Arteries Of Wistar-Kyoto Rats Research Articles

Atlas of Cell Repertoire Within Neointimal Lesions Is Metabolically Altered in Hypertensive Rats.

High blood pressure has been suggested to accelerate vascular injury-induced neointimal formation and progression. However, little is known about the intricate relationships between vascular injury and hypertension in the context of arterial remodeling. Single-cell RNA-sequencing analysis was used to depict the cell atlas of carotid arteries of Wistar Kyoto rats and spontaneously hypertensive rats with or without balloon injury. We found that hypertension significantly aggravated balloon injury-induced arterial stenosis. A total of 36 202 cells from carotid arteries with or without balloon injury were included in single-cell RNA-sequencing analysis. Cell composition analysis showed that vascular injury and hypertension independently induced distinct aortic cell phenotypic alterations including immune cells, endothelial cells (ECs), and smooth muscle cells. Specifically, our data showed that injury and hypertension-induced specific EC phenotypic alterations, and revealed a transition from functional ECs to hypermetabolic, and eventually dysfunctional ECs in hypertensive rats upon balloon injury. Importantly, our data also showed that vascular injury and hypertension-induced different smooth muscle cell phenotypic alterations, characterized by deferential expression of synthetic signatures. Interestingly, pathway analysis showed that dysregulated metabolic pathways were a common feature in monocytes/macrophages, ECs, and smooth muscle cells in response to injury and hypertension. Functionally, we demonstrate that inhibition of mitochondrial respiration significantly ameliorated injury-induced neointimal formation in spontaneously hypertensive rats. This study provides the cell landscape changes of the main aortic cell phenotypic alterations in response to injury and hypertension. Our findings suggest that targeting cellular mitochondrial respiration could be a novel therapeutic for patients with hypertension undergoing vascular angioplasty.

Impaired Trafficking of β1 Subunits Inhibits BK Channels in Cerebral Arteries of Hypertensive Rats.

Hypertension is a risk factor for cerebrovascular diseases, including stroke and dementia. During hypertension, arteries become constricted and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by smooth muscle cell (myocyte) large-conductance calcium (Ca2+)-activated potassium (BK) channels is altered during hypertension, although mechanisms involved are unclear. We tested the hypothesis that dysfunctional trafficking of pore-forming BK channel (BKα) and auxiliary β1 subunits contributes to changes in cerebral artery contractility of stroke-prone spontaneously hypertensive rats (SP-SHRs). Our data indicate that the amounts of total and surface BKα and β1 proteins are similar in unstimulated arteries of age-matched SP-SHRs and normotensive Wistar-Kyoto rats. In contrast, stimulated surface-trafficking of β1 subunits by NO or membrane depolarization is inhibited in SP-SHR myocytes. PKCα (protein kinase C α) and PKCβII total protein and activity were both higher in SP-SHR than in Wistar-Kyoto rat arteries. NO or depolarization robustly activated Rab11, a small trafficking GTPase, in Wistar-Kyoto rat arteries but weakly activated Rab11 in SP-SHRs. Bisindolylmaleimide, a PKC inhibitor, and overexpression of a PKC phosphorylation-deficient Rab11A mutant (Rab11A S177A) restored stimulated β1 subunit surface-trafficking in SP-SHR myocytes. BK channel activation by NO was inhibited in SP-SHR myocytes and restored by Rab11A S177A expression. Vasodilation to NO and lithocholate, a BKα/β1 channel activator, was inhibited in pressurized SP-SHR arteries and reestablished by bisindolylmaleimide. In summary, data indicate that spontaneously active PKC inhibits Rab11A-mediated β1 subunit trafficking in arterial myocytes of SP-SHRs, leading to dysfunctional NO-induced BK channel activation and vasodilation.

Demethoxycurcumin Preserves Renovascular Function by Downregulating COX-2 Expression in Hypertension.

Hypertension-associated endothelial dysfunction is largely due to the exaggerated vasoconstrictor generation by cyclooxygenase-2 (COX-2). COX-2 is induced under inflammatory condition. Demethoxycurcumin (DMC) is a major component of Curcuma longa L, which possesses anti-inflammatory action. This study aimed to examine whether DMC protects endothelial function in hypertension by modulating COX-2. Changes in isometric tension showed that in vivo and ex vivo treatment with DMC rescued the attenuated endothelium-dependent relaxations (EDRs) and elevated endothelium-dependent contractions (EDCs) in the renal arteries of SHR, which were also corrected by acute usage of the COX-2 inhibitor celecoxib. The restoration of renovascular activity by DMC was accompanied by the normalization of COX-2 expression. The enhanced COX-2 expression observed in the renal arteries of hypertensive patients was suppressed by incubation of excised arteries with DMC for 12 hrs. In the renal arteries of Wistar-Kyoto rats (WKY), DMC prevented the endothelial dysfunction caused by angiotensin II. The reduction in the generation of nitric oxide (NO) and expression of eNOS phosphorylation (Ser1177) in human umbilical vein endothelial cells caused by angiotensin II (Ang II) were restored by DMC or celecoxib. Our findings suggest that DMC may decrease COX-2 expression and improve endothelial function in hypertension.

Transient Receptor Potential Channel Opening Releases Endogenous Acetylcholine, which Contributes to Endothelium-Dependent Relaxation Induced by Mild Hypothermia in Spontaneously Hypertensive Rat but Not Wistar-Kyoto Rat Arteries.

Mild hypothermia causes endothelium-dependent relaxations, which are reduced by the muscarinic receptor antagonist atropine. The present study investigated whether endothelial endogenous acetylcholine contributes to these relaxations. Aortic rings of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were contracted with prostaglandin F2 α and exposed to progressive mild hypothermia (from 37 to 31°C). Hypothermia induced endothelium-dependent, Nω-nitro-l-arginine methyl ester-sensitive relaxations, which were reduced by atropine, but not by mecamylamine, in SHR but not in WKY rat aortae. The responses in SHR aortae were also reduced by acetylcholinesterase (the enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake), and vesamicol (inhibitor of acetylcholine release). The mild hypothermia-induced relaxations in both SHR and WKY rat aortae were inhibited by AMTB [N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide; the transient receptor potential (TRP) M8 inhibitor]; only those in SHR aortae were inhibited by HC-067047 [2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide; TRPV4 antagonist] while those in WKY rat aortae were reduced by HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; TRPA1 antagonist]. The endothelial uptake of extracellular choline and release of cyclic guanosine monophosphate was enhanced by mild hypothermia and inhibited by HC-067047 in SHR but not in WKY rat aortae. Compared with WKY rats, the SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but a lesser amount of vesicular acetylcholine transporter, located mainly in the endothelium. Thus, mild hypothermia causes nitric oxide-dependent relaxations by opening TRPA1 channels in WKY rat aortae. By contrast, in SHR aortae, TRPV4 channels are opened, resulting in endothelial production of acetylcholine, which, in an autocrine manner, activates muscarinic receptors on neighboring cells to elicit endothelium-dependent relaxations in response to mild hypothermia.

Different susceptibility to vascular damage induced by chronic hyperglycemia in aortic and pulmonary arteries from Sprague Dawley and Wistar Kyoto rats

In this study was evaluated the effect of chronic hyperglycemia in the vascular response of thoracic aorta and pulmonary trunk in two strain rats. Chronic hyperglycemia was induced in male Wistar Kyoto (WK) and Sprague Dawley (SD) rats for single injection of streptozotocin (60 mg/kg, ip.). After 8 weeks, body weight, glucose, systemic arterial blood pressure (SABP) and right ventricular systolic pressure (RVSP) were measured. Thoracic aorta and pulmonary trunks were removed for vascular reactivity studies. Cumulative concentration‐response curves with phenylephrine (Phe) and Acetylcholine (ACh) were made in aorta and pulmonary rings. Glucose blood levels (mg/dl) were similar in both groups, 525±2 vs. 502±3. SABP in mmHg was increased in hyperglycemic SD compared to WK rats 160±2 vs. 140±2, while RVSP was higher in WK than SD rats 41±2.6 vs. 22±1.5 (P<0.001). Maximal tension contraction (g) response with Phe was higher in hyperglycemic SD against hyperglycemic WK aortic rings (2.1±0.1 vs. 1.2±0.1, P</a><0.001), with similar response in the pulmonary rings in both groups. ACh‐induced relaxation (%) response was reduced in SD aorta and pulmonary rings, 58±3 vs 66±4, and this response was attenuated inaorta 78±3, and pronounced in pulmonary rings, 49±3, from WK rats (P<0.001). The results shown that chronic hyperglycemia induce vascular damage predominantly in pulmonary artery in WK rats as consequence pulmonary hypertension, whereas in SD rats, systemic hypertension consecutive to aorta damage is mainly developed suggesting that a genetic factor determine the vascular damage induced by chronic hyperglycemia.

Vasorelaxant effects of camel and bovine casein hydrolysates in rat thoracic aorta and mesenteric artery

We evaluated the vasorelaxant effect of camel and bovine casein tryptic hydrolysates (CTH and BTH, respectively) and their mechanism of action in aorta and mesenteric artery of Wistar Kyoto rats. CTH produced a more potent relaxation than that induced by BTH. This relaxation was endothelium-dependent and was mediated through the activation of the nitric oxide (NO)/cyclic guanosine 3,5-monophosphate (cGMP) pathway. The intracellular level of cGMP was increased by CTH. Moreover, CTH-induced relaxation was inhibited by HOE 140, a B2 bradykinin receptors antagonist, indicating that B2 bradykinin receptors play a role in the CTH effect. In endothelium-intact mesenteric artery, CTH and BTH produced a similar concentration-dependent relaxation with a lesser maximal effect than in aortic rings. Moreover, this relaxation was inhibited by Nω nitro-l-arginine methyl ester hydrochloride, a NO synthase inhibitor, but the addition of indomethacin, a cyclooxygenase inhibitor, and tetraethylammonium, a K+ channels blocker did not potentiate the inhibition.

Chronic oxidative–nitrosative stress impairs coronary vasodilation in metabolic syndrome model rats

Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative–nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr496- and Ser1177-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr496-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative–nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms.

Enhanced constriction of supplying arteries — A mechanism of femoral head Necrosis in Wistar rats?

Femoral head necrosis occurs in 8–15% of Wistar Kyoto (WKY) rats, and in up to 46% of spontaneous hypertensive (SHR) rats. For SHR rats, the etiologic factors have been described while the pathomechanism of femoral head necrosis in Wistar rats remains unclear. The aim of this study has been to compare the vasoconstrictive effect of noradrenaline on femoral arteries in common Wistar and SHR rats. Four male SHR rats 180–209 days of age, and four male Wistar rats 179–185 days of age, were used. Seven femoral artery segments were harvested from the SHR rats while 6 artery segments were harvested from the WKY rats. The arterial segments were mounted as ring preparations on a small vessel myograph for isometric force development. The arteries were stimulated cumulatively by adding noradrenaline (0.3–30 μM at 2-min intervals) to obtain the dose–response curve of isometric wall tension. Noradrenaline elicited a concentration-dependent vasocontraction in all arteries. The lumen diameter at standard passive tension L 100 was not different. The dose–response curve showed a stronger constriction of the femoral artery segments in WKY than in SHR rats. The maximal active tension of noradrenaline was 6.4±3.4 mN in the WKY, and 3.0±1.8 mN (mean±S.D), significantly lower, in the SHR group ( p<0.05). This study showed that vasoconstriction of the femoral artery in WKY rats was stronger than that of SHR rats. This may be a pathomechanical factor in femoral head necrosis of WKY rats.

Mitogen-activated protein kinase activation by hydrogen peroxide is mediated through tyrosine kinase-dependent, protein kinase C-independent pathways in vascular smooth muscle cells: upregulation in spontaneously hypertensive rats

To investigate the putative molecular mechanisms underlying mitogen-activated protein (MAP) kinase activation by hydrogen peroxide (H(2)O(2)) in vascular smooth muscle cells (VSMC) and to evaluate whether H(2)O(2)-induced actions are altered in VSMC from spontaneously hypertensive rats (SHR). VSMC from mesenteric arteries of Wistar-Kyoto rats (WKY) and SHR were stimulated with H(2)O(2) (2-30 min). The phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and p38MAP kinase was determined by immunoblotting. The involvement of tyrosine kinase and protein kinase C (PKC) was evaluated using pharmacological inhibitors, tyrphostin (A23 and A9) and GF109203X, respectively. The role of receptor tyrosine kinases (RTK) was assessed with AG1478, AG1296 and AG1024, selective inhibitors of epidermal growth factor receptor, platelet-derived growth factor receptor and insulin-like growth factor receptor, respectively. Non-receptor tyrosine kinases (NRTK) were studied using AG490 (JAK2 inhibitor) and PP2 (Src inhibitor). H(2)O(2) stimulated phosphorylation of ERK1/2 and p38MAP kinase in a time-dependent manner. This increase was significantly greater in SHR versus WKY (P < 0.01). The activation of MAP kinases was unaffected by GF109203X but was decreased by tyrphostins (P < 0.01). The inhibition of NRTK attenuated H(2)O(2)-mediated phosphorylation of ERK1/2 (P < 0.001) but not of p38MAP kinase, whereas Src and JAK2 inhibition significantly decreased phosphorylation of both MAP kinases (P < 0.01). These data indicate that H(2)O(2) increases ERK1/2 and p38MAP kinase activation through tyrosine kinase-dependent, PKC-independent mechanisms. Whereas ERK1/2 is regulated by both RTK and NRTK, p38MAP kinase is regulated by NRTK. Our findings identify an important role for tyrosine kinases, but not PKC, in H(2)O(2)-induced phosphorylation of ERK1/2 and p38MAP kinase in VSMC. The upregulation of these processes may contribute to enhanced redox-dependent MAP kinase signaling in SHR VSMC.

Dysregulation of HSG triggers vascular proliferative disorders.

Vascular proliferative disorders, such as atherosclerosis and restenosis, are the most common causes of severe cardiovascular diseases, but a common molecular mechanism remains elusive. Here, we identify and characterize a novel hyperplasia suppressor gene, named HSG (later re-named rat mitofusin-2). HSG expression was markedly reduced in hyper-proliferative vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries, balloon-injured Wistar Kyoto rat arteries, or ApoE-knockout mouse atherosclerotic arteries. Overexpression of HSG overtly suppressed serum-evoked VSMC proliferation in culture, and blocked balloon injury induced neointimal VSMC proliferation and restenosis in rat carotid arteries. The HSG anti-proliferative effect was mediated by inhibition of ERK/MAPK signalling and subsequent cell-cycle arrest. Deletion of the p21(ras) signature motif, but not the mitochondrial targeting domain, abolished HSG-induced growth arrest, indicating that rHSG-induced anti-proliferation was independent of mitochondrial fusion. Thus, rHSG functions as a cell proliferation suppressor, whereas dysregulation of rHSG results in proliferative disorders.

Role of CA(2+)-activated K+ channels in the regulation of basilar arterial tone in spontaneously hypertensive rats.

1. Ionic channels appear to play an important role in contractile responses of the cerebral arteries and, thereby, contribute to the regulation of cerebral circulation. In the present study, we investigated the role of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels in the regulation of cerebral arterial tone during chronic hypertension. 2. Ring segments of the basilar artery from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were placed in bath chambers and the isometric tension of each ring was measured. 3. Application of inhibitors of BK(Ca) channels, namely tetraethylammonium (TEA; > or = 0.1 mmol/L) and charybdotoxin (CTX; > or = 0.1 nmol/L), produced spontaneous contraction with rhythmic oscillation in the basilar artery from SHR. 4. The oscillatory contraction was not induced by 5-hydroxytryptamine (0.01-10 micromol/L) or depolarization by external high K+ (20-60 mmol/L). 5. The rhythmic contraction was completely abolished by either the removal of external Ca(2+) or the application of nicardipine (10 nmol/L). 6. The oscillation was not affected by the substitution of external Cl(-) by various equimolar anions (i.e. acetate, benezenesulphonate, bromide and isethianate). 7. The amplitude of the oscillation was dose-dependently increased by the vasodilators forskolin and sodium nitroprusside, as well as by stimulation of the endothelium with histamine and acetylcholine, whereas the frequency was decreased. 8. In contrast, the oscillation was eliminated by depletion of Ca(2+) stores by caffeine. Neither TEA (10 mmol/L) nor CTX (10 nmol/L) produced any significant contraction of the basilar artery in WKY rats. 9. These results suggest that BK(Ca) channels may play an important role in regulating the resting tone of the cerebral artery in SHR.

Increased activity of calcium channels and Rho-associated kinase in the basilar artery during chronic hypertension in vivo.

Several factors mediating vascular responses appear to play an important role in the increased resistance of cerebral blood vessels during hypertension. The objective of this study was to elucidate the mechanisms by which hypertension increases the basal tone of the basilar artery in vivo. Using a cranial window, we examined effects of inhibitors of L-type voltage-dependent calcium channels (nicardipine) and Rho-associated kinase (Y-27632) on the baseline diameter of the basilar artery in spontaneously hypertensive rats (SHR) and compared to the responses in normotensive Wistar-Kyoto (WKY) rats. Topical application of nicardipine (10(-8), 10(-7) and 10(-6) mol/l) produced dilatation of the basilar artery. Nicardipine-induced vasodilatation was enhanced in SHR compared to WKY rats. Nicardipine (10-6 mol/l) dilated the artery in WKY rats and SHR by 9 +/- 2 and 24 +/- 4%, respectively. Topical application of Y-27632 (10(-7), 10(-6) and 10(-5) mol/l) produced dilatation of the basilar artery in WKY rats in a concentration-related manner. The vasodilatation produced by Y-27632 was markedly enhanced in SHR compared to WKY rats. Y-27632 (10(-5) mol/l) dilated the artery in WKY rats and SHR by 14 +/- 2 and 45 +/- 6%, respectively. We also tested the effects of inhibitors of nitric oxide synthase (NG-nitro-l-arginine methyl ester, l-NAME), ATP-sensitive potassium channels (glibenclamide) and large-conductance calcium-activated potassium channels (tetraetyhlammonium, TEA). l-NAME and, to a lesser extent, glibenclamide produced similar constriction of the basilar artery in both strains. TEA did not affect the baseline diameter of the basilar artery in WKY rats and produced small but significant vasoconstriction in SHR. These results suggest that the activity of L-type calcium channels and Rho-associated kinase is enhanced in the basilar artery during hypertensionin vivo. The enhanced contractility may contribute to the increased resistance of the basilar artery during chronic hypertension in vivo.

Spontaneously hypertensive rat resistance artery structure related to myogenic and mechanical properties

This investigation related arterial structure to myogenic (pressure-dependent) contractile responses in resistance arteries from spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) normotensive control rats under pressurized conditions in vitro. Femoral and mesenteric resistance arteries from either strain were cannulated and pressurized in an arteriograph for the determination of pressure-diameter relationships under passive and active conditions in the range 5-200 mmHg transmural pressure. Arterial geometrical measurements were made under relaxed conditions at 100 mmHg. Media thickness/lumen diameter (M/L) ratios were significantly increased in SHR femoral (5.00+/-0.44% compared with 3.63+/-0.34%; P<0.05) and mesenteric (4.40+/-0.29% compared with 2.62+/-0.23%; P<0.001) arteries compared with those from WKY rats. Maximum myogenic contractions, assessed as minimum normalized diameters, were not significantly different in SHR and WKY rat femoral (0.41+/-0.03 and 0.40+/-0.02 respectively) or mesenteric (0.56+/-0.02 and 0.63+/-0.03 respectively) arteries. Arterial mechanical analyses demonstrated that incremental elastic modulus is reduced in SHR mesenteric arteries, but is not significantly different in SHR femoral arteries, compared with those from WKY rats. Additionally, wall stress at estimated in vivo pressures under passive and active conditions are similar in SHR and WKY rat arteries. These data demonstrate that increased M/L ratios in resistance arteries from SHRs are not associated with increased maximum pressure-dependent contractile responses. Increased M/L ratios in resistance arteries from SHRs are not accounted for by increased vessel wall stiffness, but the hypertension-associated arterial geometrical abnormalities act to normalize wall stress in the face of increased arterial pressure.

The Influence of Chloroethylclonidine-Induced Contraction in Isolated Arteries of Wistar Kyoto Rats: α 1D- and α 1A-Adrenoceptors, Protein Kinase C, and Calcium Influx

Background It has recently been reported that chloroethylclonidine (CEC) elicited contraction in tail arteries (α 1A-adrenoceptors) and aorta (α 1D-adrenoceptors) from normotensive and spontaneously hypertensive rats (SHR). This study investigated the relationship between CEC-induced contraction and the role of protein kinase C (PKC) and extracellular Ca ++ influx in tail arteries and aorta from Wistar Kyoto rats (WKY). Methods Time-course of CEC-induced contraction in endothelium-denuded arteries from Wistar, WKY, and SHR rats was evaluated. In WKY arteries, calphostin C (1 × 10 −6 M) and nitrendipine (1 × 10 −6 M) were used to determine the role of PKC and extracellular Ca +1 in the contractile response to CEC, respectively. Results Chloroethylclonidine (1 × 10 −4 M) elicited contraction in tail arteries and aorta from normotensive and hypertensive rats. Maximal response to CEC was similar in tail arteries among strains (≈30% of norepinephrine effect), while in aorta CEC elicited a higher contraction in WKY and SHR than in Wistar (59, 86, and 18% of norepinephrine effect, respectively). CEC-elicited maximal contractile responses were reached in 5 min in tail arteries and in 30–45 min in aorta irrespective of the rat strain, suggesting that different intracellular signaling pathways are involved in the contractile response to CEC in these arteries. In WKY tail arteries, calphostin C and nitrendipine blocked CEC-induced contraction while in aorta nitrendipine, but not calphostin C, inhibited CEC action. Conclusions This study confirms marked strain-dependent differences in rat aorta responsiveness to CEC and suggests a central role for PKC in response to CEC in tail arteries and for extracellular Ca +1 influx in aorta.

Endothelium-dependent vasorelaxation independent of nitric oxide and K(+) release in isolated renal arteries of rats.

1. We investigated whether K(+) can act as an endothelium-derived hyperpolarizing factor (EDHF) in isolated small renal arteries of Wistar-Kyoto rats. 2. Acetylcholine (0.001 - 3 microM) caused relaxations that were abolished by removal of the endothelium. However, acetylcholine-induced relaxations were not affected by the nitric oxide (NO) synthase inhibitor N:(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), by L-NAME plus the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 1 microM) or by L-NAME plus the cyclo-oxygenase inhibitor indomethacin (10 microM). In rings precontracted with high-K(+)(60 mM) physiological salt solution in the presence of L-NAME, acetylcholine-induced relaxations were abolished. 3. L-NAME-resistant relaxations were abolished by the large-conductance Ca(2+)-activated K(+) channel inhibitor charybdotoxin plus the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin, while the inward rectifier K(+) channel inhibitor Ba(2+) or the gap junction inhibitor 18alpha-glycyrrhetinic acid had no effect. Acetylcholine-induced relaxation was unchanged by ouabain (10 microM) but was partially inhibited by a higher concentration (100 microM). 4. In half of the tissues tested, K(+)(10 mM) itself produced L-NAME-resistant relaxations that were blocked by ouabain (10 microM) and partially reduced by charybdotoxin plus apamin, but not affected by 18alpha-glycyrrhetinic acid or Ba(2+). However, K(+) did not induce relaxations in endothelium-denuded tissues. 5. In conclusion, acetylcholine-induced relaxations in this tissue are largely dependent upon hyperpolarization mechanisms that are initiated in the endothelium but do not depend upon NO release. K(+) release cannot account for endothelium-dependent relaxation and cannot be an EDHF in this artery. However, K(+) itself can initiate endothelium-dependent relaxations via a different pathway from acetylcholine, but the mechanisms of K(+)-induced relaxations remain to be clarified.

Cholesterol enhances contractile responses in isolated small mesenteric arteries of normotensive and spontaneously hypertensive rats.

In order to examine possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular disease, we investigated the effect of cholesterol enrichment on contractility in isolated small rat mesenteric arteries. Contractile responses of cholesterol-enriched isolated small mesenteric arteries of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were compared with control groups. First- to second-order mesenteric arteries (327-349 microm internal lumen diameter) were dissected from the mesenteric bed of 10-20-week-old male WKY rats and SHR, and incubated in cholesterol-free and cholesterol-rich (150 microg/ml) medium. Isolated arteries were mounted on a Mulvany-Halpern myograph for measurement of isometric tension. Cholesterol significantly increased active wall tension and active wall pressure in WKY rat arteries and active wall tension in SHR arteries in response to potassium chloride, norepinephrine and serotonin (P < 0.05). In addition, contractile responses to all agonists were significantly higher in cholesterol-enriched SHR arteries compared with cholesterol-enriched WKY rat vessels (P < 0.05). These findings suggest that elevated cholesterol content enhances agonist-stimulated contractility in small mesenteric resistance arteries, providing a possible mechanism by which hypercholesterolemia may contribute to the development of hypertension.

Endothelium-derived relaxing, contracting and hyperpolarizing factors of mesenteric arteries of hypertensive and normotensive rats.

Differences in the acetylcholine (ACh)-induced endothelium-dependent relaxation and hyperpolarization of the mesenteric arteries of Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Relaxation was impaired in preparations from SHRSP and tendency to reverse the relaxation was observed at high concentrations of ACh in these preparations. Relaxation was partly blocked by NG-nitro-L-arginine (L-NOARG, 100 microM) and, in the presence of L-NOARG, tendency to reverse the relaxation was observed in response to higher concentrations of ACh, even in preparations from WKY. The relaxation remaining in the presence of L-NOARG was also smaller in preparations from SHRSP. The tendency to reverse the relaxation observed at higher concentrations of ACh in preparations from SHRSP or WKY in the presence of L-NOARG were abolished by indomethacin (10 microM). Elevating the K+ concentration of the incubation medium decreased relaxation in the presence of both indomethacin and L-NOARG. Relaxation in the presence of L-NOARG and indomethacin was reduced by the application of both apamin (5 microM) and charybdotoxin (0.1 microM). This suggests that the relaxation induced by ACh is brought about by both endothelium-derived relaxing factor (EDRF, nitric oxide (NO)) and hyperpolarizing factor (EDHF), which activates Ca2+-sensitive K+ channels. Electrophysiological measurement revealed that ACh induced endothelium-dependent hyperpolarization of the smooth muscle of both preparations in the presence of L-NOARG and indomethacin; the hyperpolarization being smaller in the preparation from SHRSP than that from WKY. These results suggest that the release of both NO and EDHF is reduced in preparations from SHRSP. In addition, indomethacin-sensitive endothelium-derived contracting factor (EDCF) is released from both preparations; the release being increased in preparations from SHRSP.

Chloroethylclonidine is a partial α1-adrenoceptor agonist in aorta and caudal arteries of the Wistar Kyoto rat

The interaction between chloroethylclonidine ( N- β-chloroethyl- N-methylamino-methyl-clonidine) and α 1-adrenoceptors mediating contraction in Wistar Kyoto rat arteries was examined. In caudal ( α 1A-adrenoceptors) and aorta ( α 1D-adrenoceptors) arteries, chloroethylclonidine (10 −4 M) elicited contraction with different time frames (maximal effect within 4 min in the caudal artery and at 30–45 min in aorta). Phentolamine (10 −6 M) completely prevented chloroethylclonidine-induced contraction in aorta, but partially did so in the caudal artery. Rauwolscine (10 −7 M) partially prevented the chloroethylclonidine contractile effect in both arteries. Chloroethylclonidine attenuated the contractile effect of low concentrations of norepinephrine, however, maximal contraction was observed at catecholamine concentrations above 10 −7 M in the caudal artery and 10 −6 M in the aorta. It is concluded that chloroethylclonidine interacts with caudal α 1A-adrenoceptors as an irreversible partial agonist, inducing vascular contraction probably due to Ca 2+ mobilisation, and with aorta α 1D-adrenoceptors as a partial agonist, inducing slow-onset muscular contraction.

Angiotensin II regulates vascular smooth muscle cell pH, contraction, and growth via tyrosine kinase-dependent signaling pathways.

Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II-stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2+/-0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4+/-0.1). Ang II (10(-7) mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II-elicited responses. Tyrphostin A-23 significantly attenuated Ang II-stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II-stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II-mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype-mediated contraction and growth.

Loss of acetylcholine-induced relaxation by M3-receptor activation in mesenteric arteries of spontaneously hypertensive rats.

This study investigated which subtype of muscarinic receptor in mesenteric arteries from SHRs mediates the loss of the release of nitric oxide (NO) or endothelium-derived hyperpolarizing factor (EDHF) in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. After SHRs and WKY rats were killed, their superior mesenteric arteries were isolated to perform the vascular relaxation study. Acetylcholine (ACh, 1 nM-1 microM)-induced relaxation was in a concentration-dependent manner and was impaired in mesenteric arterial rings obtained from the SHR group, whereas nitroglycerin (10 nM-1 microM)-induced relaxation was not affected in endothelium-denuded rings obtained from the hypertensive rat. In the presence of N(omega)-nitro-L-arginine (L-NNA; 0.1 mM) or methylene blue (10 microM), the ACh-induced relaxation was partially attenuated in both SHRs and WKY rats. The relaxation of ACh was partially inhibited by 4-aminopyridine (4-AP; 1 mM), apamin (APM; 1 microM), or charybdotoxin (CTX; 0.1 microM) in WKY rats, whereas that relaxation was not affected by 4-AP, APM, or CTX in SHRs. However, the L-NNA-resistant relaxation of ACh was further inhibited by APM or CTX in WKY rats but not in SHRs. When arterial rings were precontracted by 60 mM K+, the ACh-induced relaxation was not significantly different in SHRs and WKY rats. However, this relaxation was abolished by L-NNA (0.1 mM) in both strains. In addition, the M3 muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine was the most potent in inhibiting the relaxation of ACh, being more potent than pirenzepine and methoctramine, which preferentially block M1 and M2 receptors in the mesenteric artery of WKY rats, respectively. 2-Nitro-4-carboxyphenyl-N,N-diphenylcarbamate (1 microM) almost abolished the relaxation of ACh, but not of A23187, in SHRs and WKY rats. These results suggest that NO and EDHF are released from the endothelium of rat mesenteric artery on the activation of muscarinic M3-receptor by ACh, and the loss of ACh-induced relaxation in small arteries of SHRs is mainly through reducing the release or activity or both of EDHF.