Pulmonary vasoconstrictor effects of prostacyclin in rats: potential role of thromboxane receptors

Endogenous prostacyclin (PGI2; epoprostenol) is a potent endothelium-derived pulmonary vasodilator. However, the effects of exogenous PGI2 on isolated arteries could be either relaxant or contractile, depending on the species and organ studied. The present study investigated the distal pathways involved in the PGI2-induced contraction in rat intrapulmonary artery (PA) and relaxation in lamb PA. When vessels were precontracted with 30 mM K+, PGI2 (1 microM) induced relaxation in lamb PA but caused contraction in rat PA. Use of 30 mM K+, phenylephrine, serotonin, angiotensin II, or hypoxia to precontract the vessels did not alter the contractile effect of PGI2 in rat PA. Nevertheless, PGI2 produced a mild relaxation in rat PA precontracted by U-46619, a thromboxane A2 (TxA2)-receptor agonist, whereas the TxA2-receptor blocker SQ-29548 (0.1-0.5 microM) abolished the contractile response in rat PA. These data suggest that PGI2-induced contraction is mediated by activation of TxA2 receptors. The PGI2-induced modest relaxation in rat PA, which was only observed when TxA2 receptors were blocked by SQ-29548, suggests that the PGI2-mediated vasorelaxant pathway is diminished in these vessels. Simultaneous application of forskolin, an adenylate cyclase activator, and rolipram, a phosphodiesterase inhibitor, caused similar relaxation in both rat and lamb PA. This suggests that the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway is intact in rat PA and is comparable to that in lamb PA. On the basis of these data, we conclude that the pathways responsible for the paradoxical effects of PGI2 on rat and lamb PA are located upstream of the adenosine 3',5'-cyclic monophosphate-dependent relaxing pathway and that a paucity of PGI2 receptors in rat PA may be responsible.     

Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.     

Inhibition of beta-adrenergic desensitization by KCa channels in human trachealis

We examined the reduced responsiveness to beta-adrenergic receptor agonists (beta-agonists) after exposure to beta-agonists, and the mechanisms underlying this phenomenon in isolated human tracheal smooth muscle, using isometric tension records to test the hypothesis that repeated inhalation of beta-agonists leads to reduced responsiveness to beta-agonists. The inhibitory effects of isoproterenol (ISO) on contraction by spasmogens participating in asthma attacks diminished markedly after continuous exposure to ISO (0.0003 to 3 microM) for 45 min; moreover, when ISO was repeatedly applied for 10 min to tissues precontracted by methacholine every 30 min, the relaxant effects of ISO gradually attenuated after these repeated applications. In contrast, reduced beta-adrenergic relaxation after continuous and repeated exposure to agonists did not occur when tissues were preincubated with 2 microg/ ml cholera toxin (CTX), which irreversibly activates guanosine triphosphate (GTP)-binding protein (Gs) coupled with beta-adrenergic receptors, for 6 h. However, the CTX inhibition disappeared in the presence of iberiotoxin, a selective inhibitor of large conductance Ca2+-activated K+ (KCa) channels. Our results demonstrate that continuous and repeated exposure to beta-agonists leads to beta-adrenergic desensitization, and that activation of KCa channels by Gs prevents this desensitization.     

Atypical beta-adrenoceptors in the rat isolated common carotid artery

1. The possible existence of atypical beta-adrenoceptors in vascular smooth muscle of the rat common carotid artery was examined in this study. 2. Isoprenaline produced concentration-dependent relaxation of noradrenaline (10(-7) M) precontracted ring segments of the carotid artery. The relaxation was not affected by endothelial denudation. 3. Propranolol (10(-8) M-3 x 10(-7) M) shifted the isoprenaline curve to the right without suppressing the maximum response. However, the slope (0.74) of the Schild plot was significantly (P < 0.05) less than 1. 4. Salbutamol (beta 2), CGP 12177 and BRL 37344 (beta 3) also concentration-dependently relaxed noradrenaline precontracted artery segments. These relaxations were not affected by propranolol (10(-7) M). Pretreatment of the artery segments with BRL 37344 did not desensitize the tissue to the relaxant effect of isoprenaline, CGP 12177 and salbutamol. 5. It is concluded that atypical beta-adrenoceptors exist in vascular smooth muscle of the common carotid artery.     

Effects of the novel antimigraine agent, frovatriptan, on coronary and cardiac function in dogs

The effects of frovatriptan (VML 251/SB-209509) on coronary artery function were investigated in isolated coronary arteries from beagle dogs. Low concentrations of frovatriptan produced contraction with -logEC50 7.55 +/- 0.08 (n = 11). The maximal observed contraction attained was 56 +/- 7% of the control 5-hydroxytryptamine (5-HT; 10 microM) response. At high concentrations of frovatriptan (>6 microM), reversal of sumatriptan (10 microM)-induced contractions was noted. In arteries precontracted with the thromboxane mimetic, U46619, frovatriptan produced a bell-shaped concentration-response relation with a maximal response at 600 nM. Concentrations of frovatriptan >2 microM produced marked reversal of tone, with full relaxation of precontracted tissues at 200 microM. In anesthetized, open-chest mongrel dogs, intravenous (n = 5) or intracoronary (n = 5) artery administration of frovatriptan (0.0001-1 mg/kg) had no consistent effect on left ventricular end-diastolic pressure, left end-systolic pressure, cardiac contractility, aortic blood flow, systemic peripheral resistance, coronary blood flow, coronary vascular resistance, mean arterial blood pressure, or heart rate when compared with vehicle (n = 3). Intravenous sumatriptan produced minor effects on blood pressure and heart rate. Intracoronary artery administration of sumatriptan (0.0003 mg/kg) produced an increase in systemic peripheral resistance to 120.5 +/- 8.2% compared with vehicle (97.8 +/- 5.4%; p < 0.05). This dose of sumatriptan also produced a significant increase in coronary blood flow and decrease in coronary vascular resistance. Intravenous administration of sumatriptan produced a dose-related reduction in left ventricular diastolic pressure with a reduction to 58.3 +/- 8.3% and 41.7 +/- 25% of control values observed at 0.3 and 1 mg/kg, respectively; however, administration of sumatriptan by an intracoronary route had no effect. In a model of myocardial infarction, comparable doses of sumatriptan (1.0 mg/kg) or frovatriptan (0.1 mg/kg), in terms of their effect on carotid vascular resistance, had no significant effect on infarct size. Frovatriptan had no effect on coronary blood flow after reperfusion; however, sumatriptan produced a significant reduction in coronary blood flow for < or =3 h. These studies show that frovatriptan has the capability of relaxing coronary arteries in vitro, has no overall effect on cardiac function at rest with no effect on coronary hemodynamics after myocardial infarction, and has a profile superior to that of sumatriptan.     

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle. 2. At 25 degrees C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-CIATP (2.7+/-0.5 microM, n=7) >ATP (12.9+/-1.1 microM, n=9) > or =2-MeSATP (21.9+/-4.8 M, n=4). In the presence of alphabeta-methylene ATP (alphabeta-MeATP, 200 microM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited. 3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP. 4. The A2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1-100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited. 5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to alphabeta-MeATP (200 microM) but were inhibited by ZM 241385 (100 nM). 6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [gamma-32P]-ATP showed that muscular strips rapidly (10-60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57+/-11% (n=3) by 200 microM alphabeta-MeATP. 7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A-receptors.     

Helospectin/helodermin-like peptides in guinea pig lung: distribution and dilatory effects

The lower airways of guinea pigs were analysed for helospectin and helodermin using immunocytochemistry. A moderate supply of helospectin/helodermin-like immunoreactive nerve fibers and few nerve fibers displaying helodermin immunoreactivity was seen in the smooth muscle, around seromucous glands and small blood vessels in the trachea and around bronchi and pulmonary blood vessels. Helospectin I-, helospectin II- and helodermin-induced suppression of smooth muscle responses were analysed using isolated circular segments of trachea and pulmonary arteries of guinea pigs. In both airways and arteries the peptides caused a concentration-dependent relaxation of precontracted segments. The maximal relaxant activity observed was more pronounced in the airways than in the arteries. The effects of the helospectins and helodermin were compared to those of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitary adenylate cyclase activating peptide (PACAP) and acetylcholine (ACh). All peptides, with the exception of PACAP, caused a total or nearly total relaxation of the precontracted tracheal segments. In the trachea PACAP was significantly more potent than the other five peptides whereas only small potency differences were seen in the pulmonary artery. The relaxant responses to helospectin I, helospectin II and helodermin in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine and cimetidine. Conceivably, nerve fibers containing helospectin and helodermin may play a role in the regulation of airway resistance and in the regulation of local pulmonary blood flow.     

Erectile dysfunction of vasculogenic origin: ultrastructural evaluation of cavernous myo-endothelial unit as prognostic index of penile revascularization]

1. The present study examined the effects of various agents on high calcium-induced relaxation of the rabbit thoracic aorta precontracted by phenylephrine (0.1 microM) or KCl (30 mM). 2. The vascular smooth muscle relaxation caused by high calcium was not changed in the presence of endothelium, glibenclamide (3 microM), TEA (5 mM), verapamil (1 microM), lidocaine (0.1 mM) and vanadate (0.1 mM). 3. In the presence of ouabain (0.1 mM) or potassium-free medium, high calcium-induced relaxation was completely abolished. 4. When rings were precontracted by high concentrations of phenylephrine (1 microM, 10 microM) and KCl (30 mM, 45 mM, 60 mM), calcium-induced relaxation was gradually decreased. 5. A low concentration of calcium ionophore A-23187 (0.1 microM) did not change calcium-induced relaxation, but A-23187 at a high concentration (1 microM) depressed this relaxation. 6. These results suggest that Na-K-ATPase activation could be responsible for high calcium-induced relaxation.     

Potency and kinetics of nitric oxide-mediated vascular smooth muscle relaxation determined with flash photolysis of ruthenium nitrosyl chlorides

Flash photolysis of thermally stable, photolabile 'caged' precursors permits rapid and precise changes of ligand concentration at their site of action. This approach was used to determine the concentration-dependence and time course of NO-mediated relaxation of aortic smooth muscle, by use of two photolabile NO donors, trichloronitrosylruthenium (Ru(NO)Cl3) and dipotassium pentachloronitrosylruthenate (K2Ru(NO)Cl5). At concentrations up to 500 microM, both compounds were non-toxic before photolysis, and produced non-toxic by-products on photolysis. Photolytic release of NO produced relaxations of intact and endothelium-denuded aortic rings precontracted with noradrenaline (0.1-0.5 microM), with an EC50 for NO-mediated relaxations of 10.5 nM and 13 nM, respectively. NO-mediated relaxations were reversibly blocked by 1 microM oxyhaemoglobin. The time course of NO-mediated relaxation comprised a delay of 3-7 s, followed by a sigmoidal decline in tension with peak rates that were strongly dependent on NO concentration.     

Pharmacological characteristics of MJ-451, a new benzopyran- derived ATP-sensitive potassium channel opener, in guinea pig isolated trachea

In this study, we determined the pharmacological activities of MJ-451 (6-cyano-3S,4R-dihydro-2, 2-dimethyl-2H-3-hydroxy-4-[2-oxo-5S-1-hydroxmethyl)-1-pyrrolidinyl ]-1 -benzopyran) in guinea pig isolated trachea and compared its effects with those of cromakalim. MJ-451 (0.1-10 micromol/l) and cromakalim (0.01-1 micromol/l) produced concentration-dependent relaxation of guinea pig isolated trachea precontracted with carbachol (0.5 micromol/l) or histamine (1 micromol/l). MJ-451 (0.03-30 micromol/l), as well as cromakalim (0.03-30 micromol/l), caused a complete and concentration-dependent relaxation of guinea pig isolated trachea precontracted with 20 mmol/l KCl, but did not inhibit the spasmogenic effect of 80 mmol/l KCl. However, theophylline (30-3,000 micromol/l) caused a complete and concentration-dependent relaxation of guinea pig isolated trachea precontracted with either 20 or 80 mmol/l KCl. Propranolol (0.1 micromol/l) markedly antagonized the relaxant action of isoprenaline, but not that of MJ-451 in carbachol-contracted isolated trachea. 8-(p)-sulfophenyltheophylline (150 micromol/l), a selective P1 purinoceptor antagonist, had no effect against the tracheal relaxation induced by MJ-451, but markedly depressed the concentration-response curve of 5'-N-ethylcarboxamidoadenosine. Charybdotoxin (10 micromol/l), a large-conductance Ca2+-activated K+ channel blocker, failed to modify the relaxant activity of MJ-451 in carbachol-contracted isolated trachea. The ATP-sensitive K+ channel blocker, glibenclamide (0.1, 1 and 10 micromol/l) concentration-dependently antagonized the relaxant activity of MJ-451 in carbachol-contracted isolated trachea. It is concluded that MJ-451 is a selective ATP-sensitive K+ channel opener in the tracheal smooth muscle of the guinea pig.     

The effects of levcromakalim and pinacidil on the human internal mammary artery

The present study was undertaken to examine the effects of pinacidil and levcromakalim, two potassium, channel openers, on human internal mammary artery (HIMA) obtained from patients undergoing coronary artery bypass surgery, and to clarify the contribution of different K+ channel subtypes in pinacidil and levcromakalim action in this blood vessel. Pinacidil and levcromakalim induced a concentration-dependent relaxation of the precontracted arterial segments (pEC50 = 5.77 +/- 0.05 and 6.89 +/- 0.03, respectively), 4-Aminopyridine (3 mM), a non-selective blocker of K+ channels, induced significant shifts to the right of the concentration-response curves for pinacidil and levcromakalim. Tetraethylammonium (6 mM), charybdotoxin (0.4 microM) and apamin (0.1 microM), blockers of Ca(2+)-sensitive K+ channels, had no effect on the pinacidil- and levcromakalim-evoked relaxation. Glibenclamide (0.1-10 microM), a selective blocker of adenosine triphosphate (ATP)-sensitive K+ channels, competitively antagonized the response to levcromakalim (pKB = 7.92 +/- 0.07). In contrast, glibenclamide, in significantly higher concentrations (3-30 microM), non-competitively antagonized the response to pinacidil. High concentrations of pinacidil (> 10 microM) relaxed arterial rings bathed by a medium containing 100 mM K+ with maximum response 83 +/- 6%. Under the same conditions, the maximum levcromakalim-induced relaxation on HIMA was almost abolished (15 +/- 2%). It is concluded that pinacidil and levcromakalim do not relax the HIMA through the same subtype of K+ channel. ATP-sensitive K+ channels are probably involved in levcromakalim- but not in a pinacidil-induced relaxation in the HIMA. In addition, in pinacidil-induced relaxation of the HIMA, K+ channel-independent mechanisms seem to be involved.     

Relaxing actions of corticotropin-releasing factor on rat resistance arteries

1. Although it well established that corticotropin-releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150-300 microns). 2. Initial in vivo experiments confirmed that CRF (1.5 nmol.kg-1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9-41. 3. For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre-contracted with one of 3 agonists (prostaglandin F2 alpha, arginine vasopressin or noradrenaline) or with a high-potassium solution (K+). 4. With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nM, and near complete relaxation with 100 nM. Femoral vessels pre-constricted with agonists and all vessels pre-constricted with K+ were less affected by CRF. In the mesenteric vessels, with sub-maximal levels of pre-constriction, CRF caused substantial relaxation at 1 nM and could cause complete relaxation at 10 nM. 5. The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nM CRF9-41. Neither tetraethyl ammonium (30 mM) nor glibenclamide (3 microM) antagonized the relaxant effect of CRF. 6. The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium. 7. The results indicate that CRF causes an endothelial-independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.     

Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.     

Identification of two replicons in phage-plasmid P4

1 Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation. 2 Cardiac output indices of coronary artery-ligated and sham-operated rats were 125+/-8 and 189+/-10 ml min(-1) kg(-1), respectively (P<0.05), indicating the development of CHF at this period. 3 The maximal relaxant response of aortic segments to 10 microM acetylcholine in rats with CHF and sham-operated rats was 64.0+/-5.7 and 86.9+/-1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats. 4 In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 microM isoprenaline (44.5+/-6.7%) was lower that sham-operated rats (81.3+/-2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by NG-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 microM dibutyryl cyclic AMP in rats with CHF (26.8+/-2.7%) was lower than that in sham-operated rats (63.4+/-11.8%, P<0.05). 5 Trandolapril (3 mg kg(-1) day(-1)) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5+/-3.6 ml min(-1)) was lower than that of sham-operated rats (55.0+/-3.0 ml min(-1)), and this reduction was reversed (54.1+/-3.4 ml min(-1)) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7+/-1.0%; that to isoprenaline, 80.5+/-2.8%; that to dibutyryl cyclic AMP, 54.7+/-6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged. 6 The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.     

Dopamine-induced relaxation in isolated intrarenal arteries from adult stroke-prone spontaneously hypertensive rats

1. The relaxant effects of dopamine (DA) on the intrarenal arteries obtained from 6 month old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar-Kyoto (WKY) rats were pharmacologically investigated in vitro. 2. DA (10(-7)-3 x 10(-5) mol/L) produced endothelium-independent relaxation on the arterial rings which had been incubated with phenoxybenzamine (2 x 10(-6) mol/L) and precontracted with KCl. 3. DA-induced relaxation was greater in the arterial rings from SHRSP than in those from WKY. SKF 38393 (10(-8)-10(-6) mol/L) partially mimicked DA-produced relaxation in both groups. SCH 23390 dose-dependently inhibited DA-induced relaxation with pD'2 value of 9.33 for SHRSP and of 9.26 for WKY. 4. There were no significant differences between SHRSP and WKY in the relaxation caused by forskolin, dibutyryl cyclic AMP, or 3-isobutyl-1-methylxanthine. 5. These results suggested that DA1 receptor-mediated relaxation was increased in the intrarenal arteries from SHRSP, and this increase might not be associated with altered vasodilation mediated by cyclic AMP.     

Inhibition of vasoactive amine induced contractions of vascular smooth muscle by hydrogen peroxide in rabbit aorta

OBJECTIVE: The aims were to investigate the effects of H2O2 on arterial contractions induced by vasoactive amine agonists and a high concentration of potassium ions (high K+) in vitro and to explore the possible underlying mechanism(s) involved. METHODS: Isometric tension of rabbit isolated aortic strips was measured and the effects of pretreatment with H2O2 on contractions induced by phenylephrine and high K+ were compared. The effects of H2O2 on precontracted strips were determined in the presence and absence of the aortic endothelium and compared with those of acetylcholine. RESULTS: The tension developed in response to an agonist was expressed as a percentage of the contraction induced by high K+ (64.7 mM) superfusion. Pretreatment with 300 microM H2O2 reduced the mean phenylephrine (0.3 microM) induced contraction from 96.2(SEM 1.4) to 61.8(2.8)%; the effect was stable and reversed by washing out the H2O2. Hydrogen peroxide relaxed phenylphrine precontracted strips with and without endothelium but it showed no relaxant effect when the strips were precontracted by high K+, whereas acetylcholine (1 microM) induced transient relaxation of high K+ precontracted strips by 27.8(2.9)%. The relaxant effect of H2O2 was not affected by pretreatment with indomethacin (a cyclo-oxygenase inhibitor), desferrioxamine (a hydroxyl radical scavenger), or diphenylphenylenediamine (a lipophilic antioxidant). CONCLUSIONS: H2O2 inhibits vasoactive amine induced contractions of the vascular smooth muscle of rabbit aorta in vitro without affecting voltage dependent Ca2+ influx or contractile machinery. The mechanism responsible for its inhibitory effects may be related to impairments of the cellular signalling reactions initiated by the agonists.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.     

A randomized trial to assess the efficacy of surgery in addition to radiotherapy in patients with a single cerebral metastasis

1. A comparison of the effects of dietary and genetically-induced hypercholesterolaemia on the vasodilator and antiaggregatory capacity of the endothelium was made in rabbit isolated subclavian artery rings. 2. Dietary-induced hypercholesterolaemia in NZW rabbits decreased the maximum relaxation to carbachol (0.01-10 microM) and calcimycin (0.01-0.1 microM) in vessel rings precontracted with 5-hydroxytryptamine (5-HT), 0.1 microM), when compared to responses observed in rings obtained from control normocholesterolaemic NZW rabbits. The relaxant responses to SIN-1 (3-(4-morpholinyl)-sydnonimine hydrochloride) were attenuated but were not significantly different from controls. In Froxfield genetically hypercholesterolaemic (FHH) rabbits, the maximum relaxations to carbachol, calcimycin and SIN-1 were all reduced significantly. 3. Neither genetic nor dietary-induced hypercholesterolaemia modified the contractile responses of vessel rings to either KCl (10-100 mM) or 5-HT (0.01-10 microM). 4. Endothelium-dependent inhibition of collagen-induced platelet aggregation in whole blood was demonstrated by stimulation of a vessel ring, incorporated into the blood sample, with carbachol (10 microM, final blood concentration). This effect was inhibited by NG-nitro-L-arginine (L-NOARG, 100 microM). SIN-1 (10 microM, final blood concentration) also decreased whole blood platelet aggregation, but only in the presence of an unstimulated vessel ring, and this was unaffected by L-NOARG. Superoxide dismutase (150 u ml-1) did not influence the inhibition of aggregation by either a carbachol-stimulated vessel ring or by SIN-1. 5. Carbachol-stimulated artery rings from FHH rabbits inhibited platelet aggregation to a similar extent to that seen with rings from control normocholesterolaemic rabbits. Rings from hypercholesterolaemic NZW rabbits, however, did not significantly inhibit platelet aggregation when stimulated with carbachol. SIN-1 inhibited platelet aggregation in the presence of rings from either group of hypercholesterolaemic rabbits. 6. Hypercholesterolaemia induced by dietary modification induces changes in endothelial function which are characteristically different from those seen in genetically hypercholesterolaemic rabbits. It appears that dietary-induced hypercholesterolaemia primarily decreases NO release from the endothelium, while in genetically-induced hypercholesterolaemic vessel rings NO is released but there is a decreased responsiveness of the vascular smooth muscle cells to NO. This may reflect differences in the age and severity of the atherosclerotic lesions in the two groups of rabbits.