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

Angiotensin II induces carbon monoxide production in the perfused kidney: relationship to protein kinase C activation. Am J Physiol Renal Physiol 287: F914-F920, 2004. First published July 13, 2004; doi: 10.1152/ajprenal.00073.2004.--Heme oxygenase (HO)-derived carbon monoxide (CO) attenuates vascular reactivity to constrictor stimuli. ANG II produces vasoconstriction and induces HO-1 isoform expression. However, direct evidence that ANG II promotes HO product generation is lacking. Therefore, we examined the effects of ANG II on CO release and HO isoform expression in isolated rat kidneys. Kidneys were perfused with oxygenated Krebs buffer. ANG II (1 [micro]mol/l) increased (P < 0.05) perfusion pressure from 97 [+ or -] 9 to 150 [+ or -] 14 mmHg; it also increased (P < 0.05) the concentration of CO in the venous effluent (from 27.1 [+ or -] 11.9 to 45.6 [+ or -] 11.7, 62.5 [+ or -] 16.7, 94.8 [+ or -] 20.7, and 101.9 [+ or -] 13.1 nmol/l after 30, 60, 90, and 120 min, respectively). The pressor effect of ANG II was blunted (P < 0.05) in kidneys perfused with buffer containing losartan (10 [micro] mol/l) or PKC inhibitors staurosporine (0.1 [micro] mol/1) or calphostin C (1 [micro] mol/l). Kidneys perfused with buffer containing ANG II for 120 min also displayed increased (P < 0.05) HO-1 expression. Stannous mesoporphyrin (30 [micro] mol/l) decreased CO release (P < 0.05) in preparations perfused with and without ANG II; the HO inhibitor also increased (P < 0.05) perfusion pressure, more so in kidneys perfused with that without ANG II. We conclude that ANG II stimulates CO production and release in isolated, perfused rat kidneys. This action of ANG II is linked to the activation of A[T.sub.1] receptors and involves PKC activation and upregulation of renal HO-1 but not of HO-2 protein expression. The study suggests upregulation of renal HO-1 and CO release are protagonic events in a counterregulatory mechanism that attenuates ANG II-induced renal vasoconstriction. heme oxygenase; renal hemodynamics; vasoconstriction

Cytochrome P-450-4A1 (CYP4A1) is an [omega]-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4AI. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 [+ or -] 3 [micro]m) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 [+ or -] 4 [micro]m). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12, 12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40-100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product. myogenic vasoconstriction; 20-hydroxyeicosatetraenoic acid; cytochrome P-450 oxygenases; vascular reactivity

Endothelial cell dysfunction (ECD) is emerging as the common denominator for diverse and highly prevalent cardiovascular diseases. Recently, an increased number of procoagulant circulating endothelial microparticles (EMPs) has been identified in patients with acute myocardial ischemia, preeclampsia, and diabetes, which suggests that these particles represent a surrogate marker of ECD. Our previous studies showed procoagulant potential of endothelial microparticles and mobilization of microparticles by PAI-1. The aim of this study was to test the effects of isolated EMPs on the vascular endothelium. EMPs impaired ACh-induced vasorelaxation and nitric oxide production by aortic tings obtained from Sprague-Dawley rats in a concentration-dependent manner. This effect was accompanied by increased superoxide production by aortic rings and cultured endothelial cells that were coincubated with EMPs and was inhibited by a SOD mimetic and blunted by an endothelial nitric oxide synthase inhibitor. Superoxide was also produced by isolated EMP. In addition, p22(phox) subunit of NADPH-oxidase was detected in EMP. Our data strongly suggest that circulating EMPs directly affect the endothelium and thus not only act as a marker for ECD but also aggravate preexisting ECD. superoxide; nitric oxide; cell dysfunction; cardiovascular disease

Pulmonary intralobar arteries express heme oxygenase (HO)-1 and -2 and release carbon monoxide (CO) during incubation in Krebs buffer. Acute hypoxia elicits isometric tension development (0.77 [+ or -] 0.06 mN/mm) in pulmonary vascular rings treated with 15 [micro]mol/l chromium mesoporphyrin (CrMP), an inhibitor of HO-dependent CO synthesis, but has not effect in untreated vessels. Acute hypoxia also induces contraction of pulmonary vessels taken from rats injected with HO-2 antisense oligodeoxynucleotides (ODN), which decrease pulmonary HO-2 vascular expression and CO release. Hypoxia-induced contraction of vessels treated with CrMP is attenuated (P < 0.05) by endothelium removal, by CO (1-100 [micro]mol/l) in the bathing buffer, and by endothelin-1 (ET-1) receptor blockade with L-754142 (10 [micro]mol/l). CrMP increases ET-1 levels in pulmonary intralobar arteries, particularly during incubation in hypooxygenated media. CrMP also causes a leftward shift in the concentration-response curve to ET-1, which is offset by exogenous CO. In anesthetized rats, pretreatment with CrMP (40 [micro]mol/kg iv) intensifies the elevation of pulmonary artery pressure elicited by breathing a hypoxic gas mixture. However, acute hypoxia does not elicit augmentation of pulmonary arterial pressure in rats pretreated concurrently with CrMP and the ET-1 receptor antagonist L-745142 (15 mg/kg iv). These data suggest that a product of HO activity, most likely CO, inhibits hypoxia-induced pulmonary vasoconstriction by reducing ET-1 vascular levels and sensitivity. hypoxic pulmonary vasoconstriction; heme oxygenase

20-Hydroxyeicosatetraenoic acid (20-HETE), which promotes renal vasoconstriction, is formed in the rat kidney primarily by cytochrome P-450 (CYP) 4A isoforms (4A1, 4A2, 4A3, 4A8). Nitric oxide (NO) has been shown to bind to the heme moiety of the CYP4A2 protein and to inhibit 20-HETE synthesis in renal arterioles of male rats. However, it is not known whether NO interacts with and affects the activity of CYP4A1 and CYP4A3, the major renal CYP4A isoforms in female rats. Incubation of recombinant CYP4A1 and 4A3 proteins with sodium nitroprusside (SNP) shifted the absorbance at 440 nm, indicating the formation of a ferric-nitrosyl-CYP4A complex. The absorbance for CYP4A3 was about twofold higher than that of CYP4A1. Incubation of SNP or peroxynitrite (PN; 0.01-1 mM) with CYP4A recombinant membranes caused a concentration-dependent inhibition of 20-HETE synthesis, with both chemicals having a greater inhibitory effect on CYP4A3-catalyzed activity. Moreover, incubation of CYP4A1 and 4A3 proteins with PN (1 mM) resulted in nitration of tyrosine residues in both proteins. In addition, PN and SNP inhibited 20-HETE synthesis in renal microvessels from female rats by 65 and 59%, respectively. We previously showed that microvessel CYP4A1/CYP4A3 expression and 20-HETE synthesis are decreased in late pregnancy. Therefore, we investigated whether such a decrease is dependent on NO, the synthesis of which has been shown to increase in late pregnancy. Administration of [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) to pregnant rats for 6 days (days 15-20 of pregnancy) caused a significant increase in systolic blood pressure, which was prevented by concurrent treatment with the CYP4A inhibitor 1-aminobenzotriazole (ABT). Urinary N[O.sub.2]/N[O.sub.3] excretion decreased by 40 and 52% in L-NAME-and L-NAME + ABT-treated groups, respectively. Interestingly, renal microvessel 20-HETE synthesis showed a marked increase following L-NAME treatment, and this increase was diminished with coadministration of ABT. These results demonstrate that NO interacts with CYP4A proteins in a distinct manner and it interferes with renal microvessel 20-HETE synthesis, which may play an important role in the regulation of blood pressure and renal function during pregnancy. cytochrome P-450 4A; [N.sup.G]-nitro-L-arginine methyl ester

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

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits [Ca.sup.2+]-activated [K.sup.+] channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low-[K.sub.m] arachidonic acid [omega]-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4Al-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in E[C.sub.50] from 0.37 [+ or -] 0.04 [micro]M in plasmid-transfected arteries to 0.07 [+ or -] 0.01 [micro]M in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl- 12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine. 20-hydroxyeicosatetraenoic acid; arachidonic acid; phenylephrine; cytochrome P-450

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3', 5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-i gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. [[N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heine-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heine, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to Sn[Cl.sub.2], which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heine and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heine. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition. guanosine 3',5'-cyclic monophosphate; retroviral vector; soluble guanylate cyclase; heine oxygenase

Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced up-regulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide-and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries. gene microarray; mitochondria; endothelium-derived hyperpolarizing factor

Renal 20-hydroxyeicosatetraenoic acid synthesis during pregnancy. Am J Physiol Regulatory Integrative Comp Physiol 282: R383-R389, 2002.--We examined whether renal 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis is altered during gestation. Renal microsomal arachidonic acid [omega]-hydroxylase activity increased by 50 and 48% in rats on days 12 and 19 of gestation, respectively. Renal microvessel 20-HETE synthesis increased by 50 and 82% in rats on days 6 and 12 of gestation, respectively, and returned to control levels at day 19 of gestation. In contrast, 20-HETE synthesis in isolated medullary thick ascending limb was unchanged from control levels on days 6 and 12 of gestation, but it increased twofold on day 19 of gestation. This increase on day 19 of gestation was associated with a twofold increase in urinary 20-HETE excretion, and it coincided with a 23-mmHg fall in blood pressure. Moreover, change in the rate of 20-HETE synthesis in microvessels was consistent with the level of expression of cytochrome P450 (CYP)4A proteins. Administration of the CYP4A inhibitor 1-aminobenzotriazole (ABT) for 2 days on day 12 of pregnancy or for 5 days starting on day 15 of pregnancy caused a transient but significant reduction in systolic blood pressure. ABT treatment also decreased urinary sodium, urinary 20-HETE, and renal and microvessel 20-HETE synthesis. This study, to our knowledge, is the first to demonstrate that 20-HETE synthesis in the kidney is altered in time- and site-specific manners during pregnancy. The localized pattern of changes suggests that there are distinct regulatory mechanisms for 20-HETE synthesis in the kidney during pregnancy. medullary thick ascending limb; renal microvessels; 1-aminobenzotriazole

The role of carbon monoxide (CO) generated from endogenous heme oxygenase (HO) in dilating newborn cerebral arterioles has been studied. Experiments were done on one to three-day old piglets anesthesized with ketamine hydrochloride and acepromazine and maintained on alpha-chloralose. Results indicate that CO is a potent dilator of newborn cerebral circulation through its direct action on Ca2+-activated potassium channels. Cerebrovascular dilation to hypoxia but not hypercapnia is involved in HO-CO.

The role of heme oxygenase in the regulation of diameter in resistance vessels has been investigated based on the assumption that endogenous carbon monoxide subserves vasodepressor functions. To this end, large and small arteries and isolated first-order gracilis muscle arterioles of rat were analyzed. It was found that the heme oxygenase inhibitors chromium mesoporphyrin and cobalt protoporphyrin reduced the diameter of pressurized gracilis muscle arterioles, which was not observed in the case of magnesium protoporphyrin. These findings indicate that the vascular carbon monoxide system contributes to the regulation of basal tone in resistance vessels.

A study was conducted on antisense oligonucleotides (ODNs) designed after the active cytochrome P4A1 (CYP4A1) and CYP4A2/4A3 expression in Sprague-Dawley rats. The effectiveness of the antisense ODNs in inducing isoform-specific inhibition of the in vitro and in vivo synthesis of the 20-hydroxyeicosatetraenoic acid (20-HETE), the metabolite formed after the omega-hydroxylation of arachidonic acid was also investigated. The findings imply that CYP4A1 holds the potential of regulating renal function and blood pressure based on its 20-HETE synthetisizing activity in the rat kidney.

Research was conducted to examine the determinants of kinin release into the venous effluent of rat hindquarters treated with Krebs bicarbonate buffer. Basal and kininogen-induced kinin release were attenuated in preparations perfused with media containing cycloheximide. Results suggested that kinin release from perfused rat hindquarters associate with the activity of both the kinin-degrading and kinin-generating pathways. They also showed the occurrence of bradykinin in the venous effluent of isolated rat hindquarters perfused with media lacking in kininogen and kallikrein.

Renal vasoconstriction after systemic inhibition of nitric oxide synthesis constitutes an increased renal vascular resistance along with systemic arterial and renal perfusion pressures and decreased renal blood flow in control rats. The increase in renal vascular resistance is less intense in rats in which renal perfusion pressure does not increase with arterial pressure. Attenuation of the increase in renal vascular resistance is observed when tubuloglomerular feedback is inhibited by furosemide pretreatment, ureteral ligation, or both.

Carbon monoxide produced by isolated arterioles attenuates pressure-induced vasoconstriction. Am J Physiol Heart Circ Physiol 281: H350-H358, 2001.--Studies were conducted on isolated rat gracilis muscle arterioles to examine the role of vascular heme oxygenase (HO)-derived carbon monoxide (CO) on myogenic constrictor responses to stepwise increments in intraluminal pressure. The arterioles express HO-2 but not HO-1 and manufacture CO. Both HO-2 protein expression and CO production are reduced in arterioles maintained for 18 h before experimentation in media containing HO-2 antisense oligodeoxynucleotides (AS-ODN). Pressurization of arterioles mounted on a myograph over the pressure range of 40-100 mmHg elicits reduction of internal diameter. At pressures [is greater than] 40 mmHg, the internal diameter of vessels treated with either HO-2 AS-ODN, the HO inhibitor chromium mesoporphyrin (CrMP), or the [K.sup.+] channel blocker tetraethylammonium (TEA) are smaller than the corresponding control values. The inclusion of exogenous CO, but not of biliverdin, in the superfusion buffer attenuates pressure-induced vasoconstriction in CrMP-treated vessels. However, exogenous CO does not attenuate pressure-induced vasoconstriction in vessels treated with both CrMP and TEA. Collectively, these data suggest that CO of vascular origin attenuates pressure-induced arteriolar constriction via a mechanism involving a TEA-sensitive [K.sup.+] channel. heme oxygenase; potassium channels; vasodilatory mechanisms; vascular reactivity; myogenic tone

Rapacon-Baker, Michelle, Fan Zhang, Michael L. Pucci, Hui Guan, and Alberto Nasjletti. Expression of myogenic constrictor tone in the aorta of hypertensive rats. Am J Physiol Regulatory Integrative Comp Physiol 280: R968-R975, 2001.--We investigated the effect of intraluminal pressure or stretch on the development of tone in the descending thoracic aorta from rats with aortic coarctation-induced hypertension of 7-14 days duration. Increments of pressure [is greater than] 100 mmHg decreased the diameter of thoracic aortas from hypertensive but not from normotensive rats. The pressure-induced constriction was not demonstrable in vessels superfused with calcium-free buffer. Stretched rings of aorta from hypertensive rats exhibited a calcium-dependent constrictor tone accompanied by elevated calcium influx that varied in relation to the degree of stretch. Blockers of L-type calcium channels and inhibitors of protein kinase C reduced both basal tone and calcium influx in aortic rings of hypertensive rats. Hence, the thoracic aorta of hypertensive rats expresses a pressure- and stretch-activated constrictor mechanism that relies on increased calcium influx through L-type calcium channels via a protein kinase C-regulated pathway. The expression of such a constrictor mechanism is suggestive of acquired myogenic behavior. myogenic tone; protein kinase C; calcium influx; aortic smooth muscle; hypertension

Leffler, Charles W., Alberto Nasjletti, Robert A. Johnson, and Alexander L. Fedinec. Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets. Am J Physiol Heart Circ Physiol 280: H1490-H1495, 2001.--Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, [N.sup.[Omega]]-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the [K.sub.Ca] channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked. heme oxygenase; permissive, potassium channels

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR. Am J Physiol Regulatory Integrative Comp Physiol 280: R255-R261, 2001.--The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low [K.sub.m] arachidonic acid [Omega]-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 [+ or -] 3 to 121 [+ or -] 4 mmHg (P [is less than] 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE syn.thesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine ([EC.sub.50] 0.69 [+ or -] 0.17 vs. 1.77 [+ or -] 0.40 [micro]M). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR. 20-hydroxyeicosatetraenoic acid; phenylephrine; myogenic response; kidney; blood pressure

Vasopressin and [PGE.sub.2] regulate activity of apical 70 pS [K.sup.+] channel in thick ascending limb of rat kidney. Am J Physiol Cell Physiol 278: C905-C913, 2000.--Vasopressin and prostaglandin [E.sub.2] ([PGE.sub.2]) are involved in regulating NaCl reabsorption in the thick ascending limb (TAL) of the rat kidney. In the present study, we used the patch-clamp technique to study the effects of vasopressin and [PGE.sub.2] on the apical 70 pS [K.sup.+] channel in the rat TAL. Addition of vasopressin increased the channel activity, defined as [NP.sub.o], from 1.11 to 1.52 (200 pM) and 1.80 (500 pM), respectively. The effect of vasopressin can be mimicked by either forskolin (1-5 [micro]M) or 8-bromo-cAMP/ dibutyryl-cAMP (8-Br-cAMP/DBcAMP) (200-500 [micro]M). Moreover, the effects of cAMP and vasopressin were not additive and application of 10 [micro]M H-89 abolished the effect of vasopressin. This suggests that the effect of vasopressin is mediated by a cAMP-dependent pathway. Applying 10 nM [PGE.sub.2] alone had no significant effect on the channel activity. However, [PGE.sub.2] (10 nM) abolished the stimulatory effect of vasopressin. The [PGE.sub.2]-induced inhibition of the vasopressin effect was the result of decreasing cAMP production because addition of 200 [micro]M 8-Br-cAMP/DBcAMP reversed the [PGE.sub.2]-induced inhibition. In addition to antagonizing the vasopressin effect, high concentrations of [PGE.sub.2] reduced channel activity in the absence of vasopressin by 33% (500 nM) and 51% (1 [micro]M), respectively. The inhibitory effect of high concentrations of [PGE.sub.2] was not the result of decreasing cAMP production because adding the membrane-permeant cAMP analog failed to restore the channel activity. In contrast, inhibiting protein kinase C (PKC) with calphostin C (100 nM) abolished the effect of 1 [micro]M [PGE.sub.2]. We conclude that [PGE.sub.2] inhibits apical [K.sup.+] channels by two mechanisms: 1) low concentrations of [PGE.sub.2] attenuate the vasopressin-induced stimulation mainly by reducing cAMP generation, and 2) high concentrations of [PGE.sub.2] inhibit the channel activity by a PKC-dependent pathway. cyclooxygenase; cAMP; protein kinase C; patch-clamp technique; NaCl transport