Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.     

Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography — mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.     

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.     

Evidence of relaxant effect of omeprazole in rabbit corpus cavernosum in vitro

The present experiments were designed to investigate the effects of omeprazole, a H(+)-K+ ATPase inhibitor, on corporal smooth muscle tone in vitro. All spontaneous contractile activity in the corpus cavernosum was blocked following omeprazole (0.1 mM-1 mM) administration. However atropine (1 microM), Nw-nitro L-arginine methyl ester (L-NAME, 30 microM) or indomethacin (10 microM) did not affect the spontaneous contraction. Omeprazole (10 microM-1 mM) concentration-dependently induced relaxation in corporal smooth muscle precontracted with 10 microM phenylephrine or 80 mM KCl. Pretreatment of corporal tissue with L-NAME (30 microM), indomethacin (10 microM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethyl ammonium chloride (0.5 mM) or glibenclamide (1 microM) had no effect on the omeprazole induced relaxant responses. Nimodipine, an L-type Ca++ channel blocker, relaxed corporal strips precontracted with 80 mM KCl. Collectively, these results indicate that the inhibition of spontaneous contraction and the relaxation of precontracted corporal smooth muscle by omeprazole is probably mediated by the blockade of calcium channels. Further work is needed to determine the cellular mechanism(s) of action by which omeprazole acts on corpus cavernosum smooth muscle.     

NATO advanced study institute on “drugs affecting leukotrienes and other eicosanoid pathways”

Several vasodilator compounds (e.g. acetylcholine) relax isolated arteries by a mechanism dependent on an intact endotheliu. The inhibitors of phospholipase A₂, mepacrine and p-bromophenacylbromide, reversed the acetylcholine-induced relaxations of rabbit aortic strips. We therefore tested the effect of melittin, a compound which stimulates phospholipase activity. Melittin induced concentration-dependent rapid relaxations of strips of rabbit aorta, preconstricted with noradrenaline, if the endothelium was intact. Strips with noradrenaline, if the endothelium aorta, preconstricted with removed endothelium were not relaxed but rather slightly contracted by the compound. Similar to acetylcholine-induced relaxations melittin relaxations could be completely prevented by nordihydroguaiaretic acid, an inhibitor of lipoxygenases, but not by even high concentrations of indomethacin, an inhibitor of cyclooxygenase. Also the dual inhibitors of cyclooxygenase and lipoxygenases 5,8,11,14-eicosatetraynoic acid (ETYA) and 3-amino-1-(3-trifluoromethylphenly)-2-pyrazoline (BW 755C) inhibited relaxations induced by both melittin and acetylcholine.We therefore conclude that melittin induces the release and/or formation of a substance (an eicosanoid?) by endothelial cells which in turn relaxes the smooth muscle of the blood vessel. This compound is probably a lipoxygenase product of arachidonic acid. The mechanism of relaxation by melittin resembles that induced by acetylcholine.     

Inhibition of arachidonic acid-induced contraction of guinea pig lung strips

The effects of several enzyme inhibitors on arachidonic acid-induced contractions of guinea pig lung strips were studied. Varying concentrations of indomethacin, an inhibitor of cyclooxygenase, produced only a limited effect on contraction of tissue strips. By contrast, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and phenidone, which inhibit either lipoxygenase, or both lipoxygenase and cyclooxygenase, caused a dose-related antgonism of the arachidonic acid-induced contraction. The effects of these latter agents were similar to that of FPL 55712. Results indicate that the products of cyclooxygenase are predominantly involved in the early phase and the products of lipoxygenase are predominantly related to the late phase of arachidonic acid-induced contraction.     

Aortic smooth muscle cell migration caused by platelet-derived growth factor is mediated by lipoxygenase product(s) of arachidonic acid

The relation between platelet-derived growth factor (PDGF)-induced smooth muscle cell migration, measured in Boyden chambers, and cellular arachidonic acid cascade was studied by using rat aortic smooth muscle cells. Partially purified PDGF stimulated cell migration significantly at a concentration of 1.33-133.0 micrograms/ml. Treatment of the cells with 10(-4)M of 5,8,11,14-eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and 10(-4)M of caffeic acid, a specific inhibitor of lipoxygenase, caused a significant suppression of PDGF-induced cell migration. Treatment with indomethacin, an inhibitor of cyclooxygenase, did not affect cell migration. These data indicate the involvement of a lipoxygenase product(s) of arachidonic acid in PDGF-associated smooth muscle cell migration.     

Cyclooxygenase inhibitors increase canine tracheal muscle response to parasympathetic stimuli in situ

To determine whether cyclooxygenase inhibitors alter parasympathetic control of airway smooth muscle in situ, we pretreated anesthetized dogs with intravenous indomethacin, meclofenamate, or normal saline and measured the isometric contraction of tracheal muscle in response to electrical stimulation of the vagus nerves. Indomethacin and meclofenamate increase the response of airway smooth muscle to parasympathetic stimulation. In subsequent experiments to determine the site of action of cyclooxygenase inhibitors, we found that indomethacin does not alter the response of tracheal muscle to intra-arterial acetylcholine (a muscarinic agonist) but does augment the response to intra-arterial dimethylpiperaziniumiodide (a nicotinic agonist). Moreover, the response to parasympathetic stimulation after pretreatment with a combination of indomethacin and BW755C (a combined cyclooxygenase-lipoxygenase inhibitor) does not differ significantly from the response after indomethacin or meclofenamate alone. We conclude that cyclooxygenase inhibitors increase the sensitivity of the contractile response of tracheal smooth muscle to parasympathetic stimulation, that they exert their effect on the postganglionic parasympathetic neuron, and that their effect is prejunctional. The effect appears secondary to a decrease in cyclooxygenase products rather than to an increase in lipoxygenase products. These findings suggest that endogenous cyclooxygenase products may modulate parasympathetic control of airway smooth muscle in vivo. They may relate to the mechanisms that underlie airway hyperresponsiveness, by which mediators of inflammation modulate airway responsiveness and by which nonsteroidal anti-inflammatory drugs induce severe bronchoconstrictor responses in some persons who have asthma.     

Endothelial cell proliferation may be mediated via the production of endogenous lipoxygenase metabolites

Endogenous regulators of endothelial cell proliferation have not been clearly defined. We investigated whether the cyclooxygenase and/or lipoxygenase metabolites are involved in this process, and report that lipoxygenase products can modulate endothelial cell growth. Nordihydroguaiaretic acid--a lipoxygenase inhibitor, inhibited endothelial cell proliferation as well as DNA synthesis. 5,8,11,14-Eicosatetraynoic acid--an inhibitor of both lipoxygenase and cyclooxygenase also inhibited endothelial cell DNA synthesis, while indomethacin--a selective cyclooxygenase inhibitor did not affect cell proliferation or DNA synthesis. While arachidonic acid stimulated DNA synthesis, this effect was completely abolished by nordihydroguaiaretic acid. These results demonstrate that products of the lipoxygenase pathway can affect endothelial cell proliferation.     

Mechanism of action of platelet-activating factor on guinea-pig lung parenchyma strips

The contribution of thromboxane A2 to platelet-activating factor (PAF)induced contraction of guinea-pig lung parenchyma strips (GPLPS) was investigated using an experimental design that allowed us to record the contractions of the tissues in parallel with the determination of thromboxane B2 (TXB2) levels in the organ baths by enzyme immunoassay. It was found that the first injection of PAF induced the contraction of GPLPS and the release of TXB2. Following subsequent additions of PAF to the same tissue, the contractile response was abolished but TXB2 levels were not significantly reduced. Pretreatment of the tissue with the thromboxane synthetase inhibitor OKY-046 (3.5, 170, and 350 microM) strongly inhibited the release of TXB2 but had no effect on the contraction of the tissues induced by PAF. The mechanism of PAF-induced contraction of GPLPS was further investigated using several drugs that interfere with arachidonic acid metabolism. It was found that pretreatment of the tissues with the cyclooxygenase and thromboxane synthetase inhibitors indomethacin (2.8, 28, and 56 microM) and OKY-046 (170 microM) or with the thromboxane antagonist SKF-88046 (1.25 and 12.5 microM) had no significant effect on the contractile response to PAF. The compound L-655,240 (2.5, 25, and 50 microM), which acts simultaneously as an antagonist of thromboxane and inhibitor of lipoxygenase, significantly reduced GPLPS contractions induced by PAF. Another lipoxygenase inhibitor, nordihydroguaiaretic acid (33 microM), and the inhibitor of both pathways of arachidonic acid metabolism, BW775c (110 microM), both reduced PAF-induced contractions of GPLPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclooxygenase- and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries

We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10(-9)-10(-4) M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 +/- 0.5% and EC(50) of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 microM), NS-398 (10 microM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 muM), nordihydroguaiaretic acid (NDGA; 1 microM), and ebselen (1 microM). HPLC analysis showed that [(14)C]AA was metabolized by mesenteric arteries to PGI(2), PGE(2), THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI(2) and PGE(2) was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PGI(2), PGE(2), and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K(+) (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways.     

Endogenous AA metabolites and their possible role in tracheal smooth muscle tone in guinea pigs

The effects of endogenous arachidonic acid (AA) metabolites on inherent tone and histamine-induced constriction were studied in guinea pig tracheal smooth muscle. Inhibitors of either cyclooxygenase (indomethacin) or lipoxygenase (AA 861) significantly diminished the inherent tone of the muscle. Antagonists of prostaglandins (SC 19220) or leukotrienes (FPL 55712) also diminished the inherent tone, whereas an inhibitor of thromboxane synthase (OKY 046) had no significant effect. These results show that the metabolites of the lipoxygenase pathway as well as prostaglandins also participate in the maintenance of inherent tone. To reexamine the previously reported augmentation of histamine constriction induced by the inhibitors and the antagonists, we compared the active tension of the muscle measured from the maximum relaxed level as the base line to eliminate the fluctuation of inherent tone. Such comparison revealed that the inhibitors and the antagonists have no augmentative effect on either the maximum response to histamine or the concentration required to produce 50% of maximum active tension and that there is functional synergism between the exogenously added histamine and the endogenously produced AA metabolites. Therefore the zero active tension is useful as a base line to compare the contractile response of a drug-treated preparation with that of a nontreated preparation.     

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.     

Arachidonic Acid Metabolites in bFGF-, PDGF-, and Serum-Stimulated Vascular Cell Growth

The signal transduction pathways through which growth factors regulate vascular cell growth are not fully understood. Recent studies suggest that metabolites of the lipoxygenase pathway may be involved in vascular cell growth. We have measured the effect of the lipoxygenase pathway inhibitors nordihydroguiaretic acid (NDGA), 5,6-dehydroarachidonic acid, and baicalein on bovine capillary endothelial cell (EC) and aortic smooth muscle cell (SMC) growth in the presence or the absence of growth factors. NDGA totally suppressed serum-stimulated EC and SMC growth as well as growth factor-stimulated proliferation over a 9-day time course. Removal of the inhibitor revealed that the inhibitory effect of NDGA was reversible and not due to cytotoxicity. The morphology of NDGA-treated EC was changed in a reversible manner from the characteristic polygonal to spindle shape. The 5-lipoxygenase inhibitor 5,6-dehydroarachidonic acid had no effect on vascular cell proliferation, but inhibition of 12-lipoxygenase with baicalein blocked both EC and SMC cell growth in a dose-dependent manner, in the presence and the absence of growth factors. Indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect on EC and SMC proliferation. Quinacrine and oleyloxyethylphosphorycholine inhibition of the phospholipase A₂-catalyzed release of arachidonic acid from membrane phospholipids blocked growth factor- and serum-stimulated proliferation of EC and SMC. These results suggested that arachidonic acid metabolites are critical intermediaries in the regulation of vascular cell growth.     

Potentiation of contraction of rabbit airway smooth muscle by some cyclooxygenase products

An alteration in smooth muscle sensitivity may be one of the mechanisms of the airway hyperresponsiveness observed in asthma. Indomethacin inhibits experimentally induced airway hyperresponsiveness. We thus examined the effects of the cyclooxygenase products PGD2, PGF2 alpha and a thromboxane A2 analogue U46619 on contractile responses of rabbit airway smooth muscle to histamine, carbachol and electrical field stimulation (EFS). PGD2 did not potentiate any contractile responses. When PGF2 alpha (1 microM) was administered 30 min before cumulative concentration-response curves to histamine and carbachol, no potentiation was observed. However, PGF2 alpha (1 microM) added immediately before EFS and bolus doses of histamine potentiated the contractile responses. U46619 increased the cumulative concentration-responses to both histamine and carbachol. The fact that we could alter smooth muscle sensitivity in vitro with PGF2 alpha and a thromboxane analogue suggests that these mediators may be involved in the airway hyperresponsiveness observed in asthma.     

Arachidonic acid and its acyclic derivatives regulate the short-term plasticity of the cholinoreceptors of the neurons in the edible snail]

On identified Helix neurones RPa3 and LPa3 using the method of double-electrode clamp technique on the membrane the influence was shown of eicosanoids on the dynamics of inward current extinction caused by the repeated ionophoretic applications of acetylcholine to soma. Extracellular influence of arachidonic acid (50-100 microM) increased the extinction. Phospholipase A2 inhibitor quinacrine hydrochloride (100-600 microM) decreasing the content of arachidonic acid in the cell acted differently. Inhibitor of lipoxygenase oxidation of arachidonic acid (nordihydraquiaretic acid) (3-10 microM) weakened the extinction. Blockader of cyclooxygenase oxidation of arachidonic acid--indomethacin (10-50 microM) did not influence the extinction. All the studied composition decreased the amplitude of input current caused by acetylcholine. The obtained results allowed to suppose that arachidonic acid and its acyclic metabolites formed as a result of lipoxygenase oxidation regulated short-term plasticity of snail neurones cholinoreceptors. Cyclic eicosanoids formed at cyclooxygenase oxidation of arachidonic acid had no regulating influence on cholinoreceptors plasticity.     

Progesterone inhibits gallbladder motility through multiple signaling pathways

Progesterone (P) has an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since P levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of P and two metabolites on contraction in guinea pig gallbladder strips. P induced a concentration-dependent relaxation in guinea pig gallbladder strips precontracted with cholecystokinin octapeptide (CCK). Pretreatment of gallbladder strips with P (50 microM) also reduced the amount of CCK-induced tension. Nifedipine (1 microM) produced a similar effect. Pretreatment of the strips with PKA inhibitor 14--22 amide myristolated (180 nM) or the PKG inhibitor KT5823 (1.2 microM) either separately or in combination significantly reduced the amount of P-induced relaxation. Rp-cAMPs (0.1mM) or H-89 (10 microM) separately or in combination significantly reduced the P-effect; however, the combination of agents produced the largest reduction. Genistein (1 microM), an inhibitor of protein tyrosine kinases, significantly (p<0.01) reduced the amount of P-induced relaxation. The use of strontium in the Kreb's solution as a substitute for Ca(2+) significantly (p<0.01) reduced the amount of CCK-induced tension. Pretreatment of the strips with 2-APB (26 microM), an inhibitor of IP(3,) induced Ca(2+) release, produced a significant (p<0.01) reduction in P-induced relaxation. We conclude that P inhibits gallbladder motility rapidly by nongenomic actions of the hormone. Several pathways that include tyrosine kinase and PKA/cAMP activity may mediate this effect.     

Metabolism of adrenic acid to vasodilatory 1alpha,1beta-dihomo-epoxyeicosatrienoic acids by bovine coronary arteries

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 +/- 0.01 and 6.18 +/- 0.60 microg/mg protein and adrenic acid concentrations of 0.29 +/- 0.01 and 1.56 +/- 0.16 microg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10(-9)-10(-5) M) induced concentration-related relaxations (maximal relaxation = 83 +/- 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K(+) channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 microM, maximal relaxation = 53 +/- 4%), and the cytochrome P-450 inhibitor, miconazole (10 microM, maximal relaxation = 52 +/- 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10(-9)-10(-5) M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 +/- 3%). Adrenic acid (10(-6) M) and DH-16,17-EET (10(-6) M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10(-8)-10(-6) M) activated iberiotoxin-sensitive, whole cell K(+) currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K(+) channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation.     

A TRPC-like non-selective cation current activated by alpha 1-adrenoceptors in rat mesenteric artery smooth muscle cells

The TRPC family of non-selective cation channels has been suggested to play a key role in the responses to alpha1-adrenoceptor stimulation of vascular smooth muscle. However, there are still very few reports of non-selective cation currents activated by alpha1-AR in resistance arteries. Here, we examine the expression of TRPC channels and the currents activated by alpha1-adrenoceptors in rat mesenteric resistance artery smooth muscle. Messenger RNA and protein for TRPC1, TRPC3 and TRPC6 were detected within the arteries by RT-PCR and immunoblotting. Endothelial and adventitial layers were found to express the TRPC1, TRPC3 and TRPC6 proteins whereas only TRPC1 and TRPC6 were detected in the arterial smooth muscle by confocal immunofluorescence microscopy. In whole-cell patch-clamp recordings from isolated mesenteric arterial myocytes, an outwardly rectifying non-selective cation current was activated by both the alpha1-adrenoceptor agonist, phenylephrine (10 microM), and the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (100 microM). Responses to 1-oleoyl-2-acetyl-sn-glycerol were not blocked, but increased, following inhibition of protein-kinase-C with either bisindolylmaleimide-I (1 microM) or chelerythrine (1 microM). The currents activated by both phenylephrine and 1-oleoyl-2-acetyl-sn-glycerol were inhibited by Gd3+ (100 microM) but potentiated by flufenamic acid (100 microM). Collectively, these findings demonstrate for the first time the expression of TRPC1 and TRPC6 in rat mesenteric artery smooth muscle and the existence in rat isolated mesenteric arterial myocytes of a TRPC-like non-selective cation current activated by alpha1-adrenoceptor stimulation and 1-oleoyl-2-acetyl-sn-glycerol.     

Effects of TRIM on tension, intracellular calcium and nitrergic transmission in the rat anococcygeus muscle.

The effects of the putatively selective inhibitor of neuronal nitric oxide synthase (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were investigated on contractility, intracellular calcium and nitrergic relaxations in the rat anococcygeus muscle. TRIM (100-1000 microM) reduced the tension of rat anococcygeus muscles when contracted with guanethidine (10 microM) and clonidine (0.1 microM). Relaxations to TRIM persisted in the presence of the non-selective NOS inhibitor L-NAME (100 microM) and the inhibitor of soluble guanylate cyclase ODQ (1 microM). TRIM also reduced tension when muscles were contracted with phenylephrine (3 microM), noradrenaline (3 microM) or high K physiological salt solution (high KPSS; 60mM). Influx of calcium ([Ca(2+)](i)) in response to high KPSS was significantly reduced in the presence of TRIM (1mM). TRIM also inhibited the influx of (45)Ca(2+) induced by KPSS, but had no effect on the influx induced by phenylephrine (10 microM). TRIM (300 microM) had a modest, but significant, inhibitory effect on nitrergic relaxations that were evoked by electrical field stimulation (1-10 Hz, 15 V, 10s trains) in muscles contracted with guanethidine and clonidine. In contrast, L-NAME (1-100 microM) inhibited these nitrergic responses with an IC(50) of 9.31+/-0.87 microM (n=4). The results suggest that the smooth muscle relaxant effect of TRIM in the rat anococcygeus muscle may affect the entry of Ca(2+) possibly through voltage-operated calcium channels. Furthermore, the relatively modest effect of TRIM on nitrergic responses indicates that it is not a particularly reliable inhibitor of nNOS.