Soluble epoxide hydrolase inhibition modulates vascular remodeling.

The soluble epoxide hydrolase enzyme (SEH) and vascular remodeling are associated with cardiovascular disease. Although inhibition of SEH prevents smooth muscle cell proliferation in vitro, the effects of SEH inhibition on vascular remodeling in vivo and mechanisms of these effects remain unclear. Herein we determined the effects of SEH antagonism in an endothelium intact model of vascular remodeling induced by flow reduction and an endothelium denuded model of vascular injury. We demonstrated that chronic treatment of spontaneously hypertensive stroke-prone rats with 12-(3-adamantan-1-yl-ureido) dodecanoic acid, an inhibitor of SEH, improved the increment of inward remodeling induced by common carotid ligation to a level that was comparable with normotensive Wistar Kyoto rats. Similarly, mice with deletion of the gene responsible for the production of the SEH enzyme (Ephx2(-/-)) demonstrated enhanced inward vascular remodeling induced by carotid ligation. However, the hyperplastic response induced by vascular injury that denudes the endothelium was unabated by SEH inhibition or Ephx2 gene deletion. These results suggest that SEH inhibition or Ephx2 gene deletion antagonizes neointimal formation in vivo by mechanisms that are endothelium dependent. Thus SEH inhibition may have therapeutic potential for flow-induced remodeling and neointimal formation.

Epoxyeicosanoids in hypertension.

Epoxyeicosatrienoic acids (EETs) are also known as epoxyeicosanoids that have renal and cardiovascular actions. These renal and cardiovascular actions can be regulated by soluble epoxide hydrolase (sEH) that degrades and inactivates EETs. Extensive animal hypertension studies have determined that vascular, epithelial transport, and anti-inflammatory actions of EETs lower blood pressure and decrease renal and cardiovascular disease progression. Human studies have also supported the notion that increasing EET levels in hypertension could be beneficial. Pharmacological and genetic approaches to increase epoxyeicosanoids in several animal models and humans have found improved endothelial vascular function, increased sodium excretion, and decreased inflammation to oppose hypertension and associated renal and cardiovascular complications. These compelling outcomes support the concept that increasing epoxyeicosanoids via sEH inhibitors or EET analogs could be a valuable hypertension treatment.

Effect of angiotensin-converting enzyme blockade, alone or combined with blockade of soluble epoxide hydrolase, on the course of congestive heart failure and occurrence of renal dysfunction in Ren-2 transgenic hypertensive rats with aorto-caval fistula.

We showed recently that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, retarded the development of renal dysfunction and progression of aorto-caval fistula(ACF)-induced congestive heart failure (CHF) in Ren-2 transgenic hypertensive rats (TGR). In that study the final survival rate of untreated ACF TGR was only 14 % but increased to 41 % after sEH blockade. Here we examined if sEH inhibition added to renin-angiotensin system (RAS) blockade would further enhance protection against ACF-induced CHF in TGR. The treatment regimens were started one week after ACF creation and the follow-up period was 50 weeks. RAS was blocked using angiotensin-converting enzyme inhibitor (ACEi, trandolapril, 6 mg/l) and sEH with an sEH inhibitor (sEHi, c-AUCB, 3 mg/l). Renal hemodynamics and excretory function were determined two weeks post-ACF, just before the onset of decompensated phase of CHF. 29 weeks post-ACF no untreated animal survived. ACEi treatment greatly improved the survival rate, to 84 % at the end of study. Surprisingly, combined treatment with ACEi and sEHi worsened the rate (53 %). Untreated ACF TGR exhibited marked impairment of renal function and the treatment with ACEi alone or combined with sEH inhibition did not prevent it. In conclusion, addition of sEHi to ACEi treatment does not provide better protection against CHF progression and does not increase the survival rate in ACF TGR: indeed, the rate decreases significantly. Thus, combined treatment with sEHi and ACEi is not a promising approach to further attenuate renal dysfunction and retard progression of CHF.

IL6 suppression provides renal protection independent of blood pressure in a murine model of salt-sensitive hypertension.

Impaired cytochrome P450 epoxygenase enzyme (Cyp2c) regulation contributes to renal damage in angiotensin salt-sensitive hypertension (ANG/HS). We hypothesized that interleukin-6 null mice (IL6-/-) would improve Cyp2c regulation and reduce renal damage in hypertensive mice fed a high salt diet. Systolic blood pressure increased to a greater extent in ANG/HS hypertension as compared to angiotensin (ANG) hypertension but blood pressure did not differ between WT and IL6-/- hypertensive groups. Albuminuria, a marker for renal injury, increased significantly in ANG/HS hypertension in WT mice (5,113 +/- 1,050 mug/day) and was attenuated in the ANG/HS IL6-/- group (1,306 +/- 385 mug/day). Renal Cyp2c protein expression significantly decreased with ANG/HS hypertension in WT mice as compared to high salt alone. However, the ability to upregulate Cyp2c expression in response to a high salt diet was restored in the ANG/HS IL6 deficient hypertensive mice. Renal expression of soluble epoxide hydrolase, which inactivates protective epoxygenase metabolites, was significantly reduced in ANG/HS IL6-/- hypertensive mice compared to the ANG/HS WT group. These data suggest that IL6, while having no effect on blood pressure, impairs regulation of epoxygenase producing Cyp2c, which could contribute to the development of renal injury in angiotensin salt-sensitive hypertension.

Epoxyeicosatrienoic Acids and 20-Hydroxyeicosatetraenoic Acid on Endothelial and Vascular Function.

Endothelial and vascular smooth cells generate cytochrome P450 (CYP) arachidonic acid metabolites that can impact endothelial cell function and vascular homeostasis. The objective of this review is to focus on the physiology and pharmacology of endothelial CYP metabolites. The CYP pathway produces two types of eicosanoid products: epoxyeicosatrienoic acids (EETs), formed by CYP epoxygenases, and hydroxyeicosatetraenoic acids (HETEs), formed by CYP hydroxylases. Advances in CYP enzymes, EETs, and 20-HETE by pharmacological and genetic means have led to a more complete understanding of how these eicosanoids impact on endothelial cell function. Endothelial-derived EETs were initially described as endothelial-derived hyperpolarizing factors. It is now well recognized that EETs importantly contribute to numerous endothelial cell functions. On the other hand, 20-HETE is the predominant CYP hydroxylase synthesized by vascular smooth muscle cells. Like EETs, 20-HETE acts on endothelial cells and impacts importantly on endothelial and vascular function. An important aspect for EETs and 20-HETE endothelial actions is their interactions with hormonal and paracrine factors. These include interactions with the renin-angiotensin system, adrenergic system, puringeric system, and endothelin. Alterations in CYP enzymes, 20-HETE, or EETs contribute to endothelial dysfunction and cardiovascular diseases such as ischemic injury, hypertension, and atherosclerosis. Recent advances have led to the development of potential therapeutics that target CYP enzymes, 20-HETE, or EETs. Thus, future investigation is required to obtain a more complete understanding of how CYP enzymes, 20-HETE, and EETs regulate endothelial cell function.

Tumour necrosis factor-α contributes to improved cardiac ischaemic tolerance in rats adapted to chronic continuous hypoxia.

AIM: It has been demonstrated that tumour necrosis factor-alpha (TNF-α) via its receptor 2 (TNFR2) plays a role in the cardioprotective effects of preconditioning. It is also well known that chronic hypoxia is associated with activation of inflammatory response. With this background, we hypothesized that TNF-α signalling may contribute to the improved ischaemic tolerance of chronically hypoxic hearts. METHODS: Adult male Wistar rats were kept either at room air (normoxic controls) or at continuous normobaric hypoxia (CNH; inspired O2 fraction 0.1) for 3 weeks; subgroups of animals were treated with infliximab (monoclonal antibody against TNF-α; 5 mg kg(-1), i.p., once a week). Myocardial levels of oxidative stress markers and the expression of selected signalling molecules were analysed. Infarct size (tetrazolium staining) was assessed in open-chest rats subjected to acute coronary artery occlusion/reperfusion. RESULTS: CNH increased myocardial TNF-α level and expression of TNFR2; this response was abolished by infliximab treatment. CNH reduced myocardial infarct size from 50.8 ± 4.3% of the area at risk in normoxic animals to 35.5 ± 2.4%. Infliximab abolished the protective effect of CNH (44.9 ± 2.0%). CNH increased the levels of oxidative stress markers (3-nitrotyrosine and malondialdehyde), the expression of nuclear factor κB and manganese superoxide dismutase, while these effects were absent in infliximab-treated animals. CNH-elevated levels of inducible nitric oxide synthase and cyclooxygenase 2 were not affected by infliximab. CONCLUSION: TNF-α plays a role in the induction of ischaemia-resistant cardiac phenotype of CNH rats, possibly via the activation of protective redox signalling.

Inhibition of soluble epoxide hydrolase does not improve the course of congestive heart failure and the development of renal dysfunction in rats with volume overload induced by aorto-caval fistula.

The detailed mechanisms determining the course of congestive heart failure (CHF) and associated renal dysfunction remain unclear. In a volume overload model of CHF induced by creation of aorto-caval fistula (ACF) in Hannover Sprague-Dawley (HanSD) rats we explored the putative pathogenetic contribution of epoxyeicosatrienoic acids (EETs), active products of CYP-450 dependent epoxygenase pathway of arachidonic acid metabolism, and compared it with the role of the renin-angiotensin system (RAS). Chronic treatment with cis-4-[4-(3-adamantan-1-yl-ureido) cyclohexyloxy]benzoic acid (c-AUCB, 3 mg/l in drinking water), an inhibitor of soluble epoxide hydrolase (sEH) which normally degrades EETs, increased intrarenal and myocardial EETs to levels observed in sham-operated HanSD rats, but did not improve the survival or renal function impairment. In contrast, chronic angiotensin-converting enzyme inhibition (ACEi, trandolapril, 6 mg/l in drinking water) increased renal blood flow, fractional sodium excretion and markedly improved survival, without affecting left ventricular structure and performance. Hence, renal dysfunction rather than cardiac remodeling determines long-term mortality in advanced stage of CHF due to volume overload. Strong protective actions of ACEi were associated with suppression of the vasoconstrictor/sodium retaining axis and activation of vasodilatory/natriuretic axis of the renin-angiotensin system in the circulating blood and kidney tissue.

Small artery resistance increases during the development of renal hypertension.

Vascular pressures were measured in the principal (A1) arteriole and in upstream small arteries of the rat cremaster muscle to investigate vascular resistance changes associated with one-kidney, one clip Goldblatt hypertension. Pressure measurements were made at a proximal and distal site of each vessel using a servonull micropipette system. Vessel diameters were measured using video microscopy. A1 arteriole and external spermatic artery diameters were both decreased after 2 and 4 weeks of hypertension. Mean arterial pressure was elevated after 2 weeks of hypertension (106 +/- 4 mm Hg versus 140 +/- 5 mm Hg). Likewise, vascular pressures were elevated at every site: pudicepigastric artery (36%), external spermatic artery (47%), and A1 arteriole (38%). The pressure drop along the external spermatic artery was increased (87%) after 2 weeks of hypertension. Mean arterial pressure was further elevated from 2-4 weeks of hypertension (105 +/- 4 mm Hg versus 162 +/- 7 mm Hg) but only the proximal pudic-epigastric artery pressure was further elevated during this time from 2 to 4 weeks (131 +/- 5 mm Hg versus 147 +/- 7 mm Hg) of hypertension development. This was associated with an increased pressure drop (87%) along the artery compared with the situation at 2 weeks. These data indicate that small arteries upstream from the microcirculation contribute significantly to the increase in vascular resistance during hypertension. In addition, these data indicate that the increases in small artery resistance do not develop uniformly throughout all vessel branches.

Nitric oxide modulates vascular tone in preglomerular arterioles.

Blockade of nitric oxide reduces renal blood flow, but the site or sites at which nitric oxide alters renal vascular resistance are unknown. The effects of N omega-nitro-L-arginine (100 microM), an inhibitor of nitric oxide synthesis, on the pressure-diameter relation of renal arterioles was studied using a rat juxtamedullary microvascular preparation perfused in vitro with a physiological salt solution containing 5% albumin. The basal diameters of the main arcuate and interlobular arteries and the proximal and distal afferent arterioles averaged 438 +/- 26, 64 +/- 4, 30 +/- 1, and 20 +/- 1 microns, respectively, at a perfusion pressure of 80 mm Hg. The diameters of the arcuate and interlobular arteries increased by 14 +/- 2% and 7 +/- 2%, whereas the proximal and distal afferent arterioles decreased by 3 +/- 1% and 7 +/- 2% when perfusion pressure was elevated to 160 mm Hg. Nitro-arginine had no effect on the basal diameters of arcuate and interlobular arteries. Nitro-arginine reduced the diameters of afferent arterioles by 7 +/- 2% at all perfusion pressures studied. Nitro-arginine increased active vascular tone in the interlobular artery and afferent arterioles and enhanced autoregulation of glomerular capillary pressure. L-Arginine (1 mM), the precursor to nitric oxide production, reversed the effects of nitro-arginine. These findings suggest that nitric oxide modulates vascular tone of the interlobular artery and afferent arterioles of deep nephrons and influences the ability of the preglomerular vasculature to autoregulate glomerular capillary pressure.

Cyclooxygenase-2 modulates afferent arteriolar responses to increases in pressure.

This study was designed to examine the contribution of cyclooxygenase-2 (COX-2) in the afferent arteriolar autoregulatory responses to increases in perfusion pressure and its relationship with neuronal nitric oxide synthase (nNOS). In rat kidneys, afferent arteriolar diameter responses to increases in perfusion pressure were assessed in vitro with the blood-perfused juxtamedullary nephron technique. Basal afferent arteriolar diameter at 100 mm Hg averaged 21.0+/-1.2 microm (n=7), and the vasoconstrictor response to increasing perfusion pressure to 160 mm Hg averaged 18.4+/-1.2%. Superfusion with the COX-2 inhibitor NS398 (10 micromol/L) did not influence basal diameters, but it did significantly enhance the vasoconstrictor response to the increase in perfusion pressure (32.9+/-4.0%). In contrast to previous findings that the nNOS inhibitor S-methyl-L-thiocitrulline (10 micromol/L) enhanced afferent arteriolar autoregulatory responses in normal rat kidneys, in this study, administration of 10 micromol/L S-methyl-L-thiocitrulline did not further modulate the vasoconstrictor response to increases in perfusion pressure in the NS398-treated kidneys of normal rats (31.8+/-4.7%). When tubuloglomerular feedback activity was interrupted by papillectomy and the addition of 50 micromol/L furosemide to the blood perfusate (n=5 for each), the afferent arteriolar constrictor responses to increasing perfusion pressure to 160 mm Hg averaged 7.9+/-0.9% and 10.7+/-0.7%, respectively, and they were significantly attenuated compared with the responses observed in control kidneys. NS398 treatment did not modulate the afferent arteriolar autoregulatory responses in papillectomized or furosemide-treated kidneys. These results indicate that COX-2-derived metabolites contribute to the nNOS modulation of pressure-mediated afferent arteriolar autoregulatory responses.

Neuronal nitric oxide synthase-dependent afferent arteriolar function in angiotensin II-induced hypertension.

This study was designed to determine the influence of neuronal nitric oxide synthase (nNOS) in tubular flow-dependent regulation of afferent arteriolar diameter in hypertensive Sprague-Dawley rats that received 60 ng/min angiotensin II (Ang II) subcutaneously for 13 days. Systolic blood pressure of control and Ang II-infused rats averaged 122+/-2 (n=23) and 194+/-2 mm Hg (n=24). Afferent arteriolar responses to the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC; 0.1 to 10 micromol/L) and the nonselective NOS inhibitor Nomega-nitro-L-arginine (L-NNA; 1 to 100 micromol/L) were assessed in vitro using the blood-perfused juxtamedullary nephron preparation. At a perfusion pressure of 160 mm Hg, afferent arteriolar diameters from control and Ang II-infused rats averaged 18.7+/-1.1 microm (n=8) and 18.1+/-1.1 microm (n=9), respectively, and decreased by 19. 9+/-1.5% and 11.8+/-1.1%, respectively, in response to 10 micromol/L L-SMTC. The L-SMTC-induced afferent arteriolar constriction was significantly greater in control than in Ang II-infused rats. In contrast, 100 micromol/L L-NNA constricted afferent arterioles similarly in both control (n=8) and Ang II-infused (n=7) rats. After transection of the loops of Henle to interrupt flow to the macula densa, the vasoconstrictor responses to L-SMTC but not to L-NNA were reversed. Increasing distal volume delivery by addition of 10 mmol/L acetazolamide to the blood perfusate significantly enhanced the afferent arteriolar constrictor responses to 10 micromol/L L-SMTC (34.5+/-4.8%, n=7) in normotensive rats. In contrast, in Ang II-infused rats, acetazolamide treatment did not enhance the responses to L-SMTC (n=8). These results indicate that chronic Ang II infusion reduces the ability of nNOS-derived nitric oxide to counteract the afferent arteriolar response to increased distal tubular flow.

Renal accumulation of circulating angiotensin II in angiotensin II-infused rats.

Previous studies have demonstrated that low-dose angiotensin II (Ang II) infusion for 14 days mimics two-kidney, one clip Goldblatt hypertension and increases intrarenal Ang II levels. The objective of the present study was to determine whether the augmented intrarenal Ang II is due to intrarenal accumulation of the infused Ang II and/or to an increase in intrarenal formation of endogenous Ang II. Male Sprague-Dawley rats were uninephrectomized and divided into three groups: control (N=6), those infused with [Ile5]Ang II (endogenous form) (N=6), and those infused with [Val5]Ang II (n=8). [Ile5]Ang II or [Val5]Ang II was infused at 40 ng/min via an osmotic minipump implanted subcutaneously. By day 12, systolic blood pressure increased significantly in both [Val5]Ang II-infused rats (197 +/- 7 mm Hg) and [Ile5]Ang II-infused rats (173 +/- 3 mm Hg). Blood and kidney samples were harvested, subjected to high-performance liquid chromatography to separate [Val5]Ang II from [Ile5]Ang II, and then measured by radioimmunoassay. Plasma renin activity was markedly suppressed in both [Ile5]Ang II- and [Val5]Ang II-infused rats. Plasma Ang II levels were elevated in rats infused with both [Ile5]Ang II (121 +/- 24 fmol/mL) and [Val5]Ang II (119 +/- 14 fmol/mL) compared with controls (69 +/- 15 fmol/mL). Both [Ile5]Ang II- and [Val5]Ang II-infused rats exhibited an enhancement of total intrarenal Ang II. Only [Ile5]Ang II (358 +/- 53 fmol/g) was detected in the kidneys of rats infused with -Ile5-Ang II. In [Val5]Ang II-infused rats, a significant portion of total renal Ang II (371 +/- 57 fmol/g) was in the form of [Val5]Ang II (256 +/- 44 fmol/g). Renal [Ile5]Ang II levels were maintained in the [Val5]Ang II-infused rats (116 +/- 15 fmol/g) compared with control rats (116 +/- 11 fmol/g) despite marked suppression of renin release. These results support the hypothesis that infused circulating ANG II is bound to receptor or taken up intrarenally in a manner that protects against degradation.

Interactive nitric oxide-angiotensin II influences on renal microcirculation in angiotensin II-induced hypertension.

The present study was conducted to determine the contribution of nitric oxide to angiotensin II (Ang II) reactivity of afferent and efferent arterioles from Ang II-infused hypertensive rats. Experiments were performed in vitro with the blood-perfused juxtamedullary nephron technique in kidneys harvested from hypertensive Sprague-Dawley rats (181+/-1 mm Hg) that had received 60 ng/min Ang II subcutaneously for 13 days. Superfusion with 0.1, 1, and 10 nmol/L Ang II reduced afferent arteriolar diameter (18.1+/-0.6 microm; n=12) by 10.0+/-0.7%, 28.1+/-1.7%, and 52.8+/-1.9%, respectively, and efferent arteriolar diameter (17.2+/-1.4 microm; n=8) decreased by 9.3+/-0.7%, 27.0+/-1.2%, and 50.4+/-1.6%, respectively. Nitric oxide synthase inhibition with 100 micromol/L N(omega)-nitro-L-arginine (NLA) reduced resting afferent and efferent arteriolar diameters to 14.7+/-0.4 and 14.3+/-1.2 microm, respectively, and enhanced afferent but not efferent arteriolar reactivity to Ang II. The enhanced afferent arteriolar reactivity to Ang II was eliminated by addition of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP, 10 micromol/L), which reversed the NLA-induced decrease in diameter. Addition of 10 micromol/L SNAP, without NLA, blunted efferent but not afferent arteriolar reactivity to Ang II. Afferent (n=7) and efferent arteriolar diameters (n=6) decreased by 48.5+/-2.2% and 41.0+/-1.9%, respectively, in response to 10 nmol/L Ang II. These results suggest that in this model of hypertension, maintained nitric oxide production in afferent arterioles counteracts the enhanced afferent arteriolar reactivity that occurs in Ang II-induced hypertension.

Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca2+ from intracellular stores.

Renal vascular responses to angiotensin II (Ang II) and norepinephrine (NE) are reported to involve both mobilization of calcium from intracellular stores and activation of calcium influx pathways. The present study was conducted to determine the contribution of calcium release from intracellular stores to afferent and efferent arteriolar responses to Ang II and NE. Experiments were performed in vitro using the blood-perfused, juxtamedullary nephron technique combined with videomicroscopy. The responses of afferent and efferent arterioles to Ang II and NE were determined before and after depletion of intracellular calcium pools with 1 mumol/L thapsigargin. Positive control responses were obtained with 55 mmol/L KCI. Ang II concentrations of 0.1, 1.0, and 10 nmol/L decreased afferent arteriolar diameter by 10 +/- 4%, 17 +/- 4%, and 29 +/- 6%, respectively (P < .05; n = 8). NE also decreased afferent diameter by 5 +/- 1%, 13 +/- 1%, and 57 +/- 9% at concentrations of 10, 100, and 1000 nmol/L, respectively (P < .05; n = 6). Thapsigargin treatment shifted the afferent arteriolar concentration-response curves for both Ang II and NE significantly to the right. Nevertheless, KCI evoked a pronounced vasoconstriction and decreased afferent diameter by 56 +/- 7% (P < .05; n = 6). Postglomerular responses to Ang II and NE were abolished by thapsigargin. During the control period, efferent diameter decreased by 3 +/- 1%, 7 +/- 2%, and 14 +/- 4% for the three Ang II concentrations and 3 +/- 1%, 5 +/- 1%, and 15 +/- 4% in response to the three NE concentrations, respectively. These responses were completely eliminated in the presence of thapsigargin, whereas KCI evoked an efferent arteriolar vasoconstriction of 57 +/- 9% (P < .05). These data demonstrate that agonist-induced calcium release from intracellular stores represents an essential component in the afferent and efferent arteriolar response to Ang II and NE. Furthermore, they suggest that efferent arteriolar responses to these agents may rely more heavily on calcium release from this store, whereas afferent responses may include activation of other pathways.

Calcium mobilization contributes to pressure-mediated afferent arteriolar vasoconstriction.

Preglomerular responses to vasoactive agonists utilize calcium released from intracellular stores and activation of calcium influx pathways to elicit vasoconstriction. The current study was performed to determine the role of calcium release from intracellular stores on the afferent arteriolar response to increases in perfusion pressure. Experiments were performed, in vitro, using the blood perfused, juxtamedullary nephron technique combined with videomicroscopy. The response of afferent arterioles to 30 mm Hg increases in perfusion pressure was determined before and after depletion of intracellular calcium pools with a 10-minute preincubation with 1 micromol/L thapsigargin or 100 micromol/L cyclopiazonic acid. Afferent arteriolar diameter averaged 20.2+/-1.0 microm (n=19) at a control perfusion pressure of 100 mm Hg. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent caliber by 10.7+/-1.0% (P<.05 versus con) and by 24.7+/-1.6% (P<.05 versus diameter at 130 mm Hg); respectively. Thapsigargin significantly increased afferent diameter by 21+/-2% (n=6) at 100 mm Hg and prevented pressure-induced autoregulatory responses. Afferent diameter averaged 24.3+/-1.7, 24.5+/-1.8 and 24.3+/-1.8 microm at perfusion pressures of 100, 130 and 160 mm Hg; respectively. Cyclopiazonic acid treatment also inhibited autoregulatory behavior but did not alter resting vessel diameter. Afferent arteriolar diameter (n=6) averaged 21.4+/-1.9 microm at 100 mm Hg and 20.9+/-2.1 and 20.5+/-2.2 microm at 130 and 160 mm Hg; respectively. Additional studies were performed to assess the role of phospholipase C activity in pressure-mediated autoregulatory behavior of afferent arterioles. Step increases in perfusion pressure decreased afferent diameter by 10.7+/-3.8 and 21.7+/-4.1%; respectively. Administration of the phospholipase C inhibitor, U-73122, (5 micromoles/L) did not significantly alter baseline diameter but did attenuate the pressure-mediated vasoconstrictor response. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent diameter by only 6.5+/-1.5 and 10.0+/-2.0%; respectively. These data demonstrate that interruption of calcium mobilization with thapsigargin, cyclopiazonic acid, or phospholipase C inhibition markedly attenuates pressure-mediated afferent arteriolar vasoconstriction and suggests that autoregulatory adjustments in afferent arteriolar diameter involve calcium release from inositoltrisphosphate(IP3)-sensitive intracellular stores.

Elevated renovascular tone in young spontaneously hypertensive rats. Role of cytochrome P-450.

The present study examined the role of cytochrome P-450 metabolites of arachidonic acid in elevating renal vascular resistance in young spontaneously hypertensive rats (SHR). Differences in vascular tone were assessed in the preglomerular vasculature of 3- to 4-week-old prehypertensive SHR (n = 11) and normotensive Wistar-Kyoto (WKY, n = 10) and Wistar-Lewis (n = 10) rats. Pressure-diameter relations to changes in renal perfusion pressure were compared using the juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution. At a pressure of 60 mm Hg, the basal diameters of the interlobular arteries and proximal and distal afferent arterioles of the SHR averaged 43 +/- 2, 17 +/- 0.3, and 11 +/- 0.4 microns, respectively. The diameters of the interlobular arteries and afferent arterioles were 9% to 14% smaller than those of corresponding vessels in WKY and Wistar-Lewis rats. Addition of P-450 inhibitors, ketoconazole (100 mumol/L) or 7-ethoxyresorufin (1 mumol/L), to the perfusate dilated the afferent arteriole of SHR by 7% to 12%, whereas it increased the diameter by only 0% to 6% in control rats and significantly reduced the differences in the pressure-diameter relation in the preglomerular vasculature of SHR and control rats. Inhibitors of P-450 eliminated the contractile response of afferent arterioles to increases in renal perfusion pressure in all three groups. Removal of calcium from the perfusate eliminated differences in the diameters of the preglomerular vasculature in SHR and normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of renal nerves in afferent arteriolar reactivity in angiotensin-induced hypertension.

The objective of this study was to determine the contribution of renal nerves to the enhanced afferent arteriolar reactivity observed in angiotensin II (Ang II)-induced hypertension. Uninephrectomized Sprague-Dawley rats were divided into four groups: sham rats, renal-denervated rats, Ang II-infused (at 40 ng/min for 13 days) rats, and Ang II-infused+renal-denervated rats. With the use of an implanted arterial catheter, mean arterial pressure (MAP) was monitored in conscious rats. Ang II infusion resulted in a progressive increase in MAP from 98 +/- 1 (day 0) to 166 +/- 7 mm Hg (day 13). This increase in MAP was attenuated in denervated rats and averaged 136 +/- 3 mm Hg on day 13. Kidneys were harvested on day 13 for microcirculatory experiments or measurement of intrarenal Ang II levels. Basal afferent arteriolar diameter was similar in all groups, and group averages ranged from 19.6 to 20.7 microns. Chronic Ang II infusion increased intrarenal Ang II levels. Renal denervation did not alter this effect. Increasing perfusion pressure from 100 to 160 mm Hg reduced afferent arteriolar diameter significantly by 11.2 +/- 0.6% in the sham group and by a similar degree in the remaining three groups. Superfusion with Ang II (10 nmol/L) reduced afferent arteriolar diameter by 34.3 +/- 2.0% in the sham group. This response was enhanced in Ang II-infused (62.3 +/- 3.4%) but not in renal-denervated or Ang II-infused+renal-denervated rats. Additionally, the enhanced afferent arteriolar reactivity to Ang II was not influenced by adrenergic receptor blockade. The afferent arteriolar response to norepinephrine was enhanced in renal-denervated, Ang II-infused, and Ang II-infused+renal-denervated rats compared with sham controls. Administration of the calcium ionophore A23187 decreased afferent arteriolar diameter similarly in all four groups. These results indicate that renal nerves contribute to the development of hypertension and to the enhanced afferent arteriolar responsiveness to Ang II elicited by chronic Ang II infusion.

Purinoceptor-mediated calcium signaling in preglomerular smooth muscle cells.

-The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca2+]i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca2+-sensitive dye fura 2. The effect of ATP and UTP on [Ca2+]i was studied on single cells with standard microscope-based fluorescence photometry techniques. Resting [Ca2+]i averaged 80+/-3 nmol/L (n=219 single cells from 58 dispersions). ATP (100 micromol/L) increased [Ca2+]i to a peak value of 845+/-55 nmol/L (n=70 single cells from 38 dispersions) before stabilizing at 124+/-81 nmol/L. Similarly, 100 micromol/L UTP (n=39 single cells from 26 dispersions) stimulated a peak increase in [Ca2+]i of 1426+/-584 nmol/L before reaching a stable plateau of 123+/-10 nmol/L. The [Ca2+]i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 micromol/L ATP induced a transient peak increase in [Ca2+]i, with the plateau phase being totally abolished. In contrast, exposure to 100 micromol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca2+]i. The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 micromol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca2+]i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca2+]i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores.

Afferent arteriolar vasodilation to the sulfonimide analog of 11, 12-epoxyeicosatrienoic acid involves protein kinase A.

The current study determined the contribution of protein kinase-A (PKA) and protein kinase-G (PKG) to the vasodilation elicited by the N-methylsulfonimide analog of 11,12-epoxyeicosatrienoic acid (11, 12-EET). Experiments were performed, in vitro, using the juxtamedullary nephron preparation combined with videomicroscopy. The response of afferent arterioles to the sulfonimide analog of 11, 12-EET, was determined before and after inhibition of PKA, PKG, or guanylyl cyclase. Afferent arterioles, preconstricted with 0.5 micromol/L norepinephrine, averaged 18+/-1 microm (n=25) at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nmol/L of the 11,12-EET analog caused a graded increase in diameter of the afferent arteriole. Vessel diameter increased by 11+/-1% and 15+/-1%, respectively, in response to 10 and 100 nmol/L of the 11,12-EET analog. The afferent arteriolar response to 10 and 100 nmol/L of the 11,12-EET analog was significantly attenuated during inhibition of PKA with 10 micromol/L H-89 (n=7) or 5 micromol/L myristolated PKI (n=6), such that afferent arteriolar diameter increased by only 5+/-2% and 2+/-1%, respectively, in response to 100 nmol/L of the 11, 12-EET analog. In contrast, the afferent arteriolar vasodilatory response to the 11,12-EET analog was unaffected by PKG or guanylyl cyclase inhibition. In the presence of 200 micromol/L histone H2B (n=5) or 10 micromol/L ODQ (n=7), the afferent arteriolar diameter increased by 16+/-3% and 12+/-2%, respectively, in response to 100 nmol/L of the 11,12-EET analog. These results demonstrate that activation of PKA is an important mechanism responsible for the afferent arteriolar vasodilation elicited by the sulfonimide analog of 11,12-EET.