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The present study was designed to test the hypothesis that the production of superoxide (O2−·) by NAD(P)H oxidase is coupled to tubular metabolic activity through ionic activation mediated by H+ movement across cell membrane. Using dual fluorescent microscopic imaging analysis, intracellular O2−· levels and pH (pHi) in renal medullary thick ascending limb of Henle (TALH) cells were simultaneously measured. It was found that intracellular O2−· levels in these cells were increased in parallel to the elevation of pHi by outflow of H+ induced via NH4Cl loading followed by rapid removal. This increase in intracellular O2−· levels was substantially blocked by an inhibitor of Na+/H+ exchanger, methylisobutyl-amiloride (MIA; 100 μM), a chemical SOD mimetic, Tiron (1 mM) or an inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI; 100 μM). In additional groups of TALHs, a proton ionophore, carbonylcyanide m-chlorophenylhydrazone (10 μM) was used to produce H+ conductance, leading to H+ flux across cell membrane depending on extracellular pH. The efflux of H+ increased both pHi and intracellular O2−· levels, but the influx of H+ did not increase intracellular O2−· levels. The H+ efflux-induced increase in intracellular O2−· levels was completely blocked by DPI and another NAD(P)H oxidase inhibitor, apocynin (100 μM). In in invo experiments, renal medullary infusion of MIA (100 μM) was found to significantly decrease the concentrations of H2O2 in the renal medullary interstitium. These results suggest that it is the outward movements of H+ ions that activates NAD(P)H oxidase to produce O2−· in TALH cells. This H+ outflow-associated activation of NAD(P)H oxidase importantly contributes to tissue levels of reactive oxygen species in the renal medulla.

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.

The present study tested the hypothesis that endostatin stimulates superoxide (O2−·) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]iand NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]iin parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]iwas unchanged. With the conversion rate of l-[3H]arginine to l-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2−· production was enhanced in endothelial cells. O2−· scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2−· production through activation of NAD(P)H oxidase. This ceramide-O2−· signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.

Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells. Background We recently demonstrated that homocysteine (Hcys) increases superoxide (O 2 − ) production via NADH/NADPH oxidase in renal mesangial cells. This O 2 − production contributes to increased expression of tissue inhibitor of metalloproteinase (TIMP-1) and consequent deposition of collagen in response to Hcys. However, the mechanism by which Hcys activates NADH/NADPH oxidase remains unknown. Given that ceramide is an intracellular activator of this oxidase in several cell types, the present study tests the hypothesis that Hcys activates NADH/NADPH oxidase through a ceramide-mediated signaling pathway in rat mesangial (MG) cells, resulting in O 2 − production. Methods Rat MG cells were incubated with L-homocysteine (L-Hcys) to determine the mechanism by which Hcys activates NADH/NADPH oxidase. Thin layer chromatography (TLC), Western blot analysis, Rac GTPase activity pull down assay, and NADH/NADPH oxidase activity measurements were performed. Results TLC analysis demonstrated that L-Hcys increased de novo production of ceramide in MG cells. L-Hcys and increased ceramide did not alter the amount of NADH/NADPH oxidase subunit p47 phox and p67 phox in both membrane and cytosolic fractions from MG cells. However, L-Hcys or ceramide markedly increased the level of GTP-bound Rac, which was accompanied by enhanced activity of NADH/NADPH oxidase. These Hcys or ceramide-induced actions were substantially blocked by a Rac GTPase inhibitor, GDPβS, and a de novo ceramide synthesis inhibitor, fumonisin B 1 (FB 1 ). Conclusion These results indicate that Hcys activates NADH/NADPH oxidase by stimulating de novo ceramide synthesis, and subsequently enhancing Rac GTPase activity in rat MG cells. This ceramide-Rac GTPase signaling pathway may mediate Hcys-induced oxidative stress in these glomerular cells.

The 3'UTRs of mammalian HIF-1alpha and EGF mRNA contain several highly conserved AU-rich elements (ARE) known to control the turnover of labile mRNAs by binding ARE-binding proteins that regulate nucleocytoplasmic shuttling, translation, and degradation. Androgens regulate the level and subcellular shuttling of HuR, a major ARE-binding protein that stabilizes many ARE-mRNAs. Pull down of biotinylated 3'UTRs of HIF-1alpha or EGF enriches HuR on blots from Jurkat cell lysates 5-fold, and enriches the amount of RNase-protected biotinylated RNA that comigrates with HuR approximately 10-fold. Dihydrotestosterone treatment decreases the HuR-protected riboprobe pulled down from total Jurkat cell lysates by 30-40%, apparently reflecting shifts in HuR from the nucleus to the cytoplasm. Androgen treatment also changes the amount of HuR-protected riboprobe pulled down from a PC-3 clone expressing a functional androgen receptor. The shift in the amount of riboprobe bound by HuR suggests that androgen is up-regulating endogenous ARE-mRNAs that can compete for binding endogenous HuR. These changes in the shuttling and ARE-binding of endogenous HuR indicate that androgen can act posttranscriptionally to regulate ARE-mRNAs, including HIF-1alpha and EGF.

Ryanodine receptors (RyR) play an important role in the regulation of intracellular Ca2+ concentration and in the control of vascular tone. However, the mechanism regulating the activity of RyR is poorly understood. The present study determined whether protein methylation participates in the control of RyR activity. Using a planar lipid bilayer clamping system, S-adenosyl-L-methionine (SAM), a methyl donor, significantly increased the activity of a 245-pS reconstituted Ca2+ release channel from coronary arterial smooth muscle (CASM) in a concentration-dependent manner. Addition of the protein methylation blockers, 3-deazaadenosine, S-adenosylhomocysteine or sinefungin into the cis solution markedly attenuated SAM-induced activation of RyR/Ca2+ release channels. By Western blot analysis, arginine N-methyltransferase (PRMT1) and FK506 binding protein (FKBP) were detected in the SR used for reconstitution of RyR. In the presence of anti-PRMT1 antibody (1:100), SAM-induced activation of RyR/Ca2+ channel was completely abolished. In addition, this SAM-induced increase in RyR/Ca2+ channel activity was blocked by 30 µM ryanodine and by FK506 (100 µM), a ligand for the RyR accessory protein. These results suggest that protein methylation activates RyR/Ca2+ release channels and may participate in the control of intracellular Ca2+ mobilization in CASM cells by transferring a methyl group to the arginine moiety of the RyR accessory protein, FKBP 12.

Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and UNaV) in a dose-dependent manner. At a dose of 20 ng · kg–1 · min–1, it increased RBF by 20% and UV and UNaV by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of ∼200 μm, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 μM. l-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 μM) was shown to double the NO levels within the endothelium of freshly dissected small RA, and l-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA.

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2microassay, it was found that interstitial H2O2levels in the renal cortex and medulla in anesthetized rats averaged 55.91 ± 3.66 and 102.18 ± 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 μmol·min-1·kg-1) significantly increased medullary H2O2levels by 46%, and coinfusion of catalase (10 mg·min-1·kg-1) completely abolished this increase. Functionally, removal of H2O2by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2by renal medullary interstitial infusion (7.5-30 nmol·min-1· kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.

The unique role of nitric oxide (NO) in the regulation of renal medullary function is supported by the evidence summarized in this review. The impact of reduced production of NO within the renal medulla on the delivery of blood to the medulla and on the long-term regulation of sodium excretion and blood pressure is described. It is evident that medullary NO production serves as an important counterregulatory factor to buffer vasoconstrictor hormone-induced reduction of medullary blood flow and tissue oxygen levels. When NO synthase (NOS) activity is reduced within the renal medulla, either pharmacologically or genetically [Dahl salt-sensitive (S) rats], a super sensitivity to vasoconstrictors develops with ensuing hypertension. Reduced NO production may also result from reduced cellular uptake of l-arginine in the medullary tissue, resulting in hypertension. It is concluded that NO production in the renal medulla plays a very important role in sodium and water homeostasis and the long-term control of arterial pressure.

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca2+ release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 µmol/L) and oxotremorine (OXO; 2.5–80 µmol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca2+ ([Ca2+]i) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca2+]i in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca2+-free Hanks’ solution. This OXO-induced rise in [Ca2+]i was significantly reduced by pirenzepine (PIR; an M1 receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from β-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca2+ release induced by activation of M1 muscarinic receptors.

We developed an in situ assay system to simultaneously monitor intracellular Ca2+concentration ([Ca2+]i, fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 μM) stimulated a rapid increase in [Ca2+]ifollowed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 μM) induced a gradual, small increase in [Ca2+]iand a slow increase in intracellular NO levels. Removal of extracellular Ca2+and depletion of Ca2+stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca2+]iby application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca2+]iand intracellular NO production in the intact endothelium is a powerful tool to study Ca2+-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca2+in tyrosine phosphorylation-induced NO production.

Background— Hyperhomocysteinemia (hHcys) is considered an independent risk factor of cardiovascular diseases. Recent studies in our laboratory have shown that hHcys produced glomerular dysfunction and sclerosis independently of hypertension. However, the mechanism mediating these pathogenic effects of homocysteine (Hcys) is poorly understood. Because Hcys and adenosine (Ado) are simultaneously produced via hydrolysis of S -adenosylhomocysteine (SAH), we hypothesized that hHcys may produce its pathogenic effects by decrease in plasma or tissue Ado concentrations. Methods and Results— l -Hcys (1.5 μmol/min per kilogram) was infused intravenously for 60 minutes to produce acute hHcys in Sprague-Dawley rats. Plasma Hcys levels increased from 6.7±0.4 to 14.7±0.5 μmol/L, but Ado decreased from 141.7±15.1 to 52.4±6.8 nmol/L in these rats with acute hHcys. This hHcys-induced reduction of Ado was also observed in the kidney dialysate. In rats with chronic hHcys, plasma Ado levels were also significantly decreased. By kinetic analysis of the enzyme activities, decrease in renal Ado levels in hHcys was shown to be associated with inhibition of SAH hydrolase but not 5′-nucleotidase. Functionally, intravenous infusion of Hcys was found to decrease renal blood flow, glomerular filtration rate, and sodium and water excretion, which could be blocked by the Ado receptor antagonist 8-SPT. Conclusions— These results strongly suggest that hHcys decreases plasma and tissue Ado concentrations associated with inhibition of SAH hydrolase. Decrease in plasma and tissue Ado may be an important mechanism mediating the pathogenic effects of Hcys.

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng · kg−1 · min−1) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 ± 8 to 117 ± 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng · kg−1 · min−1 did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 ± 2 to 138 ± 2 mmHg ( P< 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of l-arginine (300 μg · kg−1 · min−1) into the renal medulla of Dahl S rats. Intravenous infusion ofl-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.

We recently reported that NADH oxidase is one of the major enzymes responsible for superoxide (O[Formula: see text]·) production in the rat kidney. However, the functional significance of NADH oxidase-mediated O[Formula: see text]· production and the mechanisms regulating this enzyme activity are poorly understood. Using fluorescence microscopic imaging analysis, the present study demonstrated that thick ascending limbs of Henle's loop (TALHs) exhibited red fluorescence when incubated with dihydroethidium (DHE), suggesting that O[Formula: see text]· is produced in this tubular segment. Compared with other nephron segments, TALHs from both renal cortex and medulla showed the highest fluorescence intensity. By incubating cortical TALHs (cTALHs) with the substrates of NADH oxidase, xanthine oxidase, nitric oxide synthase, arachidonic acid-metabolizing enzymes, and intramitochondrial oxidases, NADH oxidase was found to be one of the most important enzymes for O[Formula: see text]· production in this tubular segment. The NADH oxidase inhibitor diphenyleneiodonium (DPI; 100 μM) completely blocked NADH-induced O[Formula: see text]· production in cTALHs. Exposure of cTALHs to low Po2(5–10 Torr) significantly increased O[Formula: see text]· production regardless of the absence or presence of NADH. Furthermore, angiotensin II (100 nM) increased NADH oxidase activity by 32%, which was completely blocked by DPI. These results suggest that NADH oxidase is a major enzyme responsible for O[Formula: see text]· production in the TALHs and that the production of O[Formula: see text]· via NADH oxidase may be regulated by renal tissue oxygenation and circulating hormones.

Hyperhomocysteinemia (hHcys) has been recognized as a new risk factor for cardiovascular diseases independent of plasma lipid levels or other factors. However, it remains unknown whether hHcys is implicated in the target organ damages associated with hypertension. The present study first examined the possible role of hHcys in the development of glomerulosclerosis in Dahl salt-sensitive (DS) hypertensive rats. High-performance liquid chromatography showed that plasma total homocysteine (tHcys) concentration was 7.64 +/- 0.29 micromol/L in conscious DS rats on a low salt (0.4% NaCl) diet, which was higher than 5.23 +/- 0.25 micromol/L in Dahl salt-resistant normotensive rats. When these rats were exposed to a high salt (4% NaCl) diet, plasma tHcys markedly increased in DS rats (14.7 +/-1.31 micromol/L) but not in Dahl salt-resistant rats (5.34 +/- 0.54 micromol/L). An iron chelater, desferrioxamine (0.3 mg/kg IV per day), completely normalized high salt--induced elevations of plasma tHcys and significantly attenuated the sclerotic changes in the glomeruli in DS rats. To further determine whether hHcys has an independent effect in the development of glomerulosclerosis, Sprague-Dawley rats were fed drinking water containing methionine (1 g/kg per day) for 6 weeks to produce hHcys. In these rats, plasma tHcys increased to 12.5 +/- 1.9 micromol/L (versus 6.1 +/- 2.6 micromol/L in control rats), and the aorta exhibited typical sclerotic changes, but arterial pressure was not altered. Urinary protein excretion increased to 52 +/- 2 mg/24 hours (versus 17 +/- 2 mg/24 hours in control rats), and the glomerular mesangium was expanded with glomerular hypercellularity, capillary collapse, and fibrous deposition in the rats with hHcys. These results suggest that elevated plasma homocysteine may be an important pathogenic factor for glomerular damage in hypertension independent of arterial pressure.

The present study was designed to test the hypothesis that hypoxia-inducible factor-1α (HIF-1α)-mediated transcriptional activation contributes to increased expression of heme oxygenase (HO) genes in renal medullary interstitial cells (RMICs). By Northern blot analysis, HO-1 mRNA expression was found to significantly increase in response to reduction of Po2in culture medium. However, HO-2 mRNA was not altered by hypoxia. This hypoxia-induced upregulation of HO-1 mRNA was significantly blocked by HIF-1α inhibition with ferrous ammonium sulfate. To further determine the role of HIF-1α in the activation of HO-1, the inducers of HIF-1α were used to address whether induction of HIF-1α stimulates HO-1 mRNA expression. Both desferrioxamine and CoCl2markedly increased HIF-1α mRNA and protein levels and resulted in the upregulation of HO-1 mRNA but not HO-2. Furthermore, inhibition of HIF-1α degradation by CBZ-LLL, an inhibitor of ubiquitin-proteasome, significantly increased HIF-1α protein and HO-1 mRNA but not HO-2 in these cells. Using cis-element oligodeoxynucleotide transfection to specifically decoy HIF-1α and block HIF-1α binding, increased mRNA expression of HO-1 in response to hypoxia and CoCl2was attenuated. In vitro nuclear run-on assays further confirmed that hypoxia and alterations of HIF-1α mRNA or protein levels significantly affected the formation of HO-1 mRNA. Taken together, our results indicate that HO-1, but not HO-2, is transcriptionally activated by hypoxia through HIF-1α-mediated mechanism in RMICs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating increased expression of HO-1 in the renal medulla.

Abstract —Ceramide serves as a second messenger in a variety of mammalian cells. Little is known regarding the role of ceramide in the regulation of vascular endothelial function. The present study was designed to determine whether ceramide affects endothelium-dependent vasodilation in coronary arteries and to explore the mechanism of action of ceramide. In isolated and pressurized small bovine coronary arteries, cell-permeable C 2 -ceramide (10 − 5 mol/L) markedly attenuated vasodilator responses to bradykinin and A23187 (by 40% and 60%, respectively). In the presence of K G -nitro- l -arginine methyl ester, ceramide produced no further inhibition on the vasodilation induced by these vasodilators. Ceramide had no effect on DETA NONOate–induced vasodilation. By use of a fluorescence NO indicator (4,5-diaminofluorescein diacetate), intracellular NO was measured in the endothelium of freshly isolated small coronary arteries. It was found that ceramide significantly inhibited bradykinin-induced NO increase within endothelial cells. However, it had no effect on the activity of arterial or endothelial NO synthase. Pretreatment of the arteries with sodium dihydroxybenzene disulfonate (Tiron, 10 − 3 mol/L), a cell-permeable superoxide scavenger, or polyethylene glycol superoxide dismutase (100 U/mL) largely restored the inhibitory effects of ceramide on the vasodilation and NO increase induced by bradykinin or A23187. Moreover, ceramide time-dependently increased intracellular superoxide (O 2 − · ) in the endothelium, as measured by a fluorescent O 2 − · indicator, dihydroethidium. These results demonstrate that ceramide inhibits endothelium-dependent vasodilation in small coronary arteries by decreasing NO in vascular endothelial cells and that this decrease in NO is associated with increased O 2 − · but not with the inhibition of NO synthase activity within these cells.

The present study characterized the biochemical pathways responsible for superoxide (O 2 −· ) production in different regions of the rat kidney and determined the role of O 2 −· in the control of renal medullary blood flow (MBF) and renal function. By use of dihydroethidium/DNA fluorescence spectrometry with microtiter plates, the production of O 2 −· was monitored when tissue homogenate from different kidney regions was incubated with substrates for the major O 2 −· -producing enzymes, such as NADH/NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain enzymes. The production of O 2 −· via NADH oxidase was greater (P 2 −· production was higher ( P 2 −· in the kidney under this condition. Overall, the renal OM exhibited the greatest enzyme activities for O 2 −· production. In anesthetized rats, renal medullary interstitial infusion of a superoxide dismutase inhibitor, diethyldithiocarbamate, markedly decreased renal MBF and sodium excretion. Diethyldithiocarbamate (5 mg/kg per minute by renal medullary interstitial infusion [RI]) reduced the renal medullary laser-Doppler flow signal from 0.6±0.04 to 0.4±0.03 V, a reduction of 33%, and both urine flow and sodium excretion decreased by 49%. In contrast, a membrane-permeable superoxide dismutase mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (TEMPOL, 30 μmol/kg per minute RI) increased MBF and sodium excretion by 34% and 69%, respectively. These effects of TEMPOL on renal MBF and sodium excretion were not altered by pretreatment with N G -nitro- l -arginine methyl ester (10 μg/kg per minute RI). We conclude that (1) renal medullary O 2 −· is primarily produced in the renal OM; (2) both NADH oxidase and mitochondrial enzymes are responsible for the O 2 −· production in this kidney region; and (3) O 2 −· exerts a tonic regulatory action on renal MBF.

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca2+release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca2+release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs+current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NPo) of the channels in the presence of ryanodine (0.01–1 μM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 μM) and ruthenium red (40–80 μM) substantially inhibited the activity of RYR/Ca2+release channels. Caffeine (0.5–5 mM) markedly increased the NPoof these Ca2+release channels of the SR, but d- myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NPoof these Ca2+release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 μM) into the cis bath solution produced a 2.9-fold increase in the NPoof these RYR/Ca2+release channels. An eightfold increase in the NPoof the RYR/Ca2+release channels (0.0056 ± 0.001 vs. 0.048 ± 0.017) was observed at a concentration of cADPR of 1 μM. The effect of cADPR was completely abolished by ryanodine (50 μM). In the presence of cADPR, Ca2+-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca2+release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca2+release through the activation of RYRs on the SR of these smooth mucle cells.

The present study was designed to determine whether nitric oxide (NO)-induced reduction of [Ca(2+)](i) is associated with Ca(2+)-induced Ca(2+) release (CICR) in coronary arterial smooth muscle cells (CASMCs). Caffeine was used as a CICR activator to induce Ca(2+) release in these cells. The effects of NO donor, sodium nitroprusside (SNP), on caffeine-induced Ca(2+) release were examined in freshly dissociated bovine CASMCs using single cell fluorescence microscopic spectrometry. The effects of NO donor on caffeine-induced coronary vasoconstriction were examined by isometric tension recordings. Caffeine, a CICR or ryanodine receptor (RYR) activator, produced a rapid Ca(2+) release with a 330 nM increase in [Ca(2+)](i). Pretreatment of the CASMCs with SNP, CICR inhibitor tetracaine or RYR blocker ryanodine markedly decreased caffeine-induced Ca(2+) release. Addition of caffeine to the Ca(2+)-free bath solution produced a transient coronary vasoconstriction. SNP, tetracaine and ryanodine, but not guanylyl cyclase inhibitor, ODQ, significantly attenuated caffeine-induced vasoconstriction. These results suggest that CICR is functioning in CASMCs and participates in the vasoconstriction in response to caffeine-induced Ca(2+) release and that inhibition of CICR is of importance in mediating the vasodilator response of coronary arteries to NO.

Abstract —cADP-ribose (cADPR) induces the release of Ca 2+ from the intracellular stores of coronary artery smooth muscle cells. However, little is known about the role of cADPR-mediated intracellular Ca 2+ release in the control of vascular tone. The present study examined the effects of nicotinamide, a specific inhibitor of ADP-ribosylcyclase, on the vascular tone of bovine coronary arteries. A bovine coronary artery homogenate stimulated the conversion of nicotinamide guanine dinucleotide into cGDP-ribose, which is a measure of ADP-ribosylcyclase activity. Nicotinamide significantly inhibited the formation of cGDP-ribose in a concentration-dependent manner: at a concentration of 10 mmol/L, it reduced the conversion rate from 3.34±0.11 nmol · min −1 · mg −1 of protein in control cells to 1.42±0.11 nmol · min −1 · mg −1 of protein in treated cells, a 58% reduction. In U46619-precontracted coronary artery rings, nicotinamide produced concentration-dependent relaxation. Complete relaxation with nicotinamide occurred at a dose of 8 mmol/L; the median inhibitory concentration (IC 50 ) was 1.7 mmol/L. In the presence of a cell membrane–permeant cADPR antagonist, 8-bromo-cADPR, nicotinamide-induced vasorelaxation was markedly attenuated. Pretreatment of the arterial rings with ryanodine (50 μmol/L) significantly blunted the vasorelaxation response to nicotinamide. However, iloprost- and adenosine-induced vasorelaxation was not altered by 8-bromo-cADPR. Moreover, nicotinamide significantly attenuated KCl- or Bay K8644–induced vasoconstriction by 60% and 70%, respectively. These results suggest that the inhibition of cADPR formation by nicotinamide produces vasorelaxation and blunts KCl- and Bay K8644–induced vasoconstriction in coronary arteries and that the cADPR-mediated Ca 2+ signaling pathway plays a role in the control of vascular tone in coronary circulation.

Abstract —Studies were performed in conscious Sprague-Dawley rats to determine the role of the α 2 -adrenergic receptor–mediated increase in the renal medullary nitric oxide synthase (NOS) activity as a counterregulatory mechanism of blood pressure control in response to increased renal adrenergic stimulation. A subpressor dose of norepinephrine (NE, 8 μg · kg −1 · h −1 ) was infused intravenously, and NOS activity was determined with arginine-citrulline conversion by high-performance liquid chromatography in renal cortical and outer and inner medullary tissues. It was found that after 7 days of intravenous NE infusion, NOS activity was significantly higher in both the outer and inner medullary tissues (158±45 versus 30±24 pmol · mg −1 · h −1 [outer medulla] and 5.1±0.7 versus 2.0±0.5 nmol · mg −1 · h −1 [inner medulla] for NE-treated versus control rats, respectively). To determine whether the increase of NOS activity was mediated through renal medullary α 2 -receptors, the receptor antagonist rauwolscine (RAU, 1 μg · kg −1 · min −1 ) was infused via an implanted renal medullary interstitial catheter, and the consequences of intravenous NE administration were evaluated. NOS activity was significantly lower in the RAU-infused animals and did not increase with infusion of NE. To determine the systemic effects of the renal medullary α 2 -receptors, studies were performed to determine the consequences of chronic intravenous infusion of subpressor amounts of NE in the presence and absence of renal medullary α 2 -receptor inhibition. Under conditions in which RAU was continuously infused into the renal medulla, the same subpressor dose of NE caused sustained and reversible hypertension (mean arterial pressure increased from 120±3 to 131±3 mm Hg). Chronic blunting of the renal medullary NOS activity with N G -nitro- l -arginine methyl ester (75 μg · kg −1 · h −1 ) also enabled NE to produce a significant rise in mean arterial pressure (from 117±2 to 134±4 mm Hg). We conclude that the hypertensive effects of moderate elevations of renal adrenergic activity were chronically buffered by the α 2 -receptor–mediated increase in NOS activity within the renal medulla.

Abstract —A sphingomyelin metabolite, ceramide, serves as a second messenger in a variety of mammalian cells. Little is known regarding the production and actions of this novel intracellular signaling lipid molecule in the vasculature. The present study was designed to test the hypothesis that a ceramide-mediated signaling pathway is present in coronary arterial smooth muscle and that ceramide serves as an inhibitor of the large-conductance Ca 2+ -activated potassium (K Ca ) channels and mediates vasoconstriction in coronary circulation. We found that C 2 -ceramide produced a concentration-dependent decrease in K Ca channel activity in vascular smooth muscle cells from small bovine coronary arteries. The average channel activity of the K Ca channels in cell-attached patches decreased from 0.046±0.01 to 0.008±0.001 at a C 2 -ceramide concentration of 10 μmol/L. In inside-out patches, C 2 -ceramide (1 μmol/L) reduced the average channel activity of the K Ca channels from 0.06±0.007 to 0.016±0.004. Dithiothreitol, an inhibitor of acidic sphingomyelinase (1 mmol/L), increased the average channel activity of the K Ca channels in cell-attached patches from 0.05±0.02 of control to 0.26±0.04, a 5-fold increase that was reversed by addition of 1 μmol/L ceramide. Glutathione, an inhibitor of neutral sphingomyelinase, was without effect. C 2 -ceramide significantly reduced the diameter of isolated perfused small coronary arteries in a concentration-dependent manner. Addition of 1 μmol/L C 2 -ceramide decreased average arterial diameter by 28%. When 14 C-sphingomyelin was incubated with coronary arterial homogenates at pH 7.4 and pH 5.0, 14 C–choline phosphate and ceramide were produced. The conversion rates of 14 C-sphingomyelin into 14 C–choline phosphate and ceramide were 65.1±1.0 fmol/min per milligram protein at pH 7.4 and 114.6±8.3 fmol/min per milligram protein at pH 5.0. We conclude that both acidic and neutral sphingomyelinases are present in the bovine coronary arteries and that ceramide inactivates the K Ca channel in arterial smooth muscle cells and hence exerts a tonic vasoconstrictor action in coronary microcirculation.

Nitric oxide (NO) plays critical roles in the control of renal and glomerular hemodynamics, tubuloglomerular feedback response, release of renin and sympathetic transmitters, tubular ion transport, and renal water and sodium excretion. This paper explores the importance of NO in the control of renal water and sodium excretion and in the long-term control of arterial blood pressure. Synthesis of NO, characteristics of NO tissue redox forms, NO synthase activity, and NO synthase isoforms in the kidney are reviewed. To define the role of NO as a natriuretic and antihypertensive factor, the most supportive evidence is summarized, and some contradictory results are also noted. Given the evidence that high salt intake results in high NO concentrations and great NO synthase expression and activity selectively in the renal medulla of the kidney, as well as evidence of a deficiency of the NO synthase activity in Dahl salt-sensitive rats confined in the renal medulla, this report emphasizes the mechanisms by which the renal medullary l-arginine/NO system controls sodium excretion and arterial blood pressure. Other mechanisms for the action of NO on sodium homeostasis such as the action on glomerular filtration rate and the direct effect on tubules are also discussed. We conclude that there is strong evidence that under physiologic conditions, NO plays an important role in the regulation of renal blood flow to the renal medulla and in the tubular regulation of sodium excretion. It is thereby involved in the long-term control of arterial blood pressure, and inhibition or deficiency of NO synthase may result in a sustained hypertension.

Abstract —Previous studies have shown that chronic salt loading increased renal interstitial adenosine concentrations and desensitized renal effects of adenosine, a phenomenon that could facilitate sodium excretion. However, the mechanisms responsible for the increased adenosine production and decreased adenosine response are poorly understood. This study examined the effects of the dietary high salt intake on adenosine metabolism and receptor expression in the renal cortex and medulla in Sprague Dawley rats. Fluorescent high-performance liquid chromatography analyses were performed to determine adenosine levels in snap-frozen kidney tissues. Comparing rats fed a normal (1% NaCl) versus high salt (4% NaCl) diet, renal adenosine concentrations in rats fed a high salt diet were significantly higher (cortex: 43±3 versus 85±4, P P 1 receptors (antinatriuretic type), did not alter A 2A and A 2B receptors (natriuretic type), and upregulated A 3 receptors (function unknown) in both renal cortex and medulla. The data show that stimulation of adenosine production and downregulation of A 1 receptors with salt loading may play an important role in adaptation in the kidney to promote sodium excretion.

Abstract —The present study was designed to determine whether arginine vasopressin (AVP) can stimulate nitric oxide (NO) production within the renal medulla and thereby modulate renal medullary blood flow. An in vivo microdialysis/NO trapping technique was used to determine changes in medullary interstitial [NO]. AVP (2 ng/kg per minute) was delivered into the renal medullary interstitium and resulted in a significant increase in renal medullary [NO] of 35%, which was blocked by pretreatment with nitro- l -arginine methyl ester (L-NAME) (1.3 μg/kg per minute) administered into the renal medullary interstitium. The vasopressin V 2 receptor agonist 1-desamino-8- d -arginine vasopressin (dDAVP) resulted in a significant increase of 32% in renal medullary interstitial [NO]. No change in renal medullary interstitial [NO] was observed after selective vasopressin V 1 receptor stimulation. Laser-Doppler flowmetry with implanted optical fibers was performed to measure cortical and medullary blood flow changes within the kidney. Renal interstitial infusion of dDAVP in rats pretreated with a vasopressin V 1 receptor antagonist resulted in a 15% increase ( P 2 receptor stimulation, which in turn elevates blood flow to the renal medulla.

Recent studies have suggested that coronary endothelial cells produce and release nitric oxide (NO), prostaglandin I 2 , and epoxyeicosatrienoic acids (EETs). These endothelium-derived vasodilators play an important role in the control of coronary vascular tone. However, the mechanism by which these endothelium-derived vasodilators cause relaxation of coronary arterial smooth muscle has yet to be determined. This study characterized and compared the effects of NO, prostaglandin I 2 , and 11,12-EET on the two main types of potassium channels in small bovine coronary arterial smooth muscle: the large conductance Ca 2+ -activated K + channels (K Ca ) and 4-aminopyridine-sensitive delayed rectifier K + channels (K drf ). In cell-attached patches, non-oate, an NO donor, activated both K Ca and K drf channels. The open probability of both K Ca and K drf channels increased 10- to 25-fold when nonoate was added to the bath at concentrations of 10 −6 to 10 −4 mol/L. 11,12-EET (10 −8 to 10 −4 mol/L) also increased the activity of the K Ca channels in a concentration-dependent manner, but it had no effect on the activity of the K drf channels, even in the highest concentration studied (10 −4 mol/L). In contrast to the effect of 11,12-EET, iloprost, a prostaglandin I 2 analogue (10 −6 to 10 −4 mol/L), produced a concentration-dependent increase in the activity of K drf channels without affecting the K Ca channels. In conclusion, all three endothelium-derived vasodilators act to open potassium channels; however, the channel types that they affect are different. NO activates both K Ca and K drf channels; 11,12-EET activates only the K Ca channels; and prostaglandin I 2 activates only the K drf channels.

This study examined the production of nitric oxide (NO) in the renal cortex and medulla through the use of an in vivo microdialysis technique. Oxyhemoglobin (OxyHb) at a concentration of 3 μmol/L was perfused through the dialysis system to trap tissue NO. Methemoglobin (MetHb), which was formed by NO oxidation of OxyHb in the dialysate, was spectrophotometrically assayed at 401 nm. Because the oxidation of OxyHb to produce MetHb is stoichiometric with NO, the production of NO can be determined by the rate of MetHb formation. We found that NO concentration was significantly higher ( P l -arginine (3 mg/kg per minute) for 30 minutes produced a twofold to threefold increase in cortical and medullary NO; N G -nitro- l -arginine methyl ester (L-NAME) (10 μg/kg per minute) decreased NO by 33% in the renal cortex and by 46.5% in the renal medulla. We have also compared under the same conditions the degradation products of NO, nitrite, and nitrate in the renal cortex and medulla using in vivo microdialysis combined with microtiter plate colorimetry. Nitrite/nitrate concentration was significantly higher ( P l -arginine increased cortical and medullary nitrite/nitrate by 65% and 39%, respectively. L-NAME reduced cortical and medullary nitrite/nitrate by 18% and 23%, respectively. The results indicate that the OxyHb-NO microdialysis trapping technique is a highly sensitive in situ method for detecting regional tissue NO concentration and changes in the NO synthase activity in the kidney. These studies have shown that NO concentration is higher in medullary tissue than in the cortex.

The present study evaluated the effects of cytochrome P-450 inhibitors on the response of the renal microvasculature to changes in renal perfusion pressure and on autoregulation of glomerular capillary pressure using the rat juxtamedullary nephron microvascular preparation perfused in vitro with a cell-free perfusate containing 5% albumin. The basal diameters of the proximal and distal afferent arterioles averaged 28 +/- 1 (n = 32) and 18 +/- 1 micron (n = 23), respectively, at a control perfusion pressure of 80 mmHg. The diameters of these vessels decreased by 8% when perfusion pressure was elevated from 80 to 160 mmHg. After addition of cytochrome P-450 inhibitors (either 17-octadecynoic acid, 20 microM; 7-ethoxyresorufin, 10 microM; or miconazole, 20 microM) to the perfusate, the diameters of the proximal and distal afferent arterioles increased by 6% in response to the same elevation in perfusion pressure. Control glomerular capillary pressure averaged 43 +/- 1 mmHg (n = 32) at a renal perfusion pressure of 80 mmHg and increased by only 9 +/- 1 mmHg when perfusion pressure was elevated to 160 mmHg. Autoregulation of glomerular capillary pressure was impaired after addition of the cytochrome P-450 inhibitors, and it increased by 18 +/- 2 mmHg when perfusion pressure was varied over the same range. These results indicate that cytochrome P-450 inhibitors attenuate the vasoconstrictor response of afferent arterioles to elevations in renal perfusion pressure and impair autoregulation of glomerular capillary pressure, suggesting a possible role for cytochrome P-450 metabolites of arachidonic acid in these responses.

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.

We have recently reported that exaggerated oxidative stress in the renal medulla due to superoxide dismutase inhibition resulted in a reduction of renal medullary blood flow and sustained hypertension. The present study tested the hypothesis that selective scavenging of O 2 ·− in the renal medulla would prevent hypertension associated with this exaggerated oxidative stress. An indwelling, aortic catheter was implanted in nonnephrectomized Sprague-Dawley rats for daily measurement of arterial blood pressure, and a renal medullary interstitial catheter was implanted for continuous delivery of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETC, 7.5 mg · kg −1 · d −1 ) and a chemical superoxide dismutase mimetic, 4-hydroxytetramethyl piperidine-1-oxyl (TEMPOL, 10 mg · kg −1 · d −1 ). Renal medullary interstitial infusion of TEMPOL completely blocked DETC-induced accumulation of O 2 ·− in the renal medulla, as measured by the conversion rate of dihydroethidium to ethidium in the dialysate and by urinary excretion of 8-isoprostanes. However, TEMPOL infusion failed to prevent DETC-induced hypertension, unless catalase (5 mg · kg −1 · d −1 ) was coinfused. Direct infusion of H 2 O 2 into the renal medulla resulted in increases of mean arterial pressure from 115±2.5 to 131±2.1 mm Hg, which was similar to that observed in rats receiving the medullary infusion of both TEMPOL and DETC. The results indicate that sufficient catalase activity in the renal medulla is a prerequisite for the antihypertensive action of TEMPOL and that accumulated H 2 O 2 in the renal medulla associated with exaggerated oxidative stress might have a hypertensive consequence.

Recent studies in our lab and by others have indicated that cyclic ADP-ribose (cADPR) as a novel second messenger is importantly involved in vasomotor response in various vascular beds. However, the mechanism regulating cADPR production and actions remains poorly understood. The present study determined whether changes in redox status influence the production and action of cADPR in coronary arterial smooth muscle cells (CASMCs) and thereby alters vascular tone in these arteries. HPLC analyses demonstrated that xanthine (X, 40 microM)/xanthine oxidase (XO, 0.1 U/ml), a superoxide-generating system, increased the ADP-ribosyl cyclase activity by 59% in freshly isolated bovine CASMCs. However, hydrogen peroxide (H2O2, 1-100 microM) had no significant effect on ADP-ribosyl cyclase activity. In these CASMCs, X/XO produced a rapid increase in [Ca2+]i (Delta[Ca2+]i=201 nM), which was significantly attenuated by a cADPR antagonist, 8-Br-cADPR. Both inhibition of cADPR production by nicotinamide (Nicot) and blockade of Ca2+-induced Ca2+ release (CICR) by tetracaine (TC) and ryanodine (Rya) significantly reduced X/XO-induced rapid Ca2+ responses. In isolated, perfused, and pressurized small bovine coronary arteries, X at 2.5-80 microM with a fixed XO level produced a concentration-dependent vasoconstriction with a maximal decrease in arterial diameter of 45%. This X/XO-induced vasoconstriction was significantly attenuated by 8-Br-cADPR, Nicot, TC, or Rya. We conclude that superoxide activates cADPR production, and thereby mobilizes intracellular Ca2+ from the SR and produces vasoconstriction in coronary arteries.

The present study hypothesized that superoxide (O[Formula: see text]·) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1α expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O[Formula: see text]· generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl2-induced increase in HIF-1α levels and completely blocked the increase in HIF-1α levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O[Formula: see text]· dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1α levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1α levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22phox, into RMICs markedly increased HIF-1α levels. In contrast, the OH· scavenger tetramethylthiourea had no effect on the accumulation of HIF-1α in these renal cells. By Northern blot analysis, scavenging or dismutation of O[Formula: see text]· by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1α-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O[Formula: see text]· levels induce HIF-1α degradation independently of H2O2and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1α in these cells under physiological conditions.

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C2-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91phoxand p47phox. C2-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47phoxto the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C2-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.

The present study tested the hypothesis that ceramide, a sphingomylinase metabolite, serves as an second messenger for tumor necrosis factor-α (TNF-α) to stimulate superoxide production, thereby decreasing endothelium-dependent vasorelaxation in coronary arteries. In isolated bovine small coronary arteries, TNF-α (1 ng/ml) markedly attenuated vasodilator responses to bradykinin and A-23187. In the presence of NG-nitro-l-arginine methyl ester, TNF-α produced no further inhibition on the vasorelaxation induced by these vasodilators. With the use of 4,5-diaminofluorescein diacetate fluorescence imaging analysis, bradykinin was found to increase nitric oxide (NO) concentrations in the endothelium of isolated bovine small coronary arteries, which was inhibited by TNF-α. Pretreatment of the arteries with desipramine (10 μM), an inhibitor of acidic sphingomyelinase, tiron (1 mM), a superoxide scavenger, and polyethylene glycol-superoxide dismutase (100 U/ml) largely restored the inhibitory effect of TNF-α on bradykinin- and A-23187-induced vasorelaxation. In addition, TNF-α activated acidic sphingomyelinase and increased ceramide levels in coronary endothelial cells. We conclude that TNF-α inhibits NO-mediated endothelium-dependent vasorelaxation in small coronary arteries via sphingomyelinase activation and consequent superoxide production in endothelial cells.

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO]i) and stimulates superoxide (O[Formula: see text]·) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 μM), and bradykinin (2 μM) were found to increase endothelial [NO]iin freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 μM). Preincubation of the arteries with l-Hcys (20–40 μM) significantly attenuated the increase in endothelial [NO]i. However, l-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [3H]arginine to [3H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting thatl-Hcys reduces endothelial [NO]iby its scavenging action. Because other thiol compounds such asl-cysteine and glutathione were also found to reduce [NO]i, it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O[Formula: see text]· levels using dihydroethidium trapping, we found that only l-Hcys among the thiol compounds studied markedly increased O[Formula: see text]· levels in the renal endothelium. These results indicate that l-Hcys inhibits the agonist-induced NO increase but stimulates O[Formula: see text]· production within endothelial cells. These effects ofl-Hcys on [NO]iand [O[Formula: see text]·] may contribute to endothelial injury associated with hHcys.

Cyclic ADP-ribose (cADPR) is a novel Ca(2+)-mobilizing second messenger in mammalian cells including cardiomyocytes. It is unknown whether myocardial ischemia and reperfusion affect the metabolism of cADPR in the myocardium. The present study therefore examined the effects of myocardial ischemia and reperfusion on the concentrations of myocardial cADPR using high-performance liquid chromatography. Basal levels of cADPR in rat myocardium were 5.3 +/- 1.8 nmol x mg(-1) protein. Myocardial ischemia for 30 min significantly decreased cADPR concentrations to 2.1 +/- 0.4 nmol x mg(-1) protein. During reperfusion, cADPR was maintained at ischemic levels. The activity of ADP-ribosyl cyclase was expressed as the conversion rate of nicotinamide guanine dinucleotide (NGD(+)) to cyclic GDP-ribose. Myocardial ischemia and reperfusion did not alter the activity of ADP-ribosyl cyclase. However, cADPR hydrolase activity, as measured by the conversion rate of cADPR to ADP-ribose, was significantly elevated by ischemia and reperfusion. To determine the mechanism resulting in the enhancement of cADPR hydrolase activity, we examined the effects of changes in ADP, ATP, pH, and PO(2) on the conversion rate of cADPR to ADPR. Alterations of ADP, ATP, or pH in myocardial tissue had no effect on the degradation of cADPR, whereas a decrease in tissue PO(2) markedly increased the hydrolysis of cADPR. These results suggest that myocardial ischemia and reperfusion decrease cADPR in the myocardium by increasing its hydrolysis. Tissue hypoxia may be one of the important mechanisms to activate cADPR hydrolase.

Ryanodine receptors (RyR) play an important role in the regulation of intracellular Ca(2+) concentration and in the control of vascular tone. However, the mechanism regulating the activity of RyR is poorly understood. The present study determined whether protein methylation participates in the control of RyR activity. Using a planar lipid bilayer clamping system, S-adenosyl-L-methionine (SAM), a methyl donor, significantly increased the activity of a 245-pS reconstituted Ca(2+) release channel from coronary arterial smooth muscle (CASM) in a concentration-dependent manner. Addition of the protein methylation blockers, 3-deazaadenosine, S-adenosylhomocysteine or sinefungin into the cis solution markedly attenuated SAM-induced activation of RyR/Ca(2+) release channels. By Western blot analysis, arginine N-methyltransferase (PRMT1) and FK506 binding protein (FKBP) were detected in the SR used for reconstitution of RyR. In the presence of anti-PRMT1 antibody (1:100), SAM-induced activation of RyR/Ca(2+) channel was completely abolished. In addition, this SAM-induced increase in RyR/Ca(2+) channel activity was blocked by 30 microM ryanodine and by FK506 (100 microM), a ligand for the RyR accessory protein. These results suggest that protein methylation activates RyR/Ca(2+) release channels and may participate in the control of intracellular Ca(2+) mobilization in CASM cells by transferring a methyl group to the arginine moiety of the RyR accessory protein, FKBP 12.

The present study examined whether chronic increased oxidative stress within the medulla of the kidney lowers medullary blood flow and leads to hypertension. Optical fibers were implanted into the renal cortex and medulla of uninephrectomized Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, Wis) for the daily measurement of blood flow to these regions using laser-Doppler flowmetry techniques, while arterial pressure was measured from an indwelling aortic catheter. A renal medullary interstitial catheter was implanted for the continuous delivery of the superoxide dismutase (SOD) inhibitor, diethyldithiocarbamic acid (DETC), at a dose of 7.5 mg/kg/d. Renal interstitial superoxide (O 2 − ) levels were determined by perfusing an O 2 − sensitive fluorescent dye, dihydroethidium, through a microdialysis probe implanted into the medulla. Urine samples (24 hours) were collected for measurements of isoprostane excretion. The results indicate that medullary DETC infusions increased tissue O 2 − concentrations in the renal medulla (93.4±22.3,n=8, saline and 867.3±260.2, n=8, DETC; fluorescence units) and increased urinary 8-isoprostane excretion (4.1±0.4 ng/d, n=9, saline and 8.8±1.6 ng/d, n=10, DETC). Mean arterial pressure increased 24 hours after the start of intrarenal DETC infusion and remained nearly 20 mm Hg above control pressure throughout the 5 days of medullary SOD inhibition. During chronic medullary DETC infusion, medullary blood flow was significantly reduced (42.7%), whereas cortical blood flow was unchanged. Intravenous infusion of the same dose of DETC produced no changes in renal medullary or cortical blood flow or arterial blood pressure. The present experiments indicate that an increase in superoxide concentration within the renal medulla selectively reduces medullary blood flow resulting in chronic hypertension.

Substitution of chromosome 13 from Brown Norway BN/SsNHsd/Mcw (BN/Mcw) rats into the Dahl salt-sensitive SS/JrHsd/Mcw (SS/Mcw) rats resulted in substantial reduction of blood pressure salt sensitivity in this consomic rat strain designated SSBN13. In the present study, we attempted to identify genes associated with salt-sensitive hypertension by utilizing a custom, known-gene cDNA microarray to compare the mRNA expression profiles in the renal medulla (a tissue playing a pivotal role in long-term blood pressure regulation) of SS/Mcw and SSBN13 rats on either low-salt (0.4% NaCl) or high-salt (4% NaCl, 2 wk) diets. To increase the reliability of microarray data, we designed a four-way comparison experiment incorporating several levels of replication and developed a conservative yet robust data analysis method. Using this approach, from the 1,751 genes examined (representing more than 80% of all currently known rat genes), we identified 80 as being differentially expressed in at least 1 of the 4 comparisons. Substantial agreements were found between the microarray results and the results predicted on the basis of the four-way comparison as well as the results of Northern blots of 20 randomly selected genes. Analysis of the four-way comparison further indicated that ∼75% of the 80 differentially expressed genes were likely related to salt-sensitive hypertension. Many of these genes had not previously been recognized to be important in hypertension, whereas several genes/pathways known to be involved in hypertension were confirmed. These results should provide an informative source for designing future functional studies in salt-sensitive hypertension.

The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca(2+) release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 micromol/L) and oxotremorine (OXO; 2.5-80 micromol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca(2+) ([Ca(2+)](i)) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca(2+)](i) in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca(2+)-free Hanks' solution. This OXO-induced rise in [Ca(2+)](i) was significantly reduced by pirenzepine (PIR; an M(1) receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from beta-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca(2+) release induced by activation of M(1) muscarinic receptors.

Vascular smooth muscle (VSM) usually exists in a partially contracted state, from which it can contract further or relax in response to different physiological or pathological stimulations. This contracted state of blood vessels and their contracting and relaxing response to stimuli are often referred to as “vascular tone” and “vasomotor response,” respectively. Numerous studies indicate that cytosolic free calcium concentrations ([Ca2+]) in VSM cells play an essential role in mediating or modulating both vascular tone and vasomotor response in a variety of blood vessels. It is well recognized that the rise in intracellular [Ca2+] initiates and maintains contraction in VSM, which importantly determines the peripheral vascular resistance and blood pressure [1–5].

Mitochondrial ATP-sensitive potassium (mitoK ATP ) channels have been suggested as triggers and end effectors in myocardial ischemic preconditioning. However, the intracellular mechanism regulating mitoK ATP channels remains unclear. In the present study, mitoK ATP channels from bovine ventricular myocardium were reconstituted using planar lipid bilayers, and the effect of superoxide (O 2 −· ) on the activity of these reconstituted channels was examined. After incorporation, a potassium-selective current was recorded. The mean conductance of this current was 56 pS at 150 mmol/L KCl, which was substantially inhibited by 1 mmol/L MgATP. 5-Hydroxydecanoate (5-HD, 10 to 100 μmol/L), a selective mitoK ATP antagonist, reduced the open state probability (NPo) of these channels in a concentration-dependent manner, whereas diazoxide (10 μmol/L), a selective mitoK ATP agonist, significantly increased channel activity. HMR-1098 (100 μmol/L), a selective sarcolemmal K ATP antagonist, had no effect on the activity of reconstituted channels. Addition of xanthine/xanthine oxidase (100 μmol/L per 0.038 U/mL), an O 2 −· -generating system, resulted in a marked activation of mitoK ATP channels; the NPo of the channels was increased from 0.60±0.10 to 1.94±0.02. This O 2 −· -induced channel activation was completely abolished by pretreatment with 5-HD (100 μmol/L) or a sulfhydryl alkylating compound, N -ethylmaleimide (2 mmol/L). It is concluded that myocardial mitoK ATP channels can be reconstituted into lipid bilayers and that O 2 −· activates these channels. The effect of O 2 −· may be associated with its direct action on the sulfhydryl groups of the channel protein.

The purpose of this study was to determine whether superoxide anions (O[Formula: see text]·) activate 5′-nucleotidase (5′-ND), thereby increasing the production of renal adenosine and regulating renal function. Using HPLC analysis, we found that incubation of renal tissue homogenate with the O[Formula: see text]· donor KO2doubled adenosine production and increased the maximal reaction velocity of 5′-ND from 141 to 192 nmol · min−1· mg protein−1. The O[Formula: see text]·-generating system, xanthine/xanthine oxidase increased the maximal reaction velocity of 5′-ND from 122 to 204 nmol · min−1· mg protein−1. Superoxide dismutase (SOD) with catalase produced a concentration-dependent reduction of 5′-ND activity in renal tissue homogenate, while the SOD inhibitor diethyldithiocarbamic acid significantly increased 5′-ND activity. Inhibition of disulfide bond formation by thioredoxin or thioredoxin reductase significantly decreased xanthine/xanthine oxidase-induced activation of renal 5′-ND. In in vivo experiments, inhibition of SOD by diethyldithiocarbamic acid (0.5 mg · kg−1· min−1iv) enhanced renal vasoconstriction induced by endogenously produced adenosine and increased renal tissue adenosine concentrations under control condition and in ischemia and reperfusion. We conclude that oxidative stress activates 5′-ND and increases adenosine production in the kidney and that this redox regulatory mechanism of adenosine production is important in the control of renal vascular tone and glomerular perfusion.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates the oxygen-dependent expression of a number of genes. This transcription factor may contribute to the abundant expression of many genes in renal medullary cells that function normally under hypoxic conditions. The present study was designed to determine the characteristics of HIF-1α cDNA cloned from the rat kidney and the expression profile of HIF-1α in different kidney regions and to explore the mechanism activating or regulating HIF-1α expression in renal medullary cells. A 3,718-bp HIF-1α cDNA from the rat kidney was first cloned and sequenced using RT-PCR and TA cloning technique. It was found that 823 amino acids deduced from this renal HIF-1α cDNA had 99%, 96%, and 90% identity with rat, mouse, or human HIF-1α deposited in GenBank, respectively. The 3′-untranslated region of HIF-1α mRNA from the rat kidney contained seven AUUUA instability elements, five of which were found to be conserved among rat, mouse, and human HIF-1α. Northern blot analyses demonstrated a corticomedullary gradient of HIF-1α mRNA expression in the kidney, with the greatest abundance in the renal inner medulla. Western blot analyses also detected a higher HIF-1α protein level in the nuclear extracts from the renal medulla than the renal cortex. A classic loop diuretic, furosemide (10 mg/kg ip), markedly increased renal medullary Po2levels from 22.5 to 52.2 mmHg, which was accompanied by a significant reduction of HIF-1α transcripts in renal medullary tissue. In in vitro experiments, low Po2, but not elevated osmolarity, was found to significantly increase HIF-1α mRNA in renal medullary interstitial cells and inner medullary collecting duct cells. These results indicate that HIF-1α is more abundantly expressed in the renal medulla compared with the renal cortex. Increased abundance of HIF-1α mRNA in the renal medulla may represent an adaptive response of renal medullary cells to low Po2.