized rats. In control rats without any pretreatment, SQ29548 did not change blood pressure, but increased renal blood flow from 7.0 +/- 0.4 to 7.7 +/- 0.4 ml min-1 g kidney weight-1 (P < .05) and decreased renal vascular resistance from 18.1 +/- 1.0 to 16.2 +/- 0.8 mm Hg/ml min-1 g kidney weight-1 (P < .05). In contrast, SQ29548 was without effect on renal blood flow or renal vascular resistance in rats pretreated with saralasin or captopril to block angiotensin II actions and formation, respectively. SQ29548 also increased renal blood flow and decreased renal vascular resistance in rats pretreated with captopril in which the plasma concentration of angiotensin II was fixed at elevated levels by concurrently infusing the peptide at doses ranging from 5 to 80 ng/min. In this experimental setting, the administration of SQ29548 reduced preglomerular vascular resistance selectively. Because, according to previous studies, SQ29548 does not interfere with the direct vasoconstrictor actions of angiotensin II, the renal vasodilatory effect of SQ29548 in rats with elevated plasma angiotensin II is attributable to interference with the operation of mechanisms of vasoconstriction mediated by activation of thromboxane A2-prostaglandin H2 receptors. We conclude that the status of the renin-angiotensin system is a determinant of the renal vasodilatory response to SQ29548.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of nitric oxide (NO) synthesis increase blood pressure and decrease regional blood flow. We investigated whether blockade of the renin-angiotensin, sympathetic nervous, prostaglandin or vasopressin systems attenuates the effects of the NO synthesis inhibitor NG-nitro-L-arginine (L-NOARG) on mean arterial pressure and renal blood flow in anesthetized male Sprague-Dawley rats. Treatment with L-NOARG (10 mg kg-1, i.v. bolus plus infusion at 20 mg kg-1 hr-1) increased mean arterial pressure from 113 +/- 2 to 133 +/- 4 mm Hg, decreased renal blood flow from 7.7 +/- 0.6 to 4.3 +/- 0.6 ml min-1 g-1 and increased renal vascular resistance from 15.8 +/- 1.8 to 36.9 +/- 6.1 mm Hg/ml min-1 g-1. These effects were attenuated in rats pretreated with L-arginine to interfere with the inhibitory action of L-NOARG on NO synthesis, but not in rats pretreated with D-arginine. Acetylcholine did not relax aortic rings taken from rats treated with L-NOARG, consistent with inhibition of NO-mediated vasorelaxation. The pressor and renal vasoconstrictor effects of L-NOARG were not impaired in rats separately pretreated with either chlorisondamine, captopril, prazosin, indomethacin or d(CH2)5Tyr(Me)AVP, or in rats pretreated with chlorisondamine, captopril and indomethacin in combination. Collectively, these data argue against significant contribution of the sympathetic nervous system, the renin-angiotensin system, vasopressor prostanoids or vasopressin to the mechanisms of L-NOARG-induced elevation of mean arterial pressure and renal vasoconstriction in anesthetized rats.

We compared the effects of sodium meclofenamate (5 mg/kg i.v.), an inhibitor of prostaglandin synthesis, on renal function in six unanesthetized dogs maintained for 5 to 7 days before the experiment on 100 mEq/day of NaCl and after sodium depletion by furosemide administration and salt deprivation. Plasma renin activity (PRA) during sodium depletion (12.35 +/- 3.93 ng ml-1 hr-1) was higher than during sodium repletion (1.46 +/- 0.47 ng ml-1 hr-1; P less than .05). The administration of meclofenamate did not alter mean arterial pressure, renal blood flow, urine volume, excretion of sodium and potassium or PRA in the sodium replete dog. However, during sodium depletion meclofenamate reduced renal blood flow from 168 +/- 35 to 105 +/- 23 ml/min (P less than .01) and urine flow from 0.32 +/- 0.09 to 0.16 +/- 0.05 ml/min (P less than .05) but did not affect mean arterial pressure, electrolyte excretion or PRA. The meclofenamate-induced reduction in renal blood flow during sodium depletion was well correlated with control PRA. After administration of meclofenamate, the urinary excretion of immunoreactive prostaglandin E2 fell by 66% (P less than .05) in the sodium replete dog and by 72% (P less than .05) in the sodium depleted dog. This study demonstrates that the effects of meclofenamate on renal hemodynamics vary with the state of sodium balance and in relation to PRA.

In the dog anesthetized with pentobarbital, the effect of bradykinin on the lowering of renal blood flow produced by bolus injections of angiotensin II was studied. Injection into the renal artery of angiotensin II (0.06--0.50 microgram) caused vasoconstriction and decreased blood flow to the kidney in a dose-related manner. Renal arterial infusion of bradykinin (10 ng kg-1 min-1), prostaglandin (PG) E2 (4 ng kg-1 min-1) or PGI2 (4 ng kg-1 min-1) produced renal vasodilation and inhibited the vasoconstrictor effect of angiotensin II. However, renal arterial infusion of another vasodilatory peptide, substance P (2 ng kg-1 min-1), did not alter the effect of angiotensin II on the renal vasculature. Pretreatment of dogs with an inhibitor of PG synthesis, sodium meclofenamate (5 mg/kg), abolished the inhibitory effect of bradykinin on angiotensin II-induced renal vasoconstriction. In contrast, renal arterial infusion of either PGE2 or PGI2 was equally effective in reducing the renal vascular effect of angiotensin II in animals with and without meclofenamate pretreatment. These results suggest that bradykinin reduces the reactivity of the renal vasculature to angiotensin II by a mechanism dependent upon PG synthesis.

The present study was designed to investigate the effect of dexamethasone treatment for 2 weeks (2.5 mg/kg/week, subcutaneously) on the level of unesterified fatty acids, particularly arachidonic acid, in the renal medulla of rats, and to relate the observed effect to changes in the tissue concentration and the fatty acid composition of renal medulla phospholipids and triglycerides. Dexamethasone treatment caused an increase in the renal inner medulla level of unesterified fatty acids, including arachidonic acid, that was associated with a reduction of triglycerides and of arachidonic acid esterified into triglycerides, and with an increase in the rate of fatty acids esterification into triglycerides. In contrast, dexamethasone treatment did not affect the renal medulla concentration of phospholipids, the arachidonic acid content of renal medulla phospholipids, or the rate of esterification of fatty acids into renal medulla phospholipids. In the face of increased fatty acid esterification into triglycerides, the finding of reduced triglyceride levels in the renal medulla of dexamethasone-treated rats suggests excessive triglyceride breakdown. If so, fatty acids including arachidonic acid liberated from triglycerides may contribute to elevation of unesterified fatty acid levels in the renal medulla during dexamethasone treatment. The increased level of free arachidonic acid in the renal medulla of dexamethasone-treated rats may explain in part the reported effect of this steroid in increasing urinary prostaglandins.

1. Male Wistar rats were used to examine whether renal prostaglandins (PG) contribute to functional compensatory changes in the remaining kidney after uninephrectomy. Total urinary excretion of PGE2, PGF2α and 6-keto-PGF1α, a metabolite of PGI2, was measured before and during 11 days after surgery in uninephrectomized (UNX, n = 8) and sham-operated control (S, n = 9) rats. 2. Urine volume was increased for the UNX rats on days 2–8 after surgery, but not thereafter, and urine osmolality for UNX was decreased on post-surgery days 1–9; total urinary sodium excretion did not differ between the two groups. Urinary PGE2 excretion was decreased in the UNX rats to 50% of S rat values, except on post-surgery days 2 and 3, when values for the two groups were not significantly different. Urinary PGF2α excretion by the UNX rats was reduced to 50% of S rat values throughout the post-surgery period. In contrast, the urinary excretion of 6-keto-PG1α by the UNX rats did not differ significantly from that by the S rats, except on a single day. 3. Chronic renal functional adaptation to renal mass reduction does not depend on increased renal prostaglandin systems activity; however, increased intrarenal PGE2 activity in the remaining kidney might be a factor in compensatory adjustments during the first days after surgery.

Experiments were performed to determine the effect of chronic therapy with the potent and long-acting thromboxane (TX) A2/prostaglandin endoperoxide (TP) receptor antagonist, ifetroban, on hypertension development and the incidence of stroke in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP instrumented with radiotelemetry probes, for continuous monitoring of arterial blood pressure, were given 1% NaCl to drink and Stroke-Prone Rodent Diet and were chronically treated with ifetroban (20 mg/kg/day, n=l0) or vehicle (n=12) starting at 16.5 weeks of age. Ifetroban did not affect blood pressure or the development of proteinuria and cerebrovascular lesions, Chronic administration of a higher dose ifetroban (40mg/kg/day) starting at 7 weeks of age was also without effect on blood pressure and stroke in noninstrumented saline-drinking SHRSP. These results do not support a major role for TXA, and its endoperoxide precursors in the elevation of blood pressure and the development of cerebrovascular lesions in saline-drinking SHRSP.

The purpose of the present study was to determine whether porcine pituitary tissue contains kininogenase activity. Porcine anterior and posterior pituitaries were homogenized in 0.25 M sucrose containing 10 mM Tris/HCl (pH 7.5) and the homogenate subjected to differential centrifugation (10,000 x g for 20 min, then 105,000 x g for 60 min). Fractions were examined for kininogenase activity using canine plasma kininogen as the substrate; kinins were measured by bioassay or radioimmunoassay. The anterior pituitary was found to have kininogenase activity highly concentrated in the 105,000 x g particulate pellet (376 ng of bradykinin generated/mg of protein/h) with little or no activity detectable in other fractions. The porcine posterior pituitary 105,000 x g pellet contained 22% of the kininogenase activity of the anterior pituitary. Anterior pituitary kininogenase activity had a broad pH optimum (pH 7.5-9.0), was heat-sensitive, was inhibited by dithiothreitol, soybean trypsin inhibitor, aprotinin, and diisopropyl fluorophosphate, and was resistant to lima bean trypsin inhibitor, pepstatin, and hirudin. Bradykinin was found to be the principal kinin generated by the anterior pituitary kininogenase as determined by high performance liquid chromatography. The anterior pituitary kininogenase differed from other kininogenases in its sensitivity to inhibitors and substrate specificity.

The effects on kidney function of aprotinin, an inhibitor of kallikrein and other serine proteinases, were investigated in rats made diuretic by infusion of 0.9% saline. Late proximal, early distal and late distal tubular fluid samples were collected before and after aprotinin administration (20,000 kallikrein I.U./kg b.wt. i.v.). Glomerular filtration rate and urinary excretion of solute and water were assessed simultaneously. Aprotinin did not alter blood pressure or glomerular filtration rate, but reduced urine flow from 23.8 +/- 4.5 to 16.4 +/- 3.4 microliter min-1 100 g-1 (P less than .05) and urinary kinin excretion from 23.5 +/- 3.2 to 10.8 +/- 1.9 pg min-1 100 g-1 (P less than .01). Aprotinin increased the tubular fluid to plasma inulin concentration ratio at late distal tubule puncture site, but not at late proximal or early distal tubule collection sites. Estimates of fluid reabsorption in the distal convoluted tubule, expressed as a percentage of glomerular filtration rate, as a percentage of delivery to this nephron segment or as net volume transported, increased after the administration of aprotinin by 22, 24 and 23% (P less than .05), respectively. In contrast, aprotinin did not alter the estimates of fluid reabsorption in the proximal convoluted tubule, the loop of Henle or the collecting tubule. We conclude that the antidiuretic effect of aprotinin in saline-expanded rats is related to selective augmentation of fluid reabsorption in the distal convoluted tubule. This effect of aprotinin may be the expression of reduced renal kinin levels, inhibition of serine proteases other than kallikrein or other unrecognized properties of the agent.

To determine whether chronic glucocorticoid excess influences the metabolism of arachidonic acid to prostaglandins (PGs) in the renal cortex, the authors investigated the effects of dexamethasone treatment (2.5 mg/kg/week) on the metabolism of arachidonic acid by renal cortex homogenates and microsomes and by isolated glomeruli, and on the release of immunoreactive prostanoids from isolated glomeruli incubated for 30 min in buffered salt solution at 37 degrees C. Glomeruli from dexamethasone-treated rats released, during basal incubation conditions, about twice (P less than .01) as much PGE2 and PGF2 alpha as did glomeruli from vehicle-treated rats. During incubation with arachidonic acid (33 microM) or calcium ionophore, A23187 (2.0 micrograms/ml), the release of PGE2 and PGF2 alpha from glomeruli of rats receiving dexamethasone also exceeded (P less than .01) the release from glomeruli of control rats. The rate of conversion of [1-14C]arachidonic acid to PGE2 and PGF2 alpha and to less polar metabolites having the chromatographic mobility of 5-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid, by isolated glomeruli and by renal cortex homogenates and microsomes from dexamethasone-treated rats, was higher (P less than .01) than the conversion by glomeruli and renal cortex homogenates and microsomes from control rats. The metabolism of arachidonic acid to the nonpolar metabolite(s) was not inhibited by indomethacin (10 microM), suggesting that it is not catalyzed by cyclooxygenase. The authors conclude that chronic dexamethasone treatment increases the release of glomerular PGE2 and PGF2 alpha and the metabolic transformation of arachidonic acid by glomeruli and by renal cortex homogenates and microsomes via both cyclooxygenase and noncyclooxygenase pathways.

We contrasted, in normotensive and hypertensive rats, the effect of L-arginine on isometric tension of phenylephrine-contracted rings of aorta bathed in Krebs' bicarbonate buffer on cyclic guanosine monophosphate content of aortic rings and on blood pressure. L-Arginine had no effect on isometric tension or cyclic guanosine monophosphate content of aortic rings from normotensive controls. Conversely, L-arginine (10(-5)-10(-3) mol/l) induced concentration-dependent relaxation of unrubbed and rubbed rings of thoracic and abdominal aorta and, as well, L-arginine (10(-3) mol/l) increased cyclic guanosine monophosphate in unrubbed and rubbed rings of thoracic aorta taken from hypertensive rats 7 to 14 days after aortic coarctation. Similar relaxations to L-arginine were seen in rings of thoracic and abdominal aorta from rats made hypertensive by infusion of angiotensin II for 7 to 8 days but not in rings of thoracic aorta from rats with aortic coarctation-induced hypertension of 28 to 42 days. Relaxant responses to L-arginine in rings of thoracic aorta from hypertensive rats 7 to 14 days after aortic coarctation were unaffected by pretreatment of rings with dexamethasone (10(-7) mol/l) or L-arginine (10(-4) mol/l) but were blunted by NG-nitro-L-arginine methylester (3 x 10(-4) mol/l) or methylene blue (10(-5) mol/l). Our results suggest that the vasorelaxant effects of L-arginine in aortic rings from hypertensive rats 7 to 14 days after aortic coarctation and 7 to 8 days after commencing angiotensin II infusion are mediated by nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of SQ-20881, an inhibitor of the peptidyl dipeptidase that degrades kinins and converts angiotensin I to angiotensin II, on the urinary excretion of immunoreactive prostaglandin E2 (iPGE2) was studied in rats receiving either deoxycorticosterone (DOCA, 5 mg/day s.c.) or sesame oil vehicle for 10 days before and then during the study, DOCA-treated animals had higher urinary excretion of iPHE2 and kallikrein, and lower plasma renin, than did animals injected with oil only. In rats pretreated with DOCA, infusion of SQ-20881 (1.2 mg/day s.c.) for 6 days increased iPGE2 excretion from 87.3 +/- 1.9 to 150.9 +/- 14.5 ng/day (P < .05). In contrast, in rats pretreated with vehicle, SQ-20881 had no significant effect on urine iPGE2. The enzyme inhibitor did not affect the intake of fluid, the volume of urine or the urinary excretion of kallikrein and electrolytes in either DOCA- or vehicle-treated animals. The blood pressure reduction elicited by a bolus injection of bradykinin (1.0 microgram i.v.) was greater in rats receiving SQ-20881 than in vehicle-infused controls, both in the DOCA- and in the sesame oil-treated groups, suggesting inhibition of kinin degradation by the converting enzyme inhibitor. These results indicate that DOCA or the consequences of its administration are required for SQ-20881 to increase iPHE2 excretion in the rat. Such an effect of the inhibitor probably relates to stimulation of renal prostaglandin synthesis consequent to elevation of kinin levels.

Effects of porcine 1-21 endothelin (ET-1) on [Ca2+]i, [Na+]i, and [Cl-]i and on membrane potential were studied in individual mesangial (MC) and vascular smooth muscle (VSMC) cells using microspectrofluorimetry of fura-2, SBFI, SPQ, and bis-oxonol, respectively. ET-1 increased [Ca2+]i by fivefold, showing an immediate and a sustained phase of response. Ca(2+)-free medium and nifedipine pretreatment significantly curtailed the sustained phase of response to ET-1. These findings were confirmed in studies of vascular ring preparations, demonstrating that Ca2+ influx may account for at least 50% of contraction. ET-1 caused immediate and sustained depolarization of MC and VSMC. This could not be attributed to Na+ influx, since fluorescence of SBFI was not affected by ET-1 and Na(+)-free medium did not abolish the ET-1-induced membrane depolarization. Studies of SPQ fluorescence changes induced by ET-1 revealed an increase in fluorescence intensity consistent with the decrease in [Cl-]i. A Cl- channel blocker, IAA-94, abolished changes in SPQ fluorescence and curtailed sustained phases of membrane depolarization and [Ca2+]i elevation in response to ET-1, but did not affect KCl-induced [Ca2+]i transients. IAA-94 also attenuated the ET-1-induced contraction of aortic rings. Microinjection of either calcium gluconate or inositol 1,4,5-trisphosphate (IP3) in SPQ-loaded cells resulted in an increase in fluorescence mimicking the effect of ET-1. These changes were blunted by pretreatment of cells with BAPTA and incubation in Ca(2+)-free medium. When IP3 was microinjected into fura-2-loaded MC, this resulted in immediate and sustained elevation of [Ca2+]i. In conclusion, generation of IP3 results in mobilization of intracellular Ca2+ stores and activation of Cl- channels. Ensuing Cl- efflux causes membrane depolarization and, in turn, activation of voltage-dependent Ca2+ channels, resulting in sustained elevation of [Ca2+]i which is indispensable for the full-scale contraction produced by ET-1.

In pentobarbital anesthetized dogs, close arterial injections of bradykinin and kallidin elicit a dose related increase in femoral blood flow. Treatment with the kininase inhibitor BPP9αaugments the femoral blood flow responses to bradykinin and kallidin by five and threefold respectively. The sensitivity of the preparation permits the detection of 0.5-1 ng of either bradykinin or kallidin in untreated dogs and as little as 0.1 ng of kinin peptides in animals receiving BPP9α. This sensitivity and the steepness of the dose response curves make this procedure suitable for the assay of kinins.

This study was designed to contrast the effects of prolonged treatment with a thromboxane (Tx) synthase inhibitor (UK 38485 or SC 41156) and a Tx receptor antagonist (SQ 29548) on the development of angiotensin II (Ang II)-salt-induced hypertension. Ang II infusion (125 ng/min i.p. for 12 days) in rats drinking 0.15 M NaCl resulted in severe hypertension accompanied by proteinuria, reduction of urinary creatinine excretion and augmentation of urinary TxB2 excretion and TxB2 release from aortic rings and renal cortex slices. In saline-drinking rats undergoing Ang II infusion, the concomitant administration by gavage of UK 38485 (100 mg/kg/day) or SC 41156 (25 mg/kg/day) reduced serum and urinary TxB2 and TxB2 release from aortic rings and/or renal cortex slices, but it was without effect on the development of hypertension. In contrast, concomitant infusion of SQ 29548 (4.2 mg/24 hr s.c.) significantly attenuated the increase of blood pressure produced by the infusion of Ang II in saline-drinking rats. This effect of SQ 29548 may be the consequence of blockade of the actions of one or more endogenous eicosanoids that increase blood pressure by a mechanism(s) involving interaction with TxA2 receptors. This implies that pressor eicosanoids play a contributory role in the development of severe Ang II-salt hypertension.

The injection of a large bolus of angiotensin II causes a biphasic blood pressure response in the conscious rabbit. To investigate contribution of prostaglandins (PGs) to the depressor phase of the blood pressure response, we studied the blood pressure effect of i.v. bolus injections of angiotensin II before and after the administration of an inhibitor of cyclooxygenase, indomethacin or sodium meclofenamate (10 mg kg-1), and in relation to associated changes in the plasma concentration of immunoreactive PGs. In conscious rabbits, angiotensin II (0.05-5.00 microgram kg-1) produced a dose-related pressor response which at both 1.5 and 5.0 micrograms kg-1 was followed by lowering of blood pressure to below the preinjection level. Neither indomethacin nor meclofenamate affected the maximal rise in pressure produced by angiotensin II, but both cyclooxygenase inhibitors augmented the duration of the pressor phase and abolished the depressor phase of the hemodynamic response to angiotensin II at 1.5 to 5.0 micrograms kg-1. After administration of angiotensin II, 5 micrograms kg-1, the plasma concentration of 6-keto-PGF1 alpha increased (P less than .01) from 218 +/- 21 pg/ml by 221 and 235% during the pressor and the depressor phases of the blood pressure response, respectively. Increments in plasma 6-keto-PGF1 alpha correlated inversely with the duration of the pressor phase and directly with the maximal lowering of blood pressure during the depressor phase. Plasma levels of PGE2 and PGF2 alpha also were increased by the peptide but the increments were not correlated with any aspect of the blood pressure response. These data suggest that a mechanism involving PGs both curtails the pressor phase and mediates the depressor phase of the hemodynamic response to pharmacological doses of angiotensin II in the conscious rabbit.

This study was designed to investigate involvement of potassium channels in the action of nitric oxide facilitating reduction of basal tone by thromboxane A2/prostaglandin H2receptor blockade with ifetroban in rings of thoracic aorta taken from rats with aortic coarctation-induced hypertension. Ifetroban-induced reduction of basal tone in aortic rings without drug pretreatment was attenuated (P<0.05) in rings pretreated with the nitric oxide synthesis inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 3 × 10<-4mol/L; 0.55±0.09 g versus 0.23±0.07 g). The vasorelaxing effect of ifetroban also was decreased (P<0.05) in preparations pretreated with a potassium channel blocker, either tetraethylammonium (TEA; 10-2mol/L) or 4-aminopyridine (4-AP; 3 × 10-3mol/L). Ifetroban-induced reduction of basal tone was not attenuated in preparations pretreated first with L-NAME and then with sodium nitroprusside (SNP; 6±1 nmol/L) to compensate for the loss of endogenous nitric oxide. However, the facilitatory effect of SNP on ifetroban-induced relaxation of aortic rings pretreated with L-NAME alone was not demonstrable in rings pretreated with L-NAME plus TEA or 4-AP. These observations suggest that a mechanism involving nitric oxide and potassium channels facilitates the reduction in basal tone produced by ifetroban in aortic rings of rats with aortic coarctation-induced hypertension

The acute vasodepressor effect of AT1 angiotensin receptor blockers losartan and CL329167 was compared in spontaneously hypertensive rats (SHR) pretreated and not pretreated with NG-monomethyl-L-arginine (LNMMA; 15 mg/kg iv bolus plus infusion at 10 mg/kg/h), an inhibitor of nitric oxide (NO) synthesis. The antihypertensive effect of losartan (30 mg/kg, iv) in SHR pretreated with LNMMA (-13±4 mmHg) was greatly diminished (P<0.01) relative to the antihypertensive effect of losartan in SHR not pretreated with LNMMA (-44±8 mmHg). Similarly, the antihypertensive effect of CL329167 (5 mg/kg, iv) in SHR pretreated with LNMMA (-12±3 mmHg) was surpassed (P<0.01) by the antihypertensive effect in SHR not pretreated with LNMMA (-41±4 mmHg). However, pretreatment of SHR with LNMMA did not minimize the vasodepressor effect of prazosin, isoproterenol or sodium nitroprusside The impairment in

The role of renin in the regulation of its substrate was evaluated after administration of homologous renin. In normal rats, increases in plasma renin were associated with decreases in substrate. In nephrectomized rats, renin delayed but did not prevent the rise in substrate seen in nephrectomized controls, in spite of the high levels of plasma renin. An inverse relationship between renin and substrate levels was found after adrenalectomy and during pregnancy, but not after delivery. It is concluded that substrate consumption by renin plays a minor role in regulating substrate levels.

In rats administration of renin decreased plasma angiotensinogen and increased plasma renin, while administration of angiotensin had the opposite effects. However, both agents stimulated angiotensinogen formation by the liver. The stimulating effect of angiotensin II, and to a lesser extent of angiotensin I, was also demonstrated by adding these peptides to the perfusate of an isolated liver preparation. We propose a positive feedback effect of circulating angiotensin on angiotensinogen formation and release.We thank Dr. F. M. Bumpus for the supply of angiotensin I and Mrs. Kathleen Rice for technical assistance.

A simple, sensitive, and accurate method for the routine measurement of plasma renin in the rat is described. It is based on the determination of angiotensin I formed when plasma is incubated with an excess of purified homologous substrate. Plasma angiotensinase activity is inhibited by the combination of EDTA, DFP, and BAL, thus permitting complete recovery of angiotensin and renin.

The effect of alterations of dietary potassium intake on the plasma concentration and the urinary excretion of vasopressin was studied in male rats. Ingestion of a high potassium diet resulted in increases in the plasma concentrations of potassium and vasopressin, systolic blood pressure, urine flow, and urinary vasopressin excretion. Ingestion of a low potassium diet had little effect on the plasma vasopressin concentration and systolic blood pressure but caused decreases in the plasma potassium concentration and urinary vasopressin excretion. The results indicate that physiological changes in the plasma potassium concentration or some other consequence of altered dietary potassium intake can affect vasopressin release and excretion.

Administration of diethylstilbestrol to normal or nephrectomized rats causes an increase in angiotensinogen concentration in plasma associated with an increase in the pressor response to renin but no change in the pressor response to angiotensin.

In the isolated lapine kidney perfused with tyrode solution and prelabeled with [3H] norepinephrine, bradykinin (10 ng/ml) decreased the overflow of tritium elicited by sympathetic nerve stimulation both in the presence and absence of indomethacin. These observations indicate that bradykinin acts at presynaptic sites reducing release of the adrenergic transmitter in the isolated lapine kidney.

Arterial vessels express one or more heme oxygenase (HO) isoenzymes that catalyze the metabolism of heme to carbon monoxide (CO) and biliverdin. Carbon monoxide promotes vasorelaxation through mechanisms that, depending on the vessels, involve activation of soluble guanylate cyclase, stimulation of calcium-activated potassium channels, or diminished synthesis of constrictor mediators, such as, endothelin and 20-HETE. Inhibitors of HO elicit vasoconstriction in vivo and in isolated pressurized arterioles. Inhibitors of HO also enhance myogenic vasoconstriction, as well as the constriction induced by phenylephrine in several vessels. The blood pressure of awake rats is increased by acute treatment with HO inhibitors, a response that is accompanied by attenuation of baroreflex activity. All in all, it would appear that a product of HO activity manufactured by arterial vessels, presumably CO, promotes vasodilation and decreases the reactivity of vascular smooth muscle to myogenic stimuli and constrictor agonists. In doing so, CO of vascular origin may contribute to the implementation of antihypertensive mechanisms. Carbon monoxide produced in central nervous system structures, for example, the nucleus tractus solitarii, also appears to support a blood pressure-lowering mechanism linked to inhibitory modulation of baroreceptor reflex activity. Am J Hypertens 2001;14:62S–67S © 2001 American Journal of Hypertension, Ltd.