The alpha adrenoceptors on endothelial cells

Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists.     

Effects of spontaneous or induced brain ischemia on vessel reactivity: the role of inducible nitric oxide synthase

Short episodes of ischemia and reperfusion in various organs may protect the organ itself, and the heart both as an immediate and a delayed effect. The present study investigates whether a systemic protection of vascular function occurs during adaption to ischemia. Brain ischemia was induced by bilateral ligation of the internal carotid arteries in C57BL6 mice, and 24-36 hours later rings of the thoracic aorta were mounted to study in vitro relaxation and contraction, or proteins were extracted for immunoblotting for endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS). eNOS decreased, while iNOS increased in the aortic wall after carotid artery ligation. In vitro contraction to increasing concentrations of prostaglandin F(2alpha) (PGF(2alpha)) was attenuated, while relaxation to acetylcholine (ACh) was enhanced. The latter was abolished by the iNOS-inhibitor aminoguanidine. When brain ischemia was induced in iNOS deficient mice, an increase of aortic eNOS was found 24 hours later. The ischemia-induced attenuated relaxation to PGF(2alpha) and enhanced relaxation to ACh were abolished. Aortic rings from mice with severe atherosclerosis (apolipoprotein E and low density lipoprotein receptor double knockout (ApoE/LDLr KO) mice) and spontaneous ischemic events in the heart or brain in vivo were also studied. Spontaneous ischemic events in ApoE/LDLr KO animals did not influence iNOS and eNOS in the vessel wall. A reduced contraction to PGF(2alpha) was observed, but relaxation to ACh was unchanged. These findings suggest that induced brain ischemia as a model of delayed, remote preconditioning protects vessel reactivity, and this protection is mediated by iNOS.     

Vascular reactivity and endothelial NOS activity in rat thoracic aorta during and after hyperbaric oxygen exposure

Accumulating evidence suggests that hyperbaric oxygen (HBO) stimulates neuronal nitric oxide (NO) synthase (NOS) activity, but the influence on endothelial NOS (eNOS) activity and vascular NO bioavailability remains unclear. We used a bioassay employing rat aortic rings to evaluate vascular NO bioavailability. HBO exposure to 2.8 atm absolute (ATA) in vitro decreased ACh relaxation. This effect remained unchanged, despite treatment with SOD-polyethylene glycol and catalase-polyethylene glycol, suggesting that the reduction in endothelium-derived NO bioavailability was independent of superoxide production. In vitro HBO induced contraction of resting aortic rings with and without endothelium, and these contractions were reduced by the NOS inhibitor N(omega)-nitro-l-arginine. In addition, in vitro HBO attenuated the vascular contraction produced by norepinephrine, and this effect was reversed by N(omega)-nitro-l-arginine, but not by endothelial denudation. These findings indicate stimulation of extraendothelial NO production during HBO exposure. A radiochemical assay was used to assess NOS activity in rat aortic endothelial cells. Catalytic activity of eNOS in cell homogenates was not decreased by HBO, and in vivo HBO exposure to 2.8 ATA was without effect on eNOS activity and/or vascular NO bioavailability in vitro. We conclude that HBO reduces endothelium-derived NO bioavailability independent of superoxide production, and this effect seems to be unrelated to a decrease in eNOS catalytic activity. In addition, HBO increases the resting tone of rat aortic rings and attenuates the contractile response to norepinephrine by endothelium-independent mechanisms that involve extraendothelial NO production.     

Alpha 1-adrenoceptor blocking properties of spiroxatrine in rat aorta.

This preliminary study has analyzed the potential ability of the 5-HT1A ligand spiroxatrine to interact with vascular alpha 1-adrenoceptors. Norepinephrine and the selective alpha 1-adrenoceptor agonist, methoxamine, elicited concentration-dependent contractions of rat aortic rings. In contrast, (+/-)-spiroxatrine (from 10(-8) to 3.1X10(-7) M) was devoid of any effect on vascular tone per se, but shifted the concentration-response curves of norepinephrine and methoxamine to the right in a concentration-dependent manner with pA2 values of 8.48 +/- 0.22 and 8.93 +/- 0.33, respectively. Endothelium removal did not significantly affect the above pA2 values of (+/-)-spiroxatrine. These data, taken in concert, support the contention that (+/-)-spiroxatrine displays alpha 1-adrenoceptor blocking properties in rat aortic rings.     

Effects of micromolar concentrations of manganese, copper, and zinc on α1-adrenoceptor-mediating contraction in rat aorta

To determine the influences of the Mn, Cu, and Zn on α₁-adrenoceptor (AR)-mediated vasoconstriction, we investigated their effects on vasoconstriction produced by the α₁-AR agonist phenylephrine in isolated rings of rat thoracic aorta. The cumulative concentration-contraction curves for phenylephrine were obtained in the absence and presence of Mn (0.3, 1, 3 μM), Cu (1, 10, 16 μM), and Zn (0.3, 1, 10 μM). Mn, Cu, and Zn each inhibited phenylephrine-mediated contraction in a dose-dependent manner. The maximal phenylephrine-induced contraction was significantly reduced by the pretreatment of the arterial rings with 10 and 16 μM Cu (p<0.05). The results suggest that variations in the plasma concentrations of metal might lead to changes in α₁-AR-mediated constrictive response.     

Interleukin-1 impairs both vascular contraction and relaxation in rabbit isolated aorta.

Incubation of rabbit aortic rings with interleukin-1 (100 U/ml) in vitro led to a depressed contractile response to norepinephrine, whether the endothelium was present or not. In both cases norepinephrine-induced contraction was restored in the presence of NG-methyl-L-arginine (300 microM), an inhibitor of nitric oxide synthesis. In interleukin-1-treated rings precontracted with norepinephrine (1 microM), the relaxing response to acetylcholine was totally suppressed independently on the presence of endothelium. High concentrations of acetylcholine (greater than 1 microM) induced a slight contraction which was of lower amplitude than that obtained in control endothelium-denuded rings and was increased in the presence of NG-methyl-L-arginine. These results show that interleukin-1 (i) affects not only vascular contraction but also relaxation and (ii) involves both endothelial and non-endothelial factors. These observations suggest an impairment of the whole vascular reactivity during septic shock.     

Acidic reoxygenation protects against endothelial dysfunction in rat aortic rings submitted to simulated ischemia

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.     

Presynaptic beta(2)-adrenoceptors mediate nicotine-induced NOergic neurogenic dilation in porcine basilar arteries

We previously reported that nicotine-induced nitric oxide (NO)-mediated cerebral neurogenic vasodilation was dependent on intact sympathetic innervation. We hypothesized that nicotine acted on sympathetic nerve terminals to release norepinephrine (NE), which then acted on adrenoceptors located on the neighboring nitric oxidergic (NOergic) nerve terminals to release NO, resulting in vasodilation. The adrenoceptor subtype in mediating nicotine-induced vasodilation in isolated porcine basilar arterial rings denuded of endothelium was therefore examined pharmacologically and immunohistochemically. Results from using an in vitro tissue bath technique indicated that propranolol and preferential beta(2)-adrenoceptor antagonists (ICI-118,551 and butoxamine), in a concentration-dependent manner, blocked the relaxation induced by nicotine (100 microM) without affecting the relaxation elicited by transmural nerve stimulation (TNS, 8 Hz). In contrast, preferential beta(1)-adrenoceptor antagonists (atenolol and CGP-20712A) did not affect either nicotine- or TNS-induced relaxation. Results of double-labeling studies indicated that beta(2)-adrenoceptor immunoreactivities and NADPH diaphorase reactivities were colocalized in the same nerve fibers in basilar and middle cerebral arteries. These findings suggest that NE, which is released from sympathetic nerves upon application of nicotine, acts on presynaptic beta(2)-adrenoceptors located on the NOergic nerve terminals to release NO, resulting in vasodilation. In addition, nicotine-induced relaxation was enhanced by yohimbine, an alpha(2)-adrenoceptor antagonist, which, however, did not affect the relaxation elicited by TNS. Prazosin, an alpha(1)-adrenoceptor antagonist, on the other hand, did not have any effect on relaxation induced by either nicotine or TNS. The predominant facilitatory effect of beta(2)-adrenoceptors in releasing NO may be compromised by presynaptic alpha(2)-adrenoceptors.     

Apocynin normalizes hyperreactivity to phenylephrine in mesenteric arteries from cholesterol-fed mice by improving endothelium-derived hyperpolarizing factor response

We studied the relationship among endothelial function, oxidative stress, and phenylephrine (PE; alpha(1)-adrenoceptor agonist)-induced contraction in mesenteric arteries from high-cholesterol (HC)-diet-fed mice. In HC mice (vs age-matched normal-diet-fed mice): (1) PE-induced contraction in endothelium-intact rings was enhanced (endothelial denudation increased contraction in "normal-diet" rings, but did not enhance it further in "HC" rings); (2) the enhanced PE-induced contraction was further enhanced in the presence of N(G)-nitro-L-arginine (L-NNA; nitric oxide synthase inhibitor) or L-NNA plus indomethacin (cyclooxygenase inhibitor) [to preserve endothelium-derived hyperpolarizing factor (EDHF)], but unchanged in the presence of charybdotoxin plus apamin (to block EDHF); (3) ACh-induced EDHF-type relaxation was reduced; and (4) oxidative stress [indicated by the plasma 8-isoprostane level (reliable systemic marker) and aortic superoxide production] was greater. In HC mice, PE-induced contraction was normalized by apocynin [NAD(P)H oxidase inhibitor] or tempol (superoxide dismutase mimetic), but enhanced by NADH [NAD(P)H oxidase substrate]. Oral dietary supplementation with apocynin (30 mg/kg/day for 4 weeks) corrected the above abnormalities. Hence: (1) PE-induced contraction is modulated by the endothelium, and the enhanced contractility in HC mice results from defective EDHF signaling and elevated oxidative stress, and (2) apocynin normalizes PE-induced contraction in HC mice by improving EDHF signaling.     

Agonist-induced tension determines vascular reactivity during anoxia and reoxygenation.

To determine the influence of pre-existing pharmacologically-induced tension on vascular reactivity during anoxia and reoxygenation, rat aortic rings were contracted with norepinephrine, epinephrine, endothelin or KCl to 1, 2 or 4 g of tension. The rings were then exposed to anoxia (95% N2) for 10 min followed by reoxygenation (95% O2). The degree of anoxia-mediated contraction varied with the magnitude of tension before anoxia and resembled the length-tension relationship in myocardial fibers. The optimal agonist-induced tension for maximal anoxic contraction was approximately 1 to 2 g. This relationship between tension and anoxic contraction was observed in all but KCl-contracted rings. The agonist- as well as KCl-contracted rings showed normal relaxant response to acetylcholine, suggesting that a decrease in endothelium-derived relaxing factor (EDRF) alone cannot be the basis of anoxic contraction and release of endothelium-derived constricting factors (EDCFs) may relate to anoxic contraction in agonist-preconstricted rings. The relationship between the magnitude of agonist-induced tension and the extent of anoxia-mediated contraction may relate to the ability of endothelium to release EDRF and EDCFs as well as to the degree of phosphorylation in vascular smooth muscle cells. The reoxygenation-mediated contraction was noted to progressively increase in all experiments regardless of the pharmacologic agent used. This increase in reoxygenation-mediated contraction correlated with pre-existing pharmacologic tension, and may relate to calcium influx and restoration of ATP and other mediators in the vascular tissues during reoxygenation.     

Further studies on the ethanol antagonism exhibited by 2(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587)

A lipid soluble alpha 1-adrenoceptor agonist 2-(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587) dose-dependently antagonized the hypnotic, hypothermic and respiratory depressant effects of ethanol in C57B1/6 mice. This effect was present whether St 587 was given before or after ethanol. St 587 did not block the pentobarbitone-induced hypnosis. It also did not influence the elimination of ethanol. Combined treatment with a subhypnotic dose of ethanol and St 587 resulted in marked hyperactivity in mice. This effect was completely abolished by pimozide pretreatment. It was inferred that the dopamine released from brain areas by this dose of ethanol together with the norepinephrine receptor activation offered by St 587 resulted in this hyperactivity. Cirazoline, a more potent alpha 1-adrenoceptor agonist than St 587 was relatively more effective than the latter in blocking the ethanol-induced hypnosis in mice. It seems that alpha 1-adrenoceptor stimulation is a major contributing factor to the ethanol antagonism exerted by St 587. This drug might prove to be useful in the treatment of acute ethanol intoxication and in understanding the mode of action of ethanol.     

Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection

In patients with high thoracic spinal lesions that remove most of the central drive to splanchnic preganglionic neurons, visceral or nociceptive stimuli below the lesion can provoke large increases in blood pressure (autonomic dysreflexia). We have examined the effects of T4 spinal transection on isometric contractions of mesenteric arteries isolated from spinalized rats. Nerve-evoked contractions involved synergistic roles for norepinephrine and ATP. At 7 wk after spinal transection, responses to perivascular stimulation at 1-5 Hz were enhanced fivefold, whereas the alpha1-adrenoceptor antagonist prazosin (10 nM) produced a twofold larger reduction in contraction (to 20 pulses at 10 Hz) than in unoperated controls. In contrast, the reduction in nerve-evoked contractions by the P2-purinoceptor antagonist suramin (0.1 mM) and the responses to the P2-purinoceptor agonist alpha,beta-methylene ATP or to high K+ concentration did not greatly differ between groups, indicating that arteries from spinalized rats were not generally hyperreactive. Sensitivity to the alpha1-adrenoceptor agonist phenylephrine was enhanced in arteries from spinalized rats, and the difference from controls was abolished by the norepinephrine uptake blocker desmethylimipramine. Sensitivity to the alpha1-adrenoceptor agonist methoxamine, which is not a substrate for the neuronal norepinephrine transporter, was similar among the groups. Thus the increased neurally evoked response after spinal transection appeared to be due to a reduction in neuronal uptake of released norepinephrine, a mechanism that did not explain the enhanced response of tail arteries after spinal transection that we previously reported. The findings provide further support for potentiated neurovascular responses contributing to the genesis of autonomic dysreflexia.     

Vascular effects of some opioid receptor agonists.

Opioid peptides have been implicated in shock-associated hypotension. Our aim was to find out whether opioid agonists have direct vasodilator actions on vascular smooth muscle. The study was conducted on rat abdominal aortic rings. In rings precontracted with either norepinephrine, prostaglandin F2 alpha, or high potassium Krebs (HPK), the effects of the opioid agonists tested (morphine, U50488H, ethylketocyclazocine (EKC), and bremazocine) depended on the precontracting agent used. HPK-precontracted rings were relaxed by all agonists tested. In norepinephrine-precontracted rings, all caused contraction at low concentrations and relaxation at high concentrations except bremazocine, which caused only relaxation. In prostaglandin F2 alpha-precontracted rings, U50488H produced contraction at low concentrations and relaxation at high concentrations while EKC caused only relaxation and morphine or bremazocine caused only contraction. All relaxant responses were endothelium-independent and were antagonized by verapamil but not by a number of antagonists including naloxone. MR2266, propranolol, diphenhydramine, cimetidine, and indomethacin. They may reflect calcium channel blockade. Morphine-induced vasoconstriction was antagonized by high concentrations of of naloxone or mepyramine and may be due to release of histamine by a naloxone-sensitive mechanism. We conclude that (a) the opioid agonists tested exert direct actions on vascular smooth muscle; (b) the nature of the response depended not only on the agonist used and its concentration but also on the agent used to precontract the tissue; and (c) it is unlikely that direct actions of endogenous opioids contribute to the shock-associated hypotension because high doses were needed to elicit them.     

Analysis of responses to sympathetic nerve stimulation in the feline pulmonary vascular bed

The adrenergic receptor subtypes mediating the response to sympathetic nerve stimulation in the pulmonary vascular bed of the cat were investigated under conditions of controlled blood flow and constant left atrial pressure. The increase in lobar vascular resistance in response to sympathetic nerve stimulation was reduced by prazosin and to a lesser extent by yohimbine, the respective alpha 1- and alpha 2-adrenoceptor antagonists. Moreover, in animals pretreated with a beta-adrenoceptor antagonist to prevent an interaction between alpha- and beta 2-adrenoceptors, responses to nerve stimulation were reduced by prazosin, but yohimbine had no significant effect. On the other hand, in animals pretreated with a beta-adrenoceptor antagonist, yohimbine had an inhibitory effect on responses to tyramine and to norepinephrine. Propranolol had no significant effect on the response to nerve stimulation, whereas ICI 118551, a selective beta 2-adrenoceptor antagonist, enhanced responses to nerve stimulation and injected norepinephrine. The present data suggest that neuronally released norepinephrine increases pulmonary vascular resistance in the cat by acting mainly on alpha 1-adrenoceptors and to a lesser extent on postjunctional alpha 2-adrenoceptors but that this effect is counteracted by an action on presynaptic alpha 2-receptors. The present studies also suggest that neuronally released norepinephrine acts on beta 2-adrenoceptors and that the response to sympathetic nerve stimulation represents the net effect of the adrenergic transmitter on alpha 1-, alpha 2-, and beta 2-adrenoceptors in the pulmonary vascular bed.     

Higher sensitivity of cerebral arteries isolated from premature and newborn baboons to adrenergic and cholinergic stimulation

To find whether effects of adrenergic and cholinergic agents on cerebral artery were dependent on maturity, we examined responses of isolated cerebral artery strips harvested from premature, term newborn and adult baboons. Although cerebral arteries from many species are only mildly sensitive to norepinephrine, we found the perinatal cerebral arteries to be quite responsive to the amine. Cerebral arteries from premature and newborn baboons were significantly (P less than 0.001) more sensitive to norepinephrine than were arteries from adults; medium effective concentration (EC50) for norepinephrine were 3 X 10(-8), 6 X 10(-8) and 32 X 10(-8)M for prematures, newborns and adults, respectively. Arteries showed a similar age-dependence in the sensitivity of the response to phenylephrine, an alpha 1-adrenoceptor agonist. EC50 values for KC1 did not differ among groups, nor did the maximum response to norepinephrine. Arteries from premature and newborn baboons showed marked contractile response to acetylcholine (maximum tensions 5.9 +/- 0.6 and 6.4 +/- 0.8 g/mm2, respectively), whereas arteries from adult baboons showed little response (0.6 +/- 0.1 g/mm2). Arteries from premature and newborn animals showed a more marked relaxation response to isoproterenol than did arteries from adult animals; the degree of relaxation from an induced contraction was 63% (premature), 72% (newborn) and 10% (adult). There was no age-dependence in the relaxation response to sodium nitrite. We conclude that the events coupling alpha 1, beta or muscarinic receptor activation with cerebral arterial contraction or relaxation are more effective in perinatal than in adult baboons.     

Differential expression of alpha2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats

The aim of the present study was to compare vascular dysfunction between the early (12 wk old) and later (36 wk old) stages of spontaneous diabetes in Goto-Kakizaki (GK) rats. We also evaluated the aortic expression of the alpha(2D)-adrenoceptor and endothelial nitric oxide synthase (eNOS). Vascular reactivity was assessed in thoracic aortas from age-matched control rats and 12- and 36-wk GK rats. Using RT-PCR and immunoblots, we also examined the changes in expression of the alpha(2D-)adrenoceptor and eNOS. In aortas from GK rats (vs. those from age-matched control rats): 1) the relaxation response to ACh was enhanced at 12 wk but decreased at 36 wk; 2) the relaxation response to sodium nitroprusside was decreased at both 12 and 36 wk, 3) norepinephrine (NE)-induced contractility was decreased at 12 wk but not at 36 wk, 4) the expressions of alpha(1B)- and alpha(1D)-adrenoceptors were unaffected, whereas those of alpha(2D)-adrenoceptor and eNOS mRNAs were increased at both 12 and 36 wk; and 5) NE- and ACh-stimulated NO(x) (nitrite and nitrate) levels were increased at 12 wk, although at 36 wk ACh-stimulated NO(x) was lower, whereas NE-stimulated NO(x) showed no change. These results clearly demonstrate that enhanced ACh-induced relaxation and impaired NE-induced contraction, due to NO overproduction via eNOS and increased alpha(2D)-adrenoceptor expression, occur in early-stage GK rats and that the impaired ACh-induced relaxation in later-stage GK rats is due to reductions in both NO production and NO responsiveness (but not in eNOS expression).     

Continuous flow augments reactivity of rabbit carotid artery by reducing bioavailability of NO despite an increase in release of EDHF

Experiments were designed to investigate the influence of steady flow and pressure on endothelial function in the rabbit carotid artery. Increases and decreases in isometric force were compared in static rings and perfused (5 or 50 ml/min) segments of the same arteries in the presence and absence of endothelium. The alpha(1)-adrenoceptor agonist phenylephrine and the muscarinic agonist acetylcholine were applied as vasoconstrictor and vasodilator stimuli, respectively. Continuous flow (5 and 50 ml/min) reduced the cGMP content and shifted the concentration-response curve to phenylephrine to the left compared with nonperfused static rings. Removal of the endothelium abolished the differences in cGMP content and the sensitivity to phenylephrine between static rings and perfused segments. No difference in sensitivity to phenylephrine was observed in tissues treated with N(omega)-nitro-l-arginine methyl ester (l-NAME). Acetylcholine-evoked relaxations were increased in perfused segments. l-NAME nearly abolished the acetylcholine-evoked relaxation in static rings, whereas about one-half of the relaxation remained in segments exposed to flow. This remnant relaxation was blocked by inhibition of endothelial small- and intermediate-conductance calcium-activated potassium channels by apamin plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). These experiments demonstrate that continuous flow increases the constriction evoked by alpha(1)-adrenergic activation in the rabbit carotid artery through a reduced influence of basally released endothelial NO and, furthermore, that luminal flow unmasks an ability of the endothelium to release a non-NO, noncyclooxygenase vasodilator, presumably endothelium-derived hyperpolarizing factor.     

Incubation of rat aortic rings produces a specific reduction in agonist-evoked contraction: effect of age of donor

We studied the effect of age on the response of aortic rings to injury produced by three days' incubation, and the mechanism of this response. Five-mm rings of the thoracic aorta isolated from Wistar rats were incubated or not in culture medium. Isometric contraction evoked by agonists (norepinephrine or serotonin) or high [K(+)](e) was determined in the presence and absence of endothelium. Experiments were repeated in the presence of propranolol (0.3 microM), polymixin B (36 microM), pyrrolidine dithiocarbamate (50 microM) or glutathione (3 mM). Inductible NO-synthase and cyclo-oxygenase-2 mRNA were determined by real-time PCR, and glutathione-related enzymes and catalase activity by spectrophotometry. Incubation reduced the isometric contraction evoked by agonists but not by high [K(+)](e). The reduction in agonist-evoked contraction was greater in rings from adult (norepinephrine Emax-80%) than in young (-40%) rats. The removal of the endothelium had no effect. The reduction in norepinephrine-evoked contraction was not due to endotoxin contamination, beta-adrenoceptor-mediated dilation or any change in ring structure (no fibrosis or edema). Inductible NO-synthase (but not cyclo-oxygenase-2) mRNA increased on incubation. N(G)-nitro-L-arginine methyl ester partially restored contractility in rings from adult animals, further addition of an anti-oxidant restored norepinephrine-evoked contraction. Catalase fell with age and glutathione reductase increased upon incubation in rings from young donors only. In conclusion, incubation of the aorta produces a specific reduction in agonist-evoked contraction that involves induction of smooth muscle cell oxidative stress and iNOS. The reaction is greater in rings from older animals.     

Simulated microgravity upregulates an endothelial vasoconstrictor prostaglandin.

Endothelial nitric oxide contributes to the vascular hyporesponsiveness to norepinephrine (NE) observed in carotid arteries from rats exposed to simulated microgravity. The goal of the present study was to determine whether a cyclooxygenase product of arachidonic acid also influences vascular responsiveness in this setting. Microgravity was simulated in rats by hindlimb unweighting (HU). After 20 days of HU, carotid arteries were isolated from control and HU-treated rats, and vascular rings were mounted in tissue baths for the measurement of isometric contraction. Two cyclooxygenase inhibitors, indomethacin and ibuprofen, and the selective thromboxane A(2) prostanoid-receptor antagonist, SQ-29548, had no effect on the contraction to NE in control vessels but markedly reduced contraction to NE in HU vessels. When the endothelium was removed, indomethacin no longer had any effect on the NE-induced contraction in HU vessels. In endothelium-intact vessels in the presence of indomethacin, the addition of the nitric oxide synthase inhibitor, N(G)-L-nitro-arginine methyl ester, to the medium bathing HU vessels increased the contraction to NE to the level of that of the control vessels. These results indicate that HU treatment induced two endothelial changes in carotid artery that opposed each other. Nitric oxide activity was increased and was responsible for the vascular hyporesponsiveness to NE. The activity of a vasoconstrictor prostaglandin was also increased, and attenuated the vasodilating effect of nitric oxide.     

A novel cell adhesion inhibitor, K-7174, reduces the endothelial VCAM-1 induction by inflammatory cytokines, acting through the regulation of GATA

A novel inhibitor for the adhesion of monocytes to cytokine-stimulated endothelial cells, K-7174, was selected by an assay system using the cultured human monocytic cells and human endothelial cells. K-7174 inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) induced by either tumor necrosis factor alpha or interleukin-1beta, without affecting the induction of intercellular adhesion molecule-1 or E-selectin. K-7174 had no effect on the stability of VCAM-1 mRNA. Electrophoretic mobility shift assay revealed that its inhibitory effect on VCAM-1 induction was mediated by an effect on the binding to the GATA motifs in the VCAM-1 gene promoter region. K-7174 did not influence the binding to any of the following binding motifs: octamer binding protein, AP-1, SP-1, ets, NFkappaB, or interferon regulatory factor. These results suggest that the regulation of GATA binding may become a new target for anti-inflammatory drug development, acting through a mechanism independent from NFkappaB activity.