Vascular alpha1-adrenoceptors in isolated perfused rat kidney: influence of ageing.

1. The present study identifies alpha1-adrenoceptor subtype(s) involved in constrictor responses of the kidney and how ageing influences it. 2. The study was conducted on kidneys from F344BNF1 rats, which unlike F344 or Wistar rats used by many previous investigators do not exhibit glomerulonephritis at advanced age. 3. Noradrenaline (NA) and phenylephrine (PHE) (non-selective alpha1) and A61063 (selective alpha(1A)) adrenoceptor agonists elicited constriction of perfused kidneys of young and old rats. The pD2 values (index of renovascular reactivity) were significantly higher for A61603 than for either PHE or NA, and significantly decrease across age groups. 4. BMY 7378 or RS 100329, alpha(1D)- or alpha(1A)-adrenoceptor antagonists, respectively antagonized the constrictor responses and suppressed the maximal responses to all agonists in young adult rat kidneys. However, antagonism of PHE or A61063 by BMY 7378 in old rat kidneys was surmountable. 5. This study suggests that: (i) alpha(1A) and alpha(1D)-adrenoceptor subtypes mediate vasoconstriction of perfused rat kidney; (ii) alpha(1A)-adrenoceptor subtype appears to predominate in renal vasculature based on agonist relative potencies. (iii) Ageing significantly decreases alpha1-adrenoceptor-mediated vasoconstriction of rat kidney.

Acrolein induces vasodilatation of rodent mesenteric bed via an EDHF-dependent mechanism.

Acrolein is generated endogenously during lipid peroxidation and inflammation and is an environmental pollutant. Protein adducts of acrolein are detected in atherosclerotic plaques and neurons of patients with Alzheimer's disease. To understand vascular effects of acrolein exposure, we studied acrolein vasoreactivity in perfused rodent mesenteric bed. Acrolein induced endothelium-dependent vasodilatation that was more robust and more sensitive than dilation induced by 4-hydroxy-trans-2-nonenal, trans-2-hexenal, or propionaldehyde. Acrolein-induced vasodilatation was mediated by K(+)-sensitive components, e.g., it was abolished in 0 [K(+)](o) buffer or in 3 mM tetrabutylammonium, inhibited 75% in 50 microM ouabain, and inhibited 64% in 20 mM K(+) buffer. Moreover, combined treatment with the Ca(2+)-activated K(+) channel inhibitors 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 100 nM) and apamin (5 microM) significantly reduced vasodilatation without altering sensitivity to acrolein. However, acrolein-induced % dilation was unaffected by l-NAME or indomethacin pretreatment indicating mechanistic independence of NO and prostaglandins. Moreover, acrolein induced vasodilatation in cirazoline-precontracted mesenteric bed of eNOS-null mice confirming eNOS independence. Pretreatment with 6-(2-propargyloxyphenyl) hexanoic acid (PPOH 50 microM), an epoxygenase inhibitor, or the superoxide dismutase mimetic Tempol (100 microM) significantly attenuated acrolein-induced vasodilatation. Collectively, these data indicate that acrolein stimulates mesenteric bed vasodilatation due to endothelium-derived signal(s) that is K(+)-, ouabain-, PPOH-, and Tempol-sensitive, and thus, a likely endothelium-derived hyperpolarizing factor (EDHF). These data indicate that low level acrolein exposure associated with vascular oxidative stress or inflammation stimulates vasodilatation via EDHF release in medium-sized arteries--a novel function.

Endothelin-induced constriction of the ductus venosus in fetal sheep: developmental aspects and possible interaction with vasodilatory prostaglandin.

1. The ductus venosus is actively regulated in the fetus, but questions remain on the presence of a functional sphincter at its inlet. Using fetal sheep (0.6-0.7 gestation onwards), we have examined the morphology of the vessel and have also determined whether endothelin-1 (ET-1) qualifies as a natural constrictor being modulated by prostaglandins (PGs). 2. Masson's staining and alpha-actin immunohistochemistry showed a muscular, sphincter-like formation at the ductus inlet and a muscle layer within the wall of the vessel proper. This muscle cell component increased with age. 3. ET-1 contracted dose-dependently isolated sphincter and extrasphincter preparations of the ductus from term fetus. This ET-1 effect also occurred in the premature, but its threshold was higher. 4. BQ123 (1 microm) caused a rightward shift in the ET-1 dose-response curve, while indomethacin at a threshold concentration (28 nm) tended to have an opposite effect. 5. Big ET-1 also contracted the ductus sphincter but differed from ET-1 for its lesser potency and inhibition by phosphoramidon (50 microm). 6. The ductus sphincter (term and preterm) and extrasphincter (term) released 6-keto-PGF(1alpha) (hence PGI(2)) and, to a lesser degree, PGE(2) at rest and their release increased dose-dependently upon ET-1 treatment. Both basal and stimulated release was curtailed by endothelium removal. 7. BQ123 and phosphoramidon reduced slightly the contraction of ductus sphincter to indomethacin (2.8 microm). 8. We conclude that the ductus contains a contractile mechanism in the sphincter and extrasphincter regions. ET-1 lends itself to a role in the generation of contractile tone and its action may be modulated by prostaglandins.

Histamine-induced vasodilatation in the perfused mesenteric arterial bed of diabetic rats.

In this study, we have examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to histamine-induced endothelium-dependent relaxation in the perfused mesenteric arterial bed of rats treated with streptozotocin (STZ) to induce diabetes. Histamine (10(-10) to 5 x 10(-6) mol) produced dose-dependent vasodilator response in the perfused mesenteric arterial bed of both control and diabetic animals. In order to isolate the EDHF component of histamine-induced vasodilator response, NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME) (10(-4) M) and indomethacin (10(-6) M) were added to the Krebs solution throughout the experiment. Histamine induced vasodilatation in the perfused mesenteric bed in preparations from both control and diabetic rats. The vasodilator response to histamine was slightly potentiated in the diabetic rat preparations. Sodium nitroprusside (SNP)-induced relaxation was similar in diabetic and control rats. The role of EDNO in histamine-induced vasodilatation was also examined. Vascular preparations were perfused with 20 mM K(+)-Krebs solution to inhibit the EDHF contribution to histamine-induced vasodilatation. Under this condition, histamine induced a vasodilator response in preparations from both control and diabetic rats. However, relative to nondiabetic control animals, histamine-induced maximal response was significantly reduced in preparations from diabetic animals. Pretreatment with L-NAME (10(-4) M) attenuated histamine-induced vasodilatation in both preparations, indicating an NO-mediated vasodilator response. There was a significant attenuation in histamine-induced vasodilatation in the vascular preparations from diabetic rats. The vasodilator effect of calcium ionophore A23187 was investigated in preparations from control and diabetic rats to investigate receptor dysfunction associated with diabetes. A23187 (10(-11) to 10(-7) mol)-induced vasodilator response was not significantly different in the preparations from control and diabetic animals. In conclusion, our results indicated that histamine-induced vasodilation in the perfused mesenteric arterial bed of the STZ-induced diabetic rats is mediated by two vasodilator components, namely EDHF and EDNO. Under diabetic conditions, the EDHF component was potentiated, while histamine-induced vasodilation mediated by the EDNO component was attenuated.

Oestrous cycle and pregnancy alter the reactivity of the rat uterine vasculature.

Isolated uterine vascular beds from virgin and pregnant rats were used to assess vascular reactivity and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. One uterine horn from female rats in each oestrous cycle day and gestation day 17 was removed and perfused with physiological saline solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to acetylcholine (ACh) generated. Responsiveness to ACh was tested in the presence of N-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit NO synthase, cyclo-oxygenase and K+ channels respectively. Cirazoline-induced tone was smaller in the pregnant compared with the proestrous group. Sensitivity to ACh was cycle day and pregnancy dependent with pregnant > dioestrous day-1 > dioestrous day-2 > proestrous and oestrous. L-NA shifted the curve to the right in all groups except dioestrous day-1. IBU inhibited the ACh response in the pregnant group only. TBA virtually abolished the response in all groups. These results suggest that in the uterine vascular bed from pregnant rats, EDHF, along with NO and a dilator prostanoid mediate ACh-induced dilatation. In contrast, in the dioestrous day-1 group, only EDHF seems to be released by ACh in this vascular bed. In the oestrous, dioestrous day-2 and proestrous groups, ACh releases both EDHF and NO.

Influence of oestrous cycle and pregnancy on the reactivity of the rat mesenteric vascular bed.

In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.

Buspirone, a 5-HT(1A) receptor agonist, dilates the perfused rat uterine vascular bed through alpha(1)-adrenoceptor blockade.

In the perfused rat uterine vascular bed, 5-hydroxytryptamine (5-HT) produced dose-dependent vasoconstrictor responses. Buspirone, a selective 5-HT(1A) receptor agonist, was not effective at low doses but produced a response at high doses. When perfusion pressure was raised with phenylephrine, responses to 5-HT were enhanced while buspirone produced dose-dependent vasodilator responses. Buspirone did not produce vasodilation when perfusion pressure was raised with vasopressin or U46619. Buspirone-induced vasodilator responses were not affected by selective 5-HT(1A) receptor antagonists, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]-decane-7,9-dione (BMY 7378) and N-tert-butyl-3-(4-[2-methoxyphenyl]piperazin-1-yl)-2-phenylpropanamide (WAY 100478), indicating that specific 5-HT(1A) receptors might not be involved in buspirone-induced vasodilation. Buspirone (3 x 10 (-5) M) and prazosin (3 x 10(-9) M) antagonized noradrenaline-induced constriction with dose ratios of 19.1+/-2.9 and 11.7+/-2.1, respectively. The dose ratio of these antagonists in combination was 46.6+/-8.1. Since the combination ratio is closer to the sum of their individual dose ratios less 2 (i.e. DR(p)+DR(b)-2) than it is to the product of their individual dose ratios, our data suggest an interaction of buspirone with alpha(1)-adrenoceptors. Buspirone also protected adrenoceptors against inactivation by phenoxybenzamine confirming that buspirone interacted with alpha(1)-adrenoceptors. We concluded that buspirone-induced vasodilation of the perfused rat uterine vascular bed is mediated through blockade of alpha(1)-adrenoceptors rather than through 5-HT(1A) receptors.

1-Ethyl-2-benzimidazolinone stimulates endothelial K(Ca) channels and nitric oxide formation in rat mesenteric vessels.

Hyperpolarization of most blood vessels occurs by the opening of K(Ca) channels. 1-Ethyl-2-benzimidazolinone (1-EBIO) is a direct activator of K(Ca) channels in epithelial cells and is potentially valuable for studying cellular hyperpolarization. This study reports the effects of 1-EBIO on isolated rat mesenteric beds perfused with normal (4.7 mM), or high (20 or 80 mM) K+ physiological salt solution (PSS) and constricted with an alpha1-adrenoceptor agonist, cirazoline (0.3-1 microM). Arterial perfusion pressures were decreased by 1-EBIO (0.1-30 nmol) in a dose- and endothelium-dependent manner. Infusion of penitrem A (100 nM), a maxi-K+ channel blocker, or apamin (0.5 microM), a small-conductance (SK(Ca)) K+ channel blocker, produced significant increases in cirazoline-mediated tone (mm Hg): 103.3 +/- 8.7 (control) vs. 156.3 +/- 14.3 (penitrem A); or 93.0 +/- 15.8 (control) vs. 114.0 +/- 15.4 (apamin). 1-EBIO relaxations were attenuated by penitrem A, while apamin, dendrotoxin (50 nM; a Kv channel antagonist), or ouabain (100 microM; a sodium pump blocker) failed to alter the responses. I-EBIO-mediated relaxations decreased significantly with increasing extracellular [K+]: relaxations to 30 nmol were 89.3% +/- 3.2% (4.7 mM K+, normal PSS) vs. 59.5% +/- 3.4% and 19.0% +/- 3.9% for 20 and 80 mM K+ PSS, respectively. Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 microM), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM), selective inhibitors of nitric oxide synthase, and nitric oxide-sensitive guanylate cyclase, respectively, abolished 1-EBIO relaxations in vessels perfused with 20 or 80 mM K+ PSS. We conclude that: (1) maxi-K+ and SK(Ca) channels are present in rat mesenteric arterial vessels and actively contribute to vascular tone, (2) vasodilator action of 1-EBIO involves the opening of endothelial maxi-K+ channels and nitric oxide synthesis.

Histamine receptor subtypes mediating hyperpolarization in the isolated, perfused rat mesenteric pre-arteriolar bed.

Histamine is a general dilator of rat blood vessels. We investigated the relative contribution of receptor subtypes to the rat mesenteric dilator responses initiated by histamine and related agonists. Histamine initiated dose, and endothelium-dependent, dilation of constricted mesenteric beds with an ED50 of 0.4 +/- 0.1 nmol. The ED50 was increased 10-fold by 0.1 microM chlorpheniramine (a histamine H1-receptor selective antagonist). Histamine H2 receptor blockade with tiotidine (0.1 microM) slightly decreased, while thioperamide (1 microM), a selective histamine H3 receptor antagonist, did not block histamine-induced dilation. Mesenteric bed dilation initiated by histamine H2 receptor selective agonists, amthamine and dimaprit, were antagonized markedly by tiotidine. However, the dilation initiated by the putative histamine H3 receptor selective agonists, R(-)- or S(+)-alpha-methylhistamine and imetit were not affected by thioperamide (1 microM). Histamine H2- and H3-receptor mediated dilator effects were endothelium-independent and were blocked by either excess (80 mM) extracellular K+, or 1 mM tetrabutylammonium (a non-selective K+ channel blocker), as well as by 1 microM dequalinium, a non-peptide blocker of the small conductance Ca2+-activated (SKCa) K+ channels. We conclude that (i) histamine H1 receptor subtype predominantly mediates endothelium-dependent dilator effect of histamine, and (ii) vascular hyperpolarization through opening of K+ channels (SKCa) mediate the dilator responses to histamine H2 receptor (amthamine and dimaprit) and the putative histamine H3 receptor (R(-)-alpha-methylhistamine and imetit) agonists.

Calcium-dependent phospholipase A2 mediates the production of endothelium-derived hyperpolarizing factor in perfused rat mesenteric prearteriolar bed.

The isolated, perfused rat mesenteric bed releases a cytochrome P450-linked metabolite of arachidonic acid (AA) as endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine and histamine. This study assessed the relative contribution of two AA-generating pathways, phospholipase A2 (PLA2) and diacylglycerol (DAG) lipase, to EDHF-mediated dilation of the rat mesenteric bed. We tested the hypothesis that PLA2-mediated release of AA is essential for the production of EDHF. Mesenteric beds were perfused with physiological salt solution (PSS) containing indomethacin and nitro-L-arginine methyl ester to block cyclooxygenase and nitric oxide synthase, respectively, and constricted with cirazoline (an alpha1-adrenoceptor agonist). Bolus applications of acetylcholine and histamine caused dose-dependent dilation of the constricted beds. The 85-kDa PLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), at 3 microM, profoundly blunted decreases in perfusion pressure initiated by 1 nmol acetylcholine (94.3+/-1.7%) and by 100 nmol histamine (88.5+/-3.3%) to 9.6+/-7.5 and 8.6+/-6.0%, respectively. AACOCF3 also blocked cirazoline-stimulated release of 6-keto-PG1alpha, but did not alter the vasodilation initiated by sodium nitroprusside (a nitric oxide donor), cromakalim (a K+ channel activator), or by Na+/K+-ATPase activation, as measured by KCl vasodilation in preconstricted beds perfused with K+-free PSS. The 14-kDa PLA2 inhibitor, oleyloxyethyl phosphorylcholine, also blocked EDHF vasodilation and also significantly inhibited K+ channel activity. Neither the Ca2+-independent PLA2 inhibitor, HELSS [E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one], nor DAG lipase inhibitor, RHC-80267 [1,6-bis-(cyclohexyloximino-carbonylamino)-hexane] altered EDHF-mediated vasodilation. However, RHC-80267 blocked cirazoline-stimulated release of 6-keto-PGF1alpha. We conclude that Ca2+-dependent PLA2, rather than DAG lipase, generates the AA for the production of EDHF in the perfused rat mesenteric bed.

Endothelium-derived hyperpolarizing factor: characterization as a cytochrome P450 1A-linked metabolite of arachidonic acid in perfused rat mesenteric prearteriolar bed.

The isolated perfused rat mesenteric bed releases endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine (ACh) or histamine. I propose that EDHF released in the mesenteric vascular bed is a cytochrome P450 (CYP)-linked, arachidonate metabolite. In the presence of nitro-L-arginine methyl ester (L-NAME) and indomethacin, injections of ACh (0.001 to 10 nmol) or histamine (0.1 to 1,000 nmol) elicited transient, dose-dependent dilation of cirazoline (an alpha1-adrenoceptor selective agonist) preconstricted mesenteric beds. The L-NAME-resistant responses to ACh or histamine were insensitive to tetrodotoxin (1 micromol/L), thus negating its neuronal origin, but were profoundly attenuated by a K+ channel inhibitor tetrabutylammonium (0.5 mmol/L). 7-Ethoxyresorufin (a selective and competitive blocker of CYP 1A isozyme) blunted ACh and histamine mediated EDHF responses but did not alter vasodilation initiated through K+ channel activation by either cromakalim or NS-1619, or through the nitric oxide-cGMP pathway (sodium nitroprusside). Clotrimazole, an imidazole that inhibits CYP by binding to the heme moiety, attenuated ACh, histamine, and cromakalim but not sodium nitroprusside-mediated vasodilator responses. Other CYP isozyme selective inhibitors, such as metyrapone (CYP 2B), 7-pentoxyresorufin (CYP 2B1), sulfaphenazole (CYP 2C/3A), and 17-octadecynoic acid (4A-linked omega-hydroxylase inhibitor), did not alter ACh or histamine-induced EDHF response. The EDHF-mediated dilations initiated by ACh and histamine, as well as K(ATP) activation by cromakalim, were blocked by mepacrine, a nonselective phospholipase A2 inhibitor. Mepacrine did not alter K(Ca) activation by compound NS-1619. I conclude that 1) the isolated perfused rat mesenteric prearteriolar bed releases in response to ACh and histamine, an EDHF that causes vasodilation through K+ channel activation; 2) the EDHF is most likely a CYP-derived arachidonate product; 3) CYP 1A is well suited as the isozyme responsible for EDHF production in this vascular bed; and 4) PLA2 appears to mediate the release of the precursor arachidonic acid.

Antithrombotic and vasorelaxant properties of a novel synthetic RGD peptide containing nitric oxide.

We have developed a novel synthetic peptide containing both the antiadhesive Arg-Gly-Asp (RGD) amino acid sequence and a nitric oxide (NO) moiety well known for its vasorelaxant properties. The main objective of this study was to determine whether this hybrid molecule is concurrently effective with regard to antithrombotic and vasorelaxation actions. Studies of in vitro platelet adhesion and of in vivo platelet thrombus formation in the rat demonstrated that the RGD-NO peptide increased the antithrombotic characteristics of the RGD peptide alone. The RGD-NO peptide also caused relaxation of rat aortic rings, while the RGD peptide did not induce relaxation. These characteristics of Ac-RGDC-SNO suggest that this or similar compounds may have potential as effective antithrombotic agents in coronary and peripheral artery disorders.

Mechanism of bile salt vasoactivity: dependence on calcium channels in vascular smooth muscle.

1. The vasoactive mechanisms of bile salts have been investigated in rat isolated portal venous and superior mesenteric arterial rings and perfused mesentery. 2. The isolated perfused mesentery was precontracted with a selective alpha 1-adrenoceptor agonist, cirazoline. Incremental doses of tauroursodeoxycholate (TUDC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) caused a dose-dependent vasorelaxation. The order of potency of the vasodilator effect was TDC > TCDC > TUDC. 3. The effect of TDC (1.9 x 10(-8)-1.9 x 10(-6) mol) was examined before and after propranolol (3 microM), tetraethylammonium (5 mM), ouabain (10(-5) M), NG-nitro-L-arginine methyl ester (10(-4) M) and capsaicin (50 mg kg-1) to block, respectively, beta-adrenoceptors, K+ -channels, Na+, K+-ATPase, nitric oxide synthase, and primary sensory nerves. The vasodilator effect of TDC was not affected by any of these blocking agents or by denuding vascular endothelium with distilled water. 4. Infusion of TDC (1.9 x 10(-8)-1.9 x 10(-6) mol) with K+-free or high K+ (60 mM) physiological salt solution (PSS) did not affect the vasodilator effect of TDC. 5. Contractions induced by KCl (0.01-1.0 M), arginine vasopressin (AVP, 10(-10)-10(-7) M) or cirazoline (10(-7) x 10(-5) M) were all inhibited by TDC (300 microM). 6. TDC (10(-6) to 10(-3) M) also inhibited the basal tension and the development of spontaneous contractions in the isolated portal vein. 7. TDC (300 microM), however, did not affect noradrenaline-induced phasic contractions elicited in Ca(2+)-free PSS by Ca2+ release from intracellular stores. 8. We conclude that TDC inhibits Ca2+ entry through both voltage-operated and receptor-operated calcium channels, whereas intracellular Ca2+ release is not affected.

The effects of perfusion rate and NG-nitro-L-arginine methyl ester on cirazoline- and KCl-induced responses in the perfused mesenteric arterial bed of rats.

1. The purpose of this study was to characterize the effect of NG-nitro-L-arginine methyl ester (L-NAME) on the perfusion rate/pressure relations, and on the pressor responses induced to cirazoline and KCl in isolated, perfused mesenteric arterial beds from normotensive and spontaneously hypertensive rats. 2. The basal perfusion pressure of arterial beds perfused with either physiological salt solution (PSS) or PSS containing 1% polyvinylpyrrolidone increased as the perfusion rate increased. L-NAME, in concentrations up to 100 microM, failed to alter the basal pressure regardless of the perfusion rate and viscosity; however, at 5 microM, it potentiated cirazoline-induced vasoconstriction at each of the perfusion rates. 3. L-NAME but not D-NAME caused a leftward shift of cirazoline concentration-response curves with a marked increase in the maximal response. The potentiating action of L-NAME was abolished in arterial beds perfused with a Ca(2+)-free physiological salt solution and also in beds denuded of endothelium by an infusion of distilled water for 5 min. 4. In endothelium-intact and -denuded preparations, L-NAME potentiated KCl pressor responses; the endothelium-independent potentiation of KCl pressor activity was stereospecific, time-independent and was not prevented by the presence of dexamethasone (0.5 microM) in the perfusion medium. However, L-NAME failed to potentiate vasoconstriction obtained to KCl in arterial beds denervated by cold storage (4-5 degrees C) for 2 days. 5. The absence of K+ in the perfusate did not inhibit the ability of L-NAME to potentiate alpha-adrenoceptor-mediated pressor responses, and nor did L-NAME inhibit KCl-induced vasodilatation in preconstricted arteries. It was thus concluded that L-NAME does not affect Na+/K(+)-ATPase activity. 6. No differences in the potentiating ability of L-NAME on either cirazoline- or KCl-mediated pressor responses were apparent between normotensive Sprague Dawley (SD), Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats.7. Our data thus provide evidence that: the presence of a vasoconstrictor is required for basal nitricoxide (NO) release in the mesenteric arterial bed from either normotensive or spontaneously hypertensive rats; L-NAME causes potentiation of cirazoline- and KCl-induced vasoconstriction respectively by inhibiting endothelial and neuronal NO synthase(s). Furthermore, our data indicate that NO synthase activity is not impaired in the mesenteric arterial bed of spontaneously hypertensive rats.

Interactions of nitric oxide synthase inhibitors and dexamethasone with alpha-adrenoceptor-mediated responses in rat aorta.

1. The effects of NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), their D-isomers, and dexamethasone on noradrenaline (NA)-induced contractions and antagonism by alpha-adrenoceptor antagonists, have been investigated in rat isolated thoracic aortic rings with/without endothelium. 2. NA produced concentration-dependent contractions of isolated aortic rings with EC50 values of 2.41 +/- 0.54 (n = 21) and 28.00 +/- 8.50 (n = 25) nM for endothelium-denuded and -intact preparations respectively. Acetylcholine (ACh) relaxed NA-precontracted rings with intact, but not those denuded of endothelium. 3. Treatment with L-NAME (1-30 microM), or L-NMMA (10-500 microM), but not their D-isomers, resulted in an endothelium-dependent enhancement of NA-induced contractions. Pre-treatment, in vitro, with 0.5 microM dexamethasone neither directly potentiated, nor influenced L-NAME-induced potentiation of NA-mediated contractions in endothelium-intact rings; however, dexamethasone pretreatment reduced EC50 values for NA, and also prevented L-NAME-induced potentiation, in denuded rings equilibrated for 5 h under resting tension. 4. In both intact and denuded rings, phentolamine, prazosin and WB 4101 shifted NA concentration-response curves to the right; L-NAME, and also L-NMMA, but not their D-isomers, reversed the blockade as indicated by significant decreases in NA dose-ratios. In denuded rings, reversal by L-NAME or L-NMMA was prevented following pretreatment with dexamethasone. 5. Following treatment with 5 or 50 nM phenoxybenzamine (PBZ), NA concentration-response (C-R)curves were shifted to the right with marked depression of maximal responses; 100 microM L-NAME reversed the antagonism in both endothelium intact and denuded rings. However, 500 nM PBZ treatment resulted in complete abolition of the responses to NA, and contractions were not restored by either L-NAME or L-NMMA.6. 5-Hydroxytryptamine (5-HT)-induced contractions of aortic rings were potentiated by endothelium denudation and also by L-, but not D-, NAME. 5-HT-induced contractions were non-competitively antagonized by 10nM ritanserin, and 100 microM L-NAME partially reversed the antagonism in intact but not denuded rings.7. It is concluded that the inhibition of constitutive endothelial NO synthase and inducible smooth muscle NO synthase accounts for the ability of L-NAME, and L-NMMA, to potentiate the effects of agonists and reduce alpha-adrenoceptor antagonism in endothelium-intact and denuded rings. Furthermore,endothelial cell removal/damage triggers the induction of a smooth muscle NO synthase.

Varying extracellular [K+]: a functional approach to separating EDHF- and EDNO-related mechanisms in perfused rat mesenteric arterial bed.

We describe a simple, functional approach to defining the relative contribution of endothelium-dependent hyperpolarization (presumably mediated by a factor, EDHF) and endothelium-derived nitric oxide (EDNO) to acetylcholine (ACh) and histamine relaxations of isolated perfused rat mesenteric resistance arterial bed. In physiologic salt solution (PSS), ACh- and histamine-induced vasodilations of cirazoline-preconstricted mesenteric arterial bed were only partially attenuated by 50 microM Nw-nitro-L-arginine methyl ester (L-NAME). The L-NAME-resistant component was abolished by 0.5 microM apamin but not by 250 nM dendrotoxin or 10 microM glyburide, thus indicating a role for apamin-sensitive K+ channels in mediating the effects of the putative EDHF. Changing membrane potential by varying [K+] decreased L-NAME-resistant vasodilation, and showed a modest L-NAME-induced increase in the basal perfusion pressure that was not observable in normal PSS. Vasodilator responses during cirazoline-induced tonus in 20 mM K+ and normal PSS were superimposable, but responses to ACh and histamine in 20 mM K+ were profoundly more sensitive to L-NAME than were those in normal PSS media. ACh responses during 20-mM K+ PSS perfusion and presumably mediated by EDNO and those resistant to L-NAME and putatively mediated by EDHF were antagonized by graded concentrations of p-fluorohexahydro-siladifenidol (p-F-HHSiD), but not pirenzepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of some inorganic divalent cations and protein kinase C inhibitors on endothelium-dependent relaxation in rat isolated aorta and mesenteric arteries.

The effects of nitrendipine and several metal ions possessing Ca2+ antagonistic activity were examined on acetylcholine (ACh) and histamine-induced endothelium-dependent relaxations in norepinephrine (NE)-precontracted rat aortic rings and perfused mesenteric arteries. Nitrendipine (1 nM) profoundly attenuated ACh and histamine-induced relaxations of perfused mesenteric arteries but was ineffective against either agonist in aorta. The transition metal ions Co2+, Mn2+, and Ni2+, but not the nontransition ions (Cd2+, Sn2+, and Zn2+), markedly inhibited ACh and histamine relaxations in the aorta, whereas all metal ions antagonized KCl contractions. At the highest concentration devoid of effect on arterial perfusion pressure, none of the transition metal ions altered endothelium-dependent relaxations in the mesenteric arteries. Endothelium-independent relaxations induced by sodium nitroprusside (SNP) were attenuated by Mn2+ but not by Co2+ or Ni2+. Calmidazolium or W-7 inhibited ACh- and histamine-induced relaxations in both aorta and mesenteric arteries, whereas staurosporine and H-7 were ineffective against aortic relaxations; in mesenteric arteries, staurosporine but not H-7 attenuated both endothelium-dependent and -independent relaxations. We conclude (a) that the transition metal ions most likely inhibit endothelium-derived relaxing factor (EDRF) (NO) release in the aorta through endothelial receptor-operated Ca2+ channels; (b) that the effects of nitrendipine (shared by nifedipine) in mesenteric arteries result from an interaction with a site that may have structural similarities with, but is distinct from, the L-type Ca2+ channel; and (c) that the inhibitory effects of the calmodulin antagonists may reflect an action on endothelial NO synthase.

Contribution of K+ channels to arachidonic acid-induced endothelium-dependent vasodilation in rat isolated perfused mesenteric arteries.

The contribution of K+ channels and cytochrome P450 generated arachidonic acid (AA) metabolites to the endothelium-dependent vasodilation produced by this fatty acid in the perfused rat isolated mesenteric arteries was examined using a variety of compounds known to inhibit transmembrane K+ channels and cytochrome P450 enzymes. AA (1-1000 nmol) caused dose- and endothelium-dependent vasodilation in the presence of indomethacin and the effect was neither altered by lipoxygenase (AA 861) nor cytochrome P450 monooxygenase (alpha-naphthoflavone, ketoconazole and metyrapone) inhibitors indicating that AA-induced, endothelium-dependent vasodilation in this vascular bed was not mediated by product(s) of AA metabolism. The vasodilator effect of AA was also not altered by L-NG-nitro-arginine, methylene blue (50 microM), oxyhemoglobin (5 microM) or superoxide dismutase (50 U/ml), thus ruling out nitric oxide being its mediator. Conversely, arterial perfusion with K(+)-free or excess (50 mM) K+ Krebs' solution, but not ouabain infusion, minimized the vasodilator effect of AA, suggesting that this action of the fatty acid is due to changes in membrane K+ conductance that is independent of Na+/K(+)-adenosine triphosphatase activity. The vasodilator action of BRL 34915 (a K+ channel activator) was also minimized by extracellular K+ depletion or excess K+ (50 mM), but not by ouabain. Apamin (0.5 microM) and crude scorpion venom (2.5 micrograms/ml) attenuated AA- but not BRL 34915-induced vasodilation. Glyburide (inhibitor of ATP-activated K+ channel) abolished the vasodilator action of AA and BRL 34915. Procaine, a nonspecific K+ channel blocker did not affect AA-induced vasodilation even though it attenuated that caused by BRL 34915.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of vascular smooth muscle contraction by sodium fluoride in the isolated aorta of rat and rabbit.

The purpose of this study was to determine the cellular basis for fluoride ion (F-)-induced contractions of isolated aortic rings from both the rat and the rabbit. The F- contractions were not affected by endothelial denudation but were enhanced in the presence of A (0.1 or 1.0 mM) added to the bathing Krebs' solution. The contractile effect of F- also was not modified by bathing with Ca(++)-free + ethylene glycol bis(b-aminoethylether)-N,N-tetracetic acid Krebs' solution or nifedipine (10 microM), but was attenuated by inorganic (Cd++, Co++ and Ni++) Ca++ antagonists in normal and Ca(++)-free Krebs' media. Bis(o-aminophenoxy)-ethane-N-N-N'-N'-tetraacetic acid, ryanodine and intracellular Ca++ modulators, respectively, caused 36.1 +/- 6.1%, 16.4 +/- 6.8% and 52.3 +/- 7.3% inhibition of the contractile response to F- in a Ca(++)-free media while causing near complete inhibition of norepinephrine-induced contractions. F- contractions were also inhibited by the calmodulin antagonists W-7 and calmidazolium (IC50 = 23.0 +/- 7.0 and 45.0 +/- 10.0 microM, respectively). On the other hand, the protein kinase C antagonists staurosporine and H-7 potently (IC50 = 0.016 +/- 0.007 and 1.1 +/- 0.5 microM, respectively) inhibited the fluoride-induced contractions. Aortic rings from the rabbit were similarly potently antagonized by the protein kinase C inhibitors, however, K(+)-induced contractions were also equally sensitive to these agents in both rat and rabbit tissues. The putative phospholipase C inhibitor neomycin was significantly less effective (IC50 = 13.0 +/- 5.0, 0.44 +/- 0.09 and 0.89 +/- 0.40 mM) at inhibiting F- than norepinephrine and KCl contractile effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-dependent and BRL 34915-induced vasodilatation in rat isolated perfused mesenteric arteries: role of G-proteins, K+ and calcium channels.

1. In the isolated perfused, noradrenaline (NA)-constricted mesenteric arteries of the rat, acetylcholine (0.003-1 nmol), histamine (0.01-10 nmol) and the calcium ionophore A23187 (0.01-1 nmol), caused endothelium-dependent vasodilatation while the vasodilatation by the K+ channel activator BRL 34915 (0.1-1 nmol) was independent of endothelium. 2. The guanylate cyclase inhibitor, methylene blue at 10 microM did not inhibit the action of any of the vasodilators but at 50 microM reduced the vasodilator effect of acetylcholine (ACh), histamine and A23187. 3. Infusion of ouabain or perfusion with K(+)-free or excess K+ (50 mM) Krebs solution reduced the vasodilator effect of ACh, histamine and A23187, suggesting the action of these agents involves, at least in part, activation of Na+/K(+)-ATPase. The vasodilator effect of BRL 34915 was not affected by ouabain, but abolished during perfusion with Krebs solution containing excess K+ or depleted of K+. 4. Five structurally distinct K+ channel blockers (apamin, crude scorpion venom, procaine, quinidine and tetraethylammonium) attenuated the vasodilator effect of ACh, histamine and A23187. The K+ channel blockers, except apamin and crude scorpion venom, also inhibited the vasodilatation produced by BRL 34915. 5. The vasodilator effect of ACh, histamine or A23187 was not altered in mesenteric vessels of pertussis toxin-treated rats, suggesting that the K+ channels associated with the endothelium-dependent vasodilator effect of these agents are either not coupled to G-proteins or are coupled to G-proteins that are insensitive to pertussis toxin. 6. The calcium channel blockers, diltiazem (0.1 or 1 microM), nifedipine (0.01 or 0.1 microM) or nitrendipine (1 nM) attenuated the vasodilatation produced by ACh, histamine, A23187 and also that by BRL 34915. 7. We conclude that endothelium-dependent vasodilatation induced by ACh, histamine and A23187 is mediated via activation of membrane K+ channels and Na+/K+-ATPase. The K+ channels involved in the vasodilator action of these agents are not coupled to pertussis toxin-sensitive G-proteins and appear to be regulated by Ca2 +.