Regulation of endothelial BK channels by heme oxygenase-derived carbon monoxide and caveolin-1

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK) channels is present after in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. Previously, we demonstrated that inhibition of either BK channels or heme oxygenase (HO) restores vasoconstrictor reactivity after CH. Additionally, administration of the scaffolding domain of caveolin (Cav)-1 inhibits EC BK activity and restores vasoconstrictor reactivity in this setting. These results led us to hypothesize that CH exposure results in a loss in Cav-1 inhibition of EC BK channels, resulting in their activation by HO-derived carbon monoxide (CO). Experiments were conducted on freshly dispersed aortic ECs from control and CH-exposed (barometric pressure: 380 mmHg for 48 h) rats. In electrophysiology experiments, outward currents were greater in cells from CH rats as well as from cells from control rats treated with the cholesterol-depleting agent methyl-β-cyclodextrin. These enhanced currents were returned to control by HO inhibition. Channel activity could be restored by the CO donor CO-releasing molecule (CORM)-2 during HO inhibition. Administration of the Cav-1 scaffolding domain eliminated BK currents in cells from CH rats, and current was not restored by the addition of CORM-2. Colocalization experiments in ECs from control and CH rats demonstrated an association between HO-2, Cav-1, and BK. We conclude that EC BK channel activity is HO dependent in the absence of the inhibitory effect of the Cav-1 scaffolding domain.     

Superoxide mediates acute renal vasoconstriction produced by angiotensin II and catecholamines by a mechanism independent of nitric oxide

NAD(P)H oxidases (NOX) and reactive oxygen species (ROS) are involved in vasoconstriction and vascular remodeling during hypertension produced by chronic angiotensin II (ANG II) infusion. These effects are thought to be mediated largely through superoxide anion (O(2)(-)) scavenging of nitric oxide (NO). Little is known about the role of ROS in acute vasoconstrictor responses to agonists. We investigated renal blood flow (RBF) reactivity to ANG II (4 ng), norepinephrine (NE, 20 ng), and alpha(1)-adrenergic agonist phenylephrine (PE, 200 ng) injected into the renal artery (ira) of anesthetized Sprague-Dawley rats. The NOX inhibitor apocynin (1-4 mg.kg(-1).min(-1) ira, 2 min) or the superoxide dismutase mimetic Tempol (1.5-5 mg.kg(-1).min(-1) ira, 2 min) rapidly increased resting RBF by 8 +/- 1% (P < 0.001) or 3 +/- 1% (P < 0.05), respectively. During NO synthase (NOS) inhibition (N(omega)-nitro-l-arginine methyl ester, 25 mg/kg iv), the vasodilation tended to increase (apocynin 13 +/- 4%, Tempol 10 +/- 1%). During control conditions, both ANG II and NE reduced RBF by 24 +/- 4%. Apocynin dose dependently reduced the constriction by up to 44% (P < 0.05). Similarly, Tempol blocked the acute actions of ANG II and NE by up to 48-49% (P < 0.05). In other animals, apocynin (4 mg.kg(-1).min(-1) ira) attenuated vasoconstriction to ANG II, NE, and PE by 46-62% (P < 0.01). During NOS inhibition, apocynin reduced the reactivity to ANG II and NE by 60-72% (P < 0.01), and Tempol reduced it by 58-66% (P < 0.001). We conclude that NOX-derived ROS substantially contribute to basal RBF as well as to signaling of acute renal vasoconstrictor responses to ANG II, NE, and PE in normal rats. These effects are due to O(2)(-) rather than H(2)O(2), occur rapidly, and are independent of scavenging of NO.     

48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.     

Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca(2+)]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100-200 microM ID) abluminally loaded with the Ca(2+) indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca(2+)] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca(2+)] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca(2+)] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca(2+)] contributes to attenuated reactivity following CH by decreasing VSM cell Ca(2+) sensitivity.     

Inhibition of heme oxygenase augments tubular sodium reabsorption

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 μmol/kg ip), a photostable HO inhibitor, was given to control rats and N(G)-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg?1·day?1), 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.     

脂多糖诱导大鼠主动脉血红素氧合酶-1表达及其对血管反应性的影响

目的：探讨血红素－HO－1－CO－cGMP道路对内毒素血症大鼠主动脉血管张力的影响及其分子机制。方法：用离体血管环张力测定技术,观察静脉注射脂多糖（LPS）6h,大鼠胸主动脉环（TARs)对苯肾上腺素（PE）累积收缩反应。分别用一氧化碳（CO）供体正缺血红素（He),血红素氧合酶－1（HO－1）抑制剂锌原卟啉（ZnPP-IX),鸟苷酸环化酶（sGC)抑制剂亚甲兰（MB）预卵育后,测定TARs对PE收缩反应的变化。分别测定主动脉中CO含量,HO－1活性,Western blot测定HO－1蛋白含量,RT－PCR检测HO－1 mRNA表达的改变。结果：LPS组TARs对PE累积收缩反应明显降低,ZnPP-IX可部分逆转低收缩反应,MB可完全逆转低收缩反应,而用He可加重低收缩反应状态;LPS组动脉组织中CO的含量上升,HO－1活性、蛋白表达量和mRNA表达均明显增加。结论：LPS可使主动脉HO－1基因表达上调,蛋白含量及酶活性明显增加,表明启动血红素－HO－1－CO－cGMP通路,是介导ES大鼠主动脉低收缩反应重要机制之一。     

Roles of adrenomedullin 2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats

Recently, a new member of the calcitonin gene-related peptide (CGRP) family, adrenomedullin 2 (AM2) or intermedin (IMD), was identified. AM2/IMD has been shown to have a vasodilator effect in mice and rats and an effect on urine formation in rats. In the present study, we investigated the effects of intravenously infused rat AM2 (rAM2) on blood pressure (BP), heart rate (HR), renal sympathetic nerve activity (RSNA), and renal blood flow (RBF) in conscious unrestrained rats relative to the effects of rat adrenomedullin (rAM) and proadrenomedullin NH2-terminal 20 peptide (rPAMP). Intravenous infusion of rAM2 (5 nmol/kg) significantly decreased BP and increased HR, RSNA, and RBF. These hypotensive and sympathoexcitatory effects diminished after 20 min, and HR returned to control levels 30 min after cessation of the infusion. In contrast, a significant increase in RBF was still evident 60 min after cessation of the peptide infusion. The duration of BP, HR, and RSNA responses was longer with rAM (5 nmol/kg) than with rAM2 infusion, whereas the increases in RBF induced by rAM2 and rAM were similar in their amplitude and duration. Infusion of rPAMP (200 nmol/kg) increased HR and RSNA but had no effect on RBF. Baroreceptor denervation suppressed, but did not diminish, the increases in HR and RSNA to rAM2. These findings indicate that the physiological roles of rAM2 and rAM are similar and that rAM2 also has a long-lasting vasodilator action on the renal vascular bed.     

Effects of acute and chronic exercise on vasoconstrictor responsiveness of rat abdominal aorta.

Reductions in blood pressure that are associated with exercise training have been hypothesized to be the result of a sustained postexertional vascular alteration following single bouts of exercise. The purpose of this study was to determine whether a decrease in vascular sensitivity to vasoconstrictor agonists occurs after a single bout of exercise and whether this vascular alteration is sustained through various periods of exercise training. Vascular responses of abdominal aortic rings to norepinephrine (NE; 10(-9)-10(-4) M) were determined in vitro. Aortas were isolated from sedentary rats immediately after rats performed a single bout of treadmill exercise (30 m/min for 1 h); 24 h after the last exercise bout in rats exercised for 1 day; and 1, 2, 4, and 10 wk of training at 30 m/min, 60 min, 5 days/wk. Sensitivity to NE was only diminished after 10 wk of training. This diminished vascular sensitivity to NE was abolished with the removal of the endothelial cell layer. Furthermore, there were no reductions in developed tension or vascular sensitivity to the vasoconstrictor agonists KCl (10-100 mM), phenylephrine (10(-8)-10(-4) M), and arginine vasopressin (10(-9)-10(-5) M) in vessels either with or without the endothelial layer after a single bout of exercise. These data indicate that a single bout of exercise does not diminish aortic responsiveness to vasoconstrictor agonists and thus is not responsible for the diminished contractile responsiveness that occurs between 4 and 10 wk of moderate-intensity exercise training in rats. This vascular adaptation to exercise training appears to be mediated through an endothelium-dependent mechanism.     

Pulmonary PKG-1 is upregulated following chronic hypoxia

Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 microM) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-beta-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 microM) or the highly specific PKG inhibitor KT-5823 (10 microM). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMP-mediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1.     

Decreased NO signaling leads to enhanced vasoconstrictor responsiveness in skeletal muscle arterioles of the ZDF rat prior to overt diabetes and hypertension

Approximately 40% of patients with type 2 diabetes present with concurrent hypertension at the time of diabetes diagnosis. Increases in peripheral vascular resistance and correspondingly enhanced vasoconstrictor capacity could have profound implications for the development of hypertension and the progression of insulin resistance to overt diabetes. The purpose of this study was to determine whether skeletal muscle arteriolar vasoconstrictor dysfunction precedes or occurs concurrently with the onset of diabetes and hypertension. Male Zucker diabetic fatty (ZDF) rats were studied at 7, 13, and 20 wk of age to represent prediabetic and short-term and long-term diabetic states, respectively. Conscious mean arterial pressure (MAP), fasted plasma insulin and glucose, vasoconstrictor responses, and passive mechanical properties of isolated skeletal muscle arterioles were measured in prediabetic, diabetic, and age-matched control rats. Elevated MAP was manifest in short-term diabetes (control 117 +/- 1, diabetic 135 +/- 3 mmHg) and persisted with long-term diabetes (control 113 +/- 2, diabetic 135 +/- 3 mmHg). This higher MAP was preceded by augmented arteriolar vasoconstrictor responses to norepinephrine and endothelin-1 and followed by diminished beta-adrenergic vasodilation and enhanced myogenic constriction in long-term diabetes. Furthermore, we demonstrate that diminished nitric oxide (NO) signaling underlies the increases in vasoconstrictor responsiveness in arterioles from prediabetic and diabetic rats. Arteriolar stiffness was not different between control and prediabetic or diabetic rats at any time point studied. Collectively, these results indicate that increases in vasoconstrictor responsiveness resulting from diminished NO signaling in skeletal muscle arterioles precede the development of diabetes and hypertension in ZDF rats.     

Endogenous carbon monoxide is an endothelial-derived vasodilator factor in the mesenteric circulation

Chronic hypoxia (CH) is associated with both blunted agonist-induced and myogenic vascular reactivity, possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the cellular location of the HO responsible for these effects has not been clearly established. Therefore, we examined the response to administration of the substrate for HO, heme-l-lysinate (HLL), in endothelium-intact and endothelium-denuded small mesenteric arteries from CH male Sprague-Dawley rats. Mesenteric arteries were isolated and mounted on glass cannulas, pressurized to 60 mmHg, and superfused with physiological saline solution. All experiments were performed in the presence of 100 microM N(omega)-nitro-l-arginine. The vasodilator response to HLL or exogenous CO was examined. HLL experiments were performed in the presence and absence of the HO inhibitor zinc protoporphyrin IX (ZnPPIX). HLL administration resulted in a dose-dependent vasodilator response that was abolished in the presence of ZnPPIX or by endothelial removal. Exogenous CO produced a vasodilator response that was independent of an intact endothelium. Cellular localization of HO was verified through immunohistochemistry in sections of the gut and aorta from CH and control animals. Staining for HO-1, HO-2, and endothelial nitric oxide synthase was confined to the endothelium. Thus we conclude that CO is a product of HO located within the endothelium.     

Persistent oxidative stress following renal ischemia-reperfusion injury increases ANG II hemodynamic and fibrotic activity

ANG II is a potent renal vasoconstrictor and profibrotic factor and its activity is enhanced by oxidative stress. We sought to determine whether renal oxidative stress was persistent following recovery from acute kidney injury (AKI) induced by ischemia-reperfusion (I/R) injury in rats and whether this resulted in increased ANG II sensitivity. Rats were allowed to recover from bilateral renal I/R injury for 5 wk and renal blood flow responses were measured. Post-AKI rats showed significantly enhanced renal vasoconstrictor responses to ANG II relative to sham-operated controls and treatment of AKI rats with apocynin (15 mM, in the drinking water) normalized these responses. Recovery from AKI for 5 wk resulted in sustained oxidant stress as indicated by increased dihydroethidium incorporation in renal tissue slices and was normalized in apocynin-treated rats. Surprisingly, the renal mRNA expression for common NADPH oxidase subunits was not altered in kidneys following recovery from AKI; however, mRNA screening using PCR arrays suggested that post-AKI rats had decreased renal Gpx3 mRNA and an increased expression other prooxidant genes such as lactoperoxidase, myeloperoxidase, and dual oxidase-1. When rats were infused for 7 days with ANG II (100 ng·kg(-1)·min(-1)), renal fibrosis was not apparent in sham-operated control rats, but it was enhanced in post-AKI rats. The profibrotic response was significantly attenuated in rats treated with apocynin. These data suggest that there is sustained renal oxidant stress following recovery from AKI that alters both renal hemodynamic and fibrotic responses to ANG II, and may contribute to the transition to chronic kidney disease following AKI.     

Impaired blood pressure recovery to hemorrhage in obese Zucker rats with orthopedic trauma

We have shown that obese Zucker rats with orthopedic trauma (OZT) exhibit a loss of arteriolar tone in skeletal muscle. We hypothesize that the loss of arteriolar tone in OZT blunts vasoconstrictor responses to hemorrhage, resulting in an impaired blood pressure recovery. Orthopedic trauma was induced with soft tissue injury and local injection of bone components in both hindlimbs in lean (LZT) and OZT (11-13 wk). One day after the orthopedic trauma, blood pressure responses following hemorrhage were measured in conscious control lean, control obese, LZT, and OZT. In another set of experiments, the spinotrapezius muscle of control and trauma animals was prepared for microcirculatory observation. Arteriolar responses to phenylephrine (PE) or hemorrhage were determined. Hemorrhage resulted in similar blood pressure responses in control animals and LZT, but the blood pressure recovery following hemorrhage was blunted in the OZT. In the spinotrapezius, OZT exhibited decreased arteriolar tone and blunted vasoconstrictor responses to PE and hemorrhage. Treatment with glibenclamide improved the blood pressure recovery in the conscious OZT and improved the arteriolar tone, and PE induced vasoconstriction in the spinotrapezius of the OZT. Thus, ATP-dependent K(+) channel-mediated loss of arteriolar tone in OZT blunts the arteriolar constriction to hemorrhage, resulting in impaired blood pressure recovery.     

Role of NO and COX pathways in mediation of adenosine A1 receptor-induced renal vasoconstriction

The mechanism of adenosine A1 receptor-induced intrarenal vasoconstriction is unclear; it depends on sodium intake and may be mediated by changing the intrarenal activity of the nitric oxide (NO) and/or cyclooxygenase (COX) pathway of arachidonic acid metabolism. The effects of 2-chloro-N(6)-cyclopentyl-adenosine (CCPA), a selective A1 receptor agonist, on renal hemodynamics were examined in anesthetized rats maintained on high sodium (HS) or low sodium (LS) diet. Total renal (i.e., cortical) blood flow (RBF) as well as superficial cortical (CBF), outer medullary (OMBF), and inner medullary (IMBF) flows were determined by laser-Doppler. In HS rats, suprarenal aortic infusions of 8-40 nmol/kg/hr CCPA decreased IMBF (15%) and other perfusion indices (22%-27%); in LS rats, IMBF increased 3% (insignificant) and other indices decreased 13%-24%. In LS rats, pretreatment with N-nitro-L-arginine methyl ester prevented the A1 receptor-mediated decrease in RBF and CBF but not OMBF; the response in IMBF was not altered. Pretreatment with indomethacin prevented the decreases in RBF, CBF, and OMBF and did not change the response of IMBF. Thus, within the cortex the vasoconstriction that follows A1 receptor activation results both from inhibition of NO synthesis and from stimulation of vasoconstrictor products of the COX pathway. In the outer medulla, the latter products seem exclusively responsible for CCPA-induced vasoconstriction. The observation that in LS rats IMBF was not affected by stimulation of adenosine A1 receptors suggests that limiting salt intake may help protect medullary perfusion against vasoconstrictor stimuli which have the potential to disturb long-term control of arterial pressure.     

Losartan corrects abnormal frequency response of renal vasculature in congestive heart failure

In congestive heart failure, renal blood flow is decreased and renal vascular resistance is increased in a setting of increased activity of both the sympathetic nervous and renin-angiotensin systems. The renal vasoconstrictor response to renal nerve stimulation is enhanced. This is associated with an abnormality in the low-pass filter function of the renal vasculature wherein higher frequencies (> or =0.01 Hz) within renal sympathetic nerve activity are not normally attenuated and are passed into the renal blood flow signal. This study tested the hypothesis that excess angiotensin II action mediates the abnormal frequency response characteristics of the renal vasculature in congestive heart failure. In anesthetized rats, the renal vasoconstrictor response to graded frequency renal nerve stimulation was significantly greater in congestive heart failure than in control rats. Losartan attenuated the renal vasoconstrictor response to a significantly greater degree in congestive heart failure than in control rats. In control rats, the frequency response of the renal vasculature was that of a first order (-20 dB/frequency decade) low-pass filter with a corner frequency (-3 dB, 30% attenuation) of 0.002 Hz and 97% attenuation (-30 dB) at > or =0.1 Hz. In congestive heart failure rats, attenuation did not exceed 45% (-5 dB) over the frequency range of 0.001-0.6 Hz. The frequency response of the renal vasculature was not affected by losartan treatment in control rats but was completely restored to normal by losartan treatment in congestive heart failure rats. The enhanced renal vasoconstrictor response to renal nerve stimulation and the associated abnormality in the frequency response characteristics of the renal vasculature seen in congestive heart failure are mediated by the action of angiotensin II on renal angiotensin II AT1 receptors.     

Endogenous central kappa-opioid systems augment renal sympathetic nerve activity to maximally retain urinary sodium during hypotonic saline volume expansion

Intracerebroventricular injection of kappa-opioid agonists produces diuresis, antinatriuresis, and a concurrent increase in renal sympathetic nerve activity (RSNA). The present study examined whether endogenous central kappa-opioid systems contribute to the renal excretory responses produced by the stress of an acute hypotonic saline volume expansion (HSVE). Cardiovascular, renal excretory, and RSNA responses were measured during control, acute HSVE (5% body weight, 0.45 M saline over 30 min), and recovery (70 min) in conscious rats pretreated intracerebroventricularly with vehicle or the kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). In vehicle-pretreated rats, HSVE produced a marked increase in urine flow rate but only a low-magnitude and delayed natriuresis. RSNA was not significantly suppressed during the HSVE or recovery periods. In nor-BNI-treated rats, HSVE produced a pattern of diuresis similar to that observed in vehicle-treated rats. However, during the HSVE and recovery periods, RSNA was significantly decreased, and urinary sodium excretion increased in nor-BNI-treated animals. In other studies performed in chronic bilateral renal denervated rats, HSVE produced similar diuretic and blunted natriuretic responses in animals pretreated intracerebroventricularly with vehicle or nor-BNI. Thus removal of the renal nerves prevented nor-BNI from enhancing urinary sodium excretion during HSVE. These findings indicate that in conscious rats, endogenous central kappa-opioid systems are activated during hypotonic saline volume expansion to maximize urinary sodium retention by a renal sympathoexcitatory pathway that requires intact renal nerves.     

Role of NO and cytochrome P-450-derived eicosanoids in ET-1-induced changes in intrarenal hemodynamics in rats

Endothelin-1 (ET-1) produces potent renal effects that we have previously shown to be dependent on cytochrome P-450 (CYP450) metabolites of aracidonic acid (24) This study evaluated the role of these metabolites in the effects produced by ET-1 on renal blood flow (RBF), cortical blood flow (CBF), medullary blood flow (MBF), and mean arterial blood pressure (MBP). ET-1 (20-200 pmol/kg) increased MBP, renal vascular resistance (RVR), and MBF but reduced CBF and RBF in a dose-dependent manner. The decreases in CBF and RBF, and increases in MBP and RVR were blunted by BMS-182874, an ET(A) receptor antagonist or BQ-788, an ET(B) receptor antagonist. Similarly, indomethacin, an inhibitor of cyclooxygenase activity, or 12,12-dibromododecenoic acid (DBDD), a CYP450-dependent inhibitor of production of 20-hydroxyeicosatetraenoic acid (20-HETE), blunted these effects. ET-3 elicited dose-related reduction in CBF and increase in MBF. Indomethacin accentuated the reduction in CBF and attenuated the increase in MBF, as did DBDD. ET-1-induced increase in MBF was attenuated by BQ-788, N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, indomethacin, or DBDD. DBDD inhibited the hemodynamic effects of L-NAME. Miconazole, the inhibitor of CYP450-dependent epoxygenase activity, was without effect. These results indicate that hemodynamic changes produced by ET-1 are mediated by vasoconstrictor prostanoids and/or prostanoid-like substances, possibly, 20-HETE via activation of ET(A) and ET(B) receptors. However, the increase in MBF is mediated by vasodilator prostanoids or by NO via ET(B) receptor activation.     

In vivo regulation of endothelium-dependent vasodilation in the rat renal circulation and the effect of streptozotocin-induced diabetes

We assessed the relative contributions of endothelium-derived relaxing factors to renal vasodilation in vivo and determined whether these are altered in established streptozotocin-induced diabetes. In nondiabetic rats, stimulation of the endothelium by locally administered ACh or bradykinin-induced transient renal hyperemia. Neither basal renal blood flow (RBF) nor renal hyperemic responses to ACh or bradykinin were altered by blockade of prostanoid production (indomethacin) or by administration of charybdotoxin (ChTx) plus apamin to block endothelium-derived hyperpolarizing factor (EDHF). In contrast, combined blockade of nitric oxide (NO) synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), and prostanoid production reduced basal RBF and the duration of the hyperemic responses to ACh and bradykinin and revealed a delayed ischemic response to ACh. Accordingly, l-NAME and indomethacin markedly reduced integrated (area under the curve) hyperemic responses to ACh and bradykinin. Peak increases in RBF in response to ACh and bradykinin were not reduced by l-NAME and indomethacin but were reduced by subsequent blockade of EDHF. l-NAME plus indomethacin and ChTx plus apamin altered RBF responses to endothelium stimulation in a qualitatively similar fashion in diabetic and nondiabetic rats. The integrated renal hyperemic responses to ACh and bradykinin were blunted in diabetes, due to a diminished contribution of the component abolished by l-NAME plus indomethacin. We conclude that NO dominates integrated hyperemic responses to ACh and bradykinin in the rat kidney in vivo. After prior inhibition of NO synthase, EDHF mediates transient renal vasodilation in vivo. Renal endothelium-dependent vasodilation is diminished in diabetes due to impaired NO function.     

Heme oxygenase-derived carbon monoxide promotes arteriolar endothelial dysfunction and contributes to salt-induced hypertension in Dahl salt-sensitive rats

Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. After 4 wk of high-salt diet, Dahl salt-sensitive (Dahl-S) rats display hypertension, increased vascular HO-1 expression, and attenuated vasodilator responses to ACh that can be completely restored by acute treatment with an inhibitor of HO. In this study, we examined the temporal development of HO-mediated endothelial dysfunction in isolated pressurized first-order gracilis muscle arterioles, identified the HO product responsible, and studied the blood pressure effects of HO inhibition in Dahl-S rats on a high-salt diet. Male Dahl-S rats (5-6 wk) were placed on high-salt (8% NaCl) or low-salt (0.3% NaCl) diets for 0-4 wk. Blood pressure increased gradually, and responses to an endothelium-dependent vasodilator, ACh, decreased gradually with the length of high-salt diet. Flow-induced dilation was abolished in hypertensive Dahl-S rats. Acute in vitro pretreatment with an inhibitor of HO, chromium mesoporphyrin (CrMP), restored endothelium-dependent vasodilation and abolished the differences between groups. The HO product CO prevented the restoration of endothelium-dependent dilation by CrMP. Furthermore, administration of an HO inhibitor lowered blood pressure in Dahl-S rats with salt-induced hypertension but did not do so in low-salt control rats. These results suggest that hypertension and HO-mediated endothelial dysfunction develop gradually and simultaneously in Dahl-S rats on high-salt diets. They also suggest that HO-derived CO underlies the impaired endothelial dysfunction and contributes to hypertension in Dahl-S rats on high-salt diets.