Coronary myogenic constriction antagonizes EDHF-mediated dilation: role of KCa channels

In hypertension, pressure-induced myogenic constriction and impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation may contribute to increased vasomotor tone. Myogenic constriction as well as EDHF-mediated dilation may share common signaling mechanisms, and both may control KCa channel activity to set arterial tone. To investigate a potential relation between the 2 mechanisms, we studied coronary arteries of Sprague-Dawley rats for individual myogenic constriction compared with EDHF-mediated dilation of the same artery. EDHF-mediated dilation was measured as the maximal dilation to acetylcholine (100 micromol/L) after preconstriction, resistant to NO inhibition (NG-methyl-l-arginine acetate salt, L-NMMA, 100 micromol/L), and prostaglandin inhibition (indomethacin, 10 micromol/L) but abolished by charybdotoxin (100 nmol/L) plus apamin (500 nmol/L). Individual coronary myogenic constriction at an intraluminal pressure of 70 mm Hg (n=9) ranged from 6% to 44% (24+/-4%). EDHF-mediated dilation ranged from 18% to 84% (42+/-7%). Elevating pressure to 130 mm Hg (n=8) increased myogenic constriction by 2-fold (P<0.01) and decreased EDHF-mediated dilation by 2.6-fold (P<0.01). Interestingly, individual myogenic constriction inversely correlated to individual EDHF-mediated dilation (r=-0.75, P<0.001, n=17). Pretreatment with the KCa channel opener NS1619 (30 micromol/L) prevented coronary myogenic constriction and increased EDHF-mediated dilation by 2.2-fold (P<0.01), whereas the KATP channel opener cromakalim (3 micromol/L) had no effect on EDHF-mediated dilation. For comparison, in mesenteric arteries (at 70 mm Hg) low myogenic constriction (2+/-1%) was associated with high EDHF-mediated dilation (93+/-2%), and pretreatment with NS1619 had no effect. Our results demonstrate that myogenic constriction in coronary arteries antagonizes EDHF-mediated dilation. Activation of KCa channels with NS1619 reduces myogenic constriction and profoundly increases EDHF-mediated dilation, specifically in coronary arteries, suggesting a potential therapeutic impact to reduce coronary risk in hypertension.     

Signalling mechanisms underlying the myogenic response in human subcutaneous resistance arteries

OBJECTIVE: In this study we have examined for the first time the signal transduction mechanisms involved in the generation of pressure-dependent myogenic tone in human small resistance arteries from the subcutaneous vascular bed. METHODS: Myogenic responses and the subcellular mechanisms involved in the generation of this response were studied on a pressure myograph. RESULTS AND CONCLUSION: Human subcutaneous resistance arteries constricted 14.1+/-1.1% in response to an increases in intraluminal pressure from 40 to 80 mmHg and a further 3.5+/-1.7% in response to the 80-120-mmHg pressure step. Ca(2+) depletion or nifedipine abolished this response, whereas BAY K 8644 increased this response to 20.6+/-2.1% (P<0.05, response vs. control). The phospholipase C inhibitor U-73122 reduced the myogenic response to 2.5+/-1.0% at 80 mmHg (P<0.01, response vs. control) and abolished it at 120 mmHg. Diacylglycerol lipase inhibition with RHC-80267 abolished all myogenic responses to pressure. The protein kinase C (PKC) activator phorbol 12,13-dibutyrate increased the maximal myogenic response to 20.9+/-1.8% (P<0.05, response vs. control), whereas the PKC inhibitor calphostin C abolished myogenic responses. These data show that the generation of pressure-dependent myogenic tone in human subcutaneous arteries is dependent on Ca(2+) influx via voltage operated Ca(2+) channels (VOCCs) and a concomitant requirement for the activation of phospholipase C (PLC), diacylglycerol, and PKC.     

An endothelium-derived hyperpolarizing factor-like factor moderates myogenic constriction of mesenteric resistance arteries in the absence of endothelial nitric oxide synthase-derived nitric oxide

Myogenic tone is an important determinant of vascular tone and blood flow in small resistance arteries of certain vascular beds. The role of the endothelium in myogenic responses is unclear. We hypothesized that endothelium-derived NO release modulates myogenic constriction in small resistance arteries and that mesenteric small arteries from mice with targeted disruption of the gene for endothelial NO synthase (eNOS) (knockout mice) demonstrate greater myogenic tone than do wild-type mice. Third-order mesenteric arteries (approximately 200 micrometer) were isolated and mounted in a pressure myograph. Internal diameter was recorded over a pressure range of 10 to 80 mm Hg. Removal of the endothelium significantly (P<0.05) enhanced the magnitude of myogenic constriction in wild-type mice. Similarly, pretreatment of arteries with N(G)-nitro-L-arginine methyl ester (L-NAME; 300 micromol/L) produced a comparable significant (P<0.05) increase in myogenic tone, whereas indomethacin (5 micromol/L) had no effect. eNOS knockout arteries also exhibited myogenic constriction. Neither L-NAME nor indomethacin had any effect on myogenic tone in the arteries of eNOS knockout mice. However, blockade of potential endothelium-derived hyperpolarizing factor-like mechanisms via inhibition of K(+) flux using either apamin (100 nmol/L) with charybdotoxin (100 nmol/L), Ba(2+) (30 micromol/L) with ouabain (1 mmol/L), or 18alpha-glycyrrhetinic acid (100 micromol/L) significantly (P<0.01) enhanced myogenic constriction. This study demonstrates that basal endothelium-derived NO modulates myogenic tone in mesenteric small arteries of wild-type mice. However, eNOS knockout arteries display normal myogenic responsiveness despite the absence of basal NO activity. The data suggest that this compensatory effect is due to the activity of an endothelium-derived hyperpolarizing factor to normalize vascular tone.     

20-HETE contributes to myogenic activation of skeletal muscle resistance arteries in Brown Norway and Sprague-Dawley rats

OBJECTIVE: To evaluate the role of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of arachidonic acid omega-hydroxylation via cytochrome P450 (CP450) 4A enzymes, in regulating myogenic activation of skeletal muscle resistance arteries from normotensive Brown Norway (BN) and Sprague-Dawley (SD) rats. METHODS: Gracilis arteries (GA) were isolated from each animal, viewed via television microscopy, and vessel diameter responses to elevated transmural pressure were measured with a video micrometer under control conditions and following pharmacological inhibition of the CP450 4A enzyme system. RESULTS: Under control conditions, GA from both rat groups exhibited strong, endothelium-independent myogenic activation, which was impaired following treatment with either 17-octadecynoic acid (17-ODYA) or dibromo-dodecenylmethylsulfimide (DDMS), two mechanistically different inhibitors of 20-HETE production. The addition of tetraethylammonium (KCa channel inhibitor) to 17-ODYA-treated GA restored myogenic reactivity to levels comparable to those under control conditions. Treatment of GA from BN and SD rats with 6(Z),15(Z)-20-HEDE, a selective antagonist for 20-HETE receptors, mimicked the effects of 17-ODYA and DDMS treatment on myogenic reactivity. CONCLUSIONS: These results suggest that the production of 20-HETE via CP450 4A enzymes contributes to the myogenic activation of skeletal muscle resistance arteries from normotensive BN and SD rats. 20-HETE may act through a receptor-mediated process to block vascular smooth muscle KCa channels in response to the elevated transmural pressure.     

Contribution of 20-HETE to augmented myogenic constriction in coronary arteries of endothelial NO synthase knockout mice

Previous studies suggested an important role for 20-HETE in the regulation of myogenic responses. Thus, pressure-diameter relationships were investigated in isolated, cannulated coronary arteries (approximately 100 microm) from male endothelial NO synthase knockout (eNOS-KO) and wild-type (WT) mice. All arteries constricted in response to step increases in perfusate pressure from 20 to 100 mm Hg. This constriction was significantly enhanced from 40 to 100 mm Hg in arteries of eNOS-KO compared with those of WT mice. For example, at 60 and 100 mm Hg, respectively, the normalized diameter (expressed as a percentage of the corresponding passive diameter) of arteries of eNOS-KO mice was 10% and 12% smaller than that of WT mice. Removal of the endothelium did not significantly affect the responses of vessels from either strain of mice. However, N-methylsulfonyl-12,12-dibromododec-11-enamide (5x10(-6) M), an inhibitor of cytochrome P-450 (CYP)/omega-hydroxylase, significantly attenuated the greater myogenic constriction of arteries from eNOS-KO mice by approximately 12% at each pressure step but did not significantly affect responses of those from WT mice, leading to a comparable myogenic response in the 2 strains. Western blot analysis demonstrated a comparable CYP4A protein content in coronary arteries of the 2 strains of mice. However, production of 20-HETE, measured by fluorescent high-performance liquid chromatography assay was approximately 2.7-fold greater in eNOS-KO compared to WT mice. Thus, as a function of eNOS deficiency, the enhanced coronary artery constriction to pressure is attributable to an increased activity of omega-hydroxylase, which, consequently, increases the synthesis of 20-HETE in vascular smooth muscle.     

Role of prostanoids and 20-HETE in mediating oxygen-induced constriction of skeletal muscle resistance arteries.

This study determined the contribution of cytochrome P450 (CP450) 4A enzyme metabolites of arachidonic acid in mediating the constriction of isolated rat skeletal muscle resistance arteries in response to elevated PO2. Gracilis arteries (GA) were viewed via television microscopy and constrictor responses to elevated PO2 were measured with a video micrometer. Endothelium removal and treatment of GA with 17-octadecynoic acid (17-ODYA; suicide substrate inhibitor of CP450 4A enzymes) impaired oxygen-induced constriction of the vessels; treatment of endothelium-denuded GA with 17-ODYA eliminated responses to elevated PO2. NOS inhibition and inhibition of EET production had no effect on oxygen-induced constriction of the vessels, although cyclooxygenase inhibition with indomethacin impaired GA responses to elevated PO2. Treatment of GA with dibromododecenyl methylsulfimide (DDMS; inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) production) or 6(Z),15(Z)-20-HEDE (antagonist for 20-HETE receptors) mimicked the effects of 17-ODYA on GA responses to elevated PO2. Treatment of vessels with iberiotoxin or glibenclamide reduced the constriction of the vessels in response to elevated PO2 while treatment with both K+ channel blockers eliminated oxygen-induced constriction of the vessels. Following treatment of GA with indomethacin and 20-HETE, the vessels failed to respond to elevated PO2. These results suggest that oxygen-induced constriction of skeletal muscle resistance arteries represents the combined effects of reduced prostanoid release from the vascular endothelium and enhanced 20-HETE production in vascular smooth muscle cells.     

In vitro analysis of alpha-adrenoceptor interactions with the myogenic response in resistance vessels.

The interaction of alpha 1- and alpha 2-adrenoceptor constriction of isolated rat cremaster small arteries (mean diameter +/- SEM, 114 +/- 5 microns) with the myogenic mechanism was examined with in vitro videomicroscopy. Microdissected vessels were double-cannulated with glass micropipets in a tissue bath, and intraluminal pressure was set at 65 mm Hg baseline pressure. Norepinephrine (NE) concentration-response curves were obtained alone and in the presence of prazosin or rauwolscine to establish the concentration of NE required to selectively stimulate alpha 1- or alpha 2-adrenoceptors. A bimodal response was produced by NE alone, indicating interaction with more than one receptor population. Rauwolscine and prazosin each produced dextral displacement at low (< 0.1 microM) NE concentrations. However, while prazosin exhibited competitive antagonism, rauwolscine did not antagonize NE at concentrations > or = EC50 value. Thus, both alpha 1- and alpha 2-adrenoceptors mediate constriction at low NE concentrations, but only alpha 1-receptors contributed to constriction at intermediate and high concentrations. These data are in contrast to previous findings for the same vessels studied in vivo. Diameter responses to increases and decreases in transmural pressure from baseline were examined in the relaxed (passive) state with nitroprusside present, during spontaneous intrinsic tone, and during induced alpha 1- or alpha 2-tone (EC20 concentration of NE plus rauwolscine or prazosin). Myogenic constriction during increases in lumen pressure and myogenic inhibition of tone during decreases in pressure were greatest during alpha 2-tone, less during intrinsic tone and least during alpha 1-tone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelial dysfunction and hypertension in rats transduced with CYP4A2 adenovirus

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114+/-1 to 133+/-1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2-transduced rats produced more 20-HETE (129+/-10 versus 97+/-7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2+/-1.6 versus 8.4+/-1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.     

Altered myogenic constriction and endothelium-derived hyperpolarizing factor-mediated relaxation in small mesenteric arteries of hypertensive subtotally nephrectomized rats

OBJECTIVES: Chronic renal failure (CRF) is associated with altered systemic arterial tone and hypertension. Myogenic constriction and endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation represent major vasoregulatory mechanisms in small systemic arteries. Elevated myogenic response and impaired EDHF might participate in the development of essential hypertension; however, their role in CRF-related hypertension is unknown. We investigated whether myogenic response and EDHF are altered in subtotally nephrectomized (sNX) rats and whether these changes are modifiable by chronic treatment with angiotensin-converting enzyme (ACE) inhibitor. METHODS: In a pressure arteriograph, myogenic constriction and EDHF-mediated relaxation were evaluated in small mesenteric arteries isolated from male Wistar rats 15 weeks after either sham operation (n = 7) (SHAM), sNX (n = 12) or sNX followed by 9 weeks of treatment with lisinopril (sNX + LIS, 2.5 mg/kg, n = 13). RESULTS: Surprisingly, myogenic response was reduced in hypertensive CRF rats (maximal myogenic tone: 37 +/- 2 and 18 +/- 4%, P < 0.01; peak myogenic index: -0.80 +/- 0.08 and -0.40 +/- 0.12%/mmHg, P < 0.05 in SHAM and sNX respectively). At the same time EDHF-mediated relaxation was also impaired (maximal response: 92 +/- 2 and 77 +/- 5%, P < 0.01; pD2: 6.5 +/- 0.1 and 5.9 +/- 0.1, P < 0.05). Both myogenic response and EDHF were inversely related to the severity of renal failure and restored by treatment with lisinopril to levels found in SHAM animals. CONCLUSION: Major constrictive (myogenic) and dilatory (EDHF) mechanisms of small systemic arteries are impaired in hypertensive CRF rats. These alterations do not seem to participate in the development of hypertension, being rather directly related to the severity of renal impairment. Both systemic vascular changes might be restored by renoprotective treatment with ACE inhibitor.     

Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure

Rats with chronic heart failure (CHF) develop increased myogenic constriction in mesenteric resistance arteries. Here we investigated increased myogenic constriction in relation to alterations in EDHF- and NO-mediated dilatation in CHF-rats. Male Spraque-Dawley rats were subjected to myocardial-infarction or sham-surgery. At 9-10 weeks after surgery, isolated mesenteric artery ring preparations were studied in a wire-myograph. Stretch-induced myogenic constriction was obtained by stepwise increase of the internal circumference diameter (0.5-1.2 L100). Cyclooxygenase- and eNOS-inhibitors were employed to study NO- and EDHF-mediated dilatation in response to acetylcholine. Rats with CHF (n=8), but not sham-rats (n=6), developed significant myogenic constriction. In addition, the contribution of endothelial dilator mediators was significantly altered in CHF-rats, with increased dependency on NO and decreased EDHF-mediated dilatation. Moreover, EDHF-mediated dilatation was inversely correlated with myogenic constriction in individual CHF-rats (r=-0.74, p=0.04). These data demonstrate increased myogenic constriction in mesenteric arteries of rats with CHF post-MI to be correlated to decreased EDHF-mediated dilatation. These findings extend the previous observation that myogenic constriction antagonizes EDHF-mediated dilatation in rat coronary artery under normal conditions, and suggests this relationship also to become functional in mesenteric arteries under pathophysiological conditions of CHF.     

20-HETE and circulating insulin in essential hypertension with obesity

Analogous to observations in Dahl salt-sensitive (SS) rats, we have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is involved in the pathogenesis of SS essential hypertension. A strong negative correlation between urine 20-HETE and body mass index (BMI) remains unexplained. We measured BP, urine sodium (UNaV), and 20-HETE in obese hypertensive subjects during a 24-hour salt load (160 mmol NaCl diet+2 L intravenous saline). We classified them into insulin-resistant (IR) (n=14) and insulin-sensitive (IS) (n=12), with the average insulin sensitivity index (SI=22.5x[fasting glucose x insulin](-1)) of 3 days (cutoff for IR, SI <0.161 mL x L/microU x mmol). IR were older (50+/-1 versus 44+/-2, P<0.03), more obese (BMI 38.2+/-1.4 versus 32.0+/-1.5 kg/m2, P<0.01), and had higher insulin (39.2+/-2.3 versus 22.0+/-1.1 microU/mL, P<0.0001) and lower SI (0.084+/-0.009 versus 0.222+/-0.013, P<0.0001) than IS. Blood pressure, UNaV, and sodium balance did not differ between groups. SI correlated negatively with age (r=-0.39, P<0.05) and BMI (r=-0.53, P<0.01). Urine 20-HETE was less in IR than in IS when normalized by serum insulin (0.91+/-0.25 versus 2.24+/-0.46 microg. 24 hours(-1)/microU x mL(-1), P<0.02), but not if uncorrected. Urinary 20-HETE excretion correlated negatively with insulin (r=-0.40, P<0.04), whereas the relationship between 20-HETE and SI was not statistically significant. Our data suggest that increased circulating insulin, not the state of insulin resistance, suppresses urine 20-HETE excretion in obese hypertensive subjects. Findings in experimental models suggest that an inhibitory effect of insulin on cytochrome P4504A, rather than effects of insulin on membrane-bound arachidonic acid or on its release to the cytosol, may explain our observation.     

Perspective: physiological role(s) of the vascular myogenic response

The vascular myogenic response is an inherent property of VSM in the walls of small arteries and arterioles, allowing these principal resistance segments of the microcirculation to respond to changes in transmural pressure. Elevated intraluminal pressure leads to myogenic constriction, whereas reduced pressure leads to myogenic dilation. This review focuses on the physiological significance of the myogenic response in microvascular networks. First, historical concepts related to the detection of stretch by the vessel wall are reviewed, including the wall tension hypothesis, and the implications of the proposal that the arteriolar network responds to Pp changes as a system of series-coupled myogenic effectors. Next, the role of the myogenic response in the local regulation of blood flow and/or Pc is examined. Finally, the interaction of myogenic constriction and dilation with other local control mechanisms, including metabolic, neural and shear-dependent mechanisms, is discussed. Throughout the review, an attempt is made to integrate historical and current literature with an emphasis on the physiological role, rather than the underlying signaling mechanisms, of this important component of vascular control.     

Substance P targets sympathetic control neurons in the paraventricular nucleus

The paraventricular nucleus (PVN) contains spinally-projecting neurons implicated in fine-tuning the cardiovascular system. In vivo activity of "presympathetic" parvocellular neurons is suppressed by tonic inhibition from GABA-ergic inputs, inhibition of which increases sympathetic pressor activity and heart rate. Targeting of this specific neuronal population could potentially limit elevations of heart rate and blood pressure associated with disease. Here we show, for the first time, that "presympathetic" PVN neurons are disinhibited by the neuropeptide substance P (SP) acting via tachykinin NK1 receptor inhibition of GABA(A) currents. Application of SP to the paraventricular nucleus of rats increases heart rate and blood pressure. In in vitro brain slice experiments, in the presence of GABA, 1 micromol/L SP increased action current frequency by a factor of 2.7+/-0.6 (n=5, P< or =0.05, ANOVA). Furthermore, 1 micromol/L SP inhibited GABA(A) currents by 70+/-8% (n=8, P< or =0.005 paired t test). These effects were abolished by NK1 antagonists, but not NK2 and NK3 antagonists. GABA(A) inhibition was not reproduced by NK2 or NK3 agonists. The inhibition of parvocellular GABA(A) currents by SP was also abolished by a protein kinase C (PKC) inhibitor peptide and mimicked by application of phorbol-12-myristate-13-acetate (PMA), implicating a PKC-dependent mechanism. Single-channel analysis indicates that SP acts through reduction of channel mean open-time (cmot): GABA(A) cmot being reduced by approximately 60% by SP (P< or =0.05 ANOVA, Bonferroni). These data suggest that tachykinins mediate their pressor activity by increasing the excitability of spinally-projecting neurons and identifies NK1 receptors as potential targets for therapeutic modulation of the cardiovascular system.     

A novel mechanism contributing to development of Dahl salt-sensitive hypertension: role of the transient receptor potential vanilloid type 1

To determine the role of the transient receptor potential vanilloid type 1 (TRPV1) channels in development of hypertension in Dahl salt-sensitive (DS) rats fed a high-salt diet (HS), male DS and Dahl salt-resistant (DR) rats were maintained on a low-salt diet (LS) or HS for 3 weeks. HS significantly increased systolic blood pressure in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Intravenous bolus injection of capsazepine (3 mg/kg), a selective TRPV1 antagonist, significantly increased mean arterial pressure in conscious DR+HS rats compared with DR+LS, DS+/-HS, and DS+/-LS rats. In contrast, capsaicin (10 or 30 microg/kg), a selective TRPV1 agonist, dose-dependently decreased mean arterial pressure in all of the groups with the most profound magnitude in DR+HS rats compared with the other 3 groups. TRPV1 expression in mesenteric resistance arteries and the renal cortex and medulla, calcitonin gene-related peptide levels in dorsal root ganglia, and calcitonin gene-related peptide-positive sensory nerve density in mesenteric resistance arteries were significantly decreased in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Taken together, our data indicate that the TRPV1 receptor is activated and its expression upregulated during HS intake in DR rats, which acts to prevent salt-induced increases in blood pressure. In contrast, TRPV1 expression and function are impaired in DS rats, which renders DS rats sensitive to salt load in terms of blood pressure regulation.     

RhoA activation and interaction with Caveolin-1 are critical for pressure-induced myogenic tone in rat mesenteric resistance arteries

OBJECTIVE: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. METHODS: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118+/-15 microm internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. RESULTS: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3+/-2.8 microm and 9.1+/-3.7 microm in +/+ and -/- mice, respectively, n = 5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26+/-13% merging at 25 mmHg versus 97+/-21% at 150 mmHg, n = 5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. CONCLUSION: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development.     

Decreased levels of cytochrome P450 2E1-derived eicosanoids sensitize renal arteries to constrictor agonists in spontaneously hypertensive rats

We compared renal interlobar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in terms of cytochrome P450 (CYP) 4A and CYP2E1 protein expression; levels of 20-HETE, 19-HETE, and 18-HETE; and responsiveness to phenylephrine in the absence and presence of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 mumol/L), a CYP4A inhibitor. Relative to data in WKY, arteries of SHR exhibited diminished (P<0.05) CYP2E1 and levels of 19-HETE (66.7+/-6.0 versus 44.9+/-2.8 pmol/mg) and 18-HETE (13.8+/-1.6 versus 7.9+/-0.5 pmol/mg), whereas CYP4A and 20-HETE levels (99.3+/-9.1 versus 98.9+/-12.8 pmol/mg) were unchanged. Phenylephrine contracted vascular rings of SHR and WKY; the R(max) was similar in both strains, but SHR vessels were more sensitive as denoted by the lower (P<0.05) EC50 (0.28+/-0.07 versus 0.71+/-0.12 mumol/L). DDMS decreased 20-HETE and, to a lesser extent, 19-HETE, while increasing (P<0.05) the EC50 for phenylephrine by 475% and 54% in vessels of SHR and WKY, respectively. The desensitizing effect of DDMS was reversed by 20-HETE. Notably, the minimal concentration of 20-HETE that decreased the EC50 for phenylephrine in DDMS-treated vessels was smaller in SHR (0.1 micromol/L) than WKY (10 micromol/L), and the sensitizing effect of 20-HETE was blunted (P<0.05) by the (R) stereoisomers of 19-HETE and 18-HETE. We conclude that the increased sensitivity to phenylephrine in arteries of SHR is attributable to a vasoregulatory imbalance produced by a deficit in vascular CYP2E1-derived products, most likely 19(R)-HETE and 18(R)-HETE, which condition amplification of the sensitizing action of 20-HETE.     

Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries

The role of cytochrome P-450 in the myogenic response of isolated, perfused renal arcuate arteries of dogs to elevations in transmural pressure was examined. The phospholipase A2 inhibitor oleyloxyethylphosphorylcholine (1 and 10 microM) inhibited the greater than threefold increase in active wall tension in these arteries after an elevation in perfusion pressure from 80 to 160 mm Hg. Inhibition of cyclooxygenase activity with indomethacin (1 or 10 microM) had no effect on this response. The cytochrome P-450 inhibitors ketoconazole (10 and 100 microM) and beta-diethyl-aminoethyldiphenylpropylacetate (SKF 525A, 10 and 100 microM) also inhibited the myogenic response. At a pressure of 160 mm Hg, SKF 525A (10 microM) and ketoconazole (100 microM) reduced active wall tension in renal arteries by approximately 70%. Partial inhibition of the myogenic response was obtained after perfusion of the vessels with mechanism-based inhibitors of P-450, 1-aminobenzotriazole (75 microM) and 12-hydroxy-16-heptadecynoic acid (20 microM). The thromboxane receptor antagonist SQ 29,548 (1 or 10 microM) had no effect on the pressure-induced increase in active wall tension in renal arteries. Arachidonic acid (50 microM) constricted isolated perfused renal arteries and potentiated the myogenic response in the presence of indomethacin. This response was completely reversed by ketoconazole (100 microM) or SKF 525A (100 microM). Microsomes (1 mg/ml) prepared from small renal arteries (200-500 microns) and incubated with [1-14C]arachidonic acid (0.5 mu Ci, 50 microM) produced a metabolite that coeluted with 20-hydroxyeicosatetraenoic acid (20-HETE) during reversed-phase high-performance liquid chromatography. The formation of this product was inhibited by both ketoconazole and SKF 525A at concentrations of 10 and 100 microM. These results are consistent with the involvement of the vasoconstrictor 20-HETE and other cytochrome P-450 metabolites of endogenous fatty acids in the myogenic response.     

Impaired vasodilation in response to perivascular nerve stimulation in mesenteric arteries of TRPV1-null mutant mice

BACKGROUND: The role of the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in perivascular sensory nerves in the regulation of vascular reactivity is largely unknown. This study was designed to test the hypothesis that vasodilation induced by electrical field stimulation (EFS) of perivascular sensory nerves is mediated by the TRPV1 via release of sensory neurotransmitters in wild-type (WT) mice, and this effect is abolished in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. METHODS: Isolated mesenteric resistance arteries from WT and TRPV1(-/-)) mice were perfused and pretreated with guanethedine and atropine to block sympathetic and parasympathetic nerve activity, respectively. After precontracting with phenylephrine, changes of vascular diameters induced by EFS were monitored in the absence or presence of the TRPV1 receptor antagonist capsazepine; the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37; or the substance P (SP) receptor antagonist, RP67580. RESULTS: EFS-induced vasodilation was significantly reduced in arteries in TRPV1(-/-)) mice when compared to that of WT mice. Capsazepine and CGRP8-37 attenuated vasodilation induced by EFS in WT but not TRPV1(-/-)) mice. In contrast, RP67580 had no effect on the EFS-induced vasodilation in WT or TRPV1(-/-)) mice. The release of CGRP in the face of EFS challenge was significantly increased in both WT and TRPV1(-/-)) arteries, which was attenuated by capsazepine in WT but not TRPV1(-/-)) arteries. Exogenous CGRP caused dose-dependent vasodilation to a similar degree in WT and TRPV1(-/-)) arteries. CONCLUSIONS: Our data show that in WT mice transmural stimulation of perivascular sensory nerves activates the TRPV1, leading to CGRP release from sensory nerve endings; and blockade of CGRP, but not SP, receptors abolishes TRPV1-mediated vasodilation during EFS. All these effects are impaired in TRPV1(-/-)) mice, indicating that TRPV1 plays a key role in modulating perivascular sensory nerve-mediated vasodilation.     

Key role of Kv1 channels in vasoregulation

Small arteries play an essential role in the regulation of blood pressure and organ-specific blood flow by contracting in response to increased intraluminal pressure, ie, the myogenic response. The molecular basis of the myogenic response remains to be defined. To achieve incremental changes in arterial diameter, as well as blood pressure or organ-specific blood flow, the depolarizing influence of intravascular pressure on vascular smooth muscle membrane potential that elicits myogenic contraction must be precisely controlled by an opposing hyperpolarizing influence. Here we use a dominant-negative molecular strategy and pressure myography to determine the role of voltage-dependent Kv1 potassium channels in vasoregulation, specifically, whether they act as a negative-feedback control mechanism of the myogenic response. Functional Kv1 channel expression was altered by transfection of endothelium-denuded rat middle cerebral arteries with cDNAs encoding c-myc epitope-tagged, dominant-negative mutant or wild-type rabbit Kv1.5 subunits. Expression of mutant Kv1.5 dramatically enhanced, whereas wild-type subunit expression markedly suppressed, the myogenic response over a wide range of intraluminal pressures. These effects on arterial diameter were associated with enhanced and reduced myogenic depolarization by mutant and wild-type Kv1.5 subunit expression, respectively. Expression of myc-tagged mutant and wild-type Kv1.5 subunit message and protein in transfected but not control arteries was confirmed, and isolated myocytes of transfected but not control arteries exhibited anti-c-myc immunofluorescence. No changes in message encoding other known, non-Kv1 elements of the myogenic response were apparent. These findings provide the first molecular evidence that Kv1-containing delayed rectifier K+ (K(DR)) channels are of fundamental importance for control of arterial diameter and, thereby, peripheral vascular resistance, blood pressure, and organ-specific blood flow.