Activation of multiple sites by adenosine analogues in the rat isolated aorta

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM). 2. In the prostatic half of the vas deferens the A1 selective adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A1/A2 adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC50+/-s.e.mean values 6.15+/-0.24, 5.99+/-0.26 and 5.51+/-0.24, respectively). The responses to CPA were blocked by the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM). 3. In the epididymal half of the vas deferens NECA potentiated (at < or = 100 nM) and inhibited (at > or = 1 microM) electrically-evoked contractions. In the presence of the non-selective alpha-adrenoceptor antagonist phentolamine (3 microM), the alpha1-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC50 values 6.05+/-0.25, 5.97+/-0.29 and 5.71 +/-0.27, respectively). CPA (at 10 microM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC50 6.14+/-0.67); this effect was antagonized by DPCPX (30 nM, apparent pK(B) 8.26+/-0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 microM), CPA (up to 1 microM) potentiated electrically-evoked contractions. 4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC50 values 7.49+/-0.62, 7.65+/-0.74 and 5.84+/-0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK(B) value of 7.64+/-0.64. 5. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 microM) potentiated responses to phenylephrine (< or = 1 microM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 microM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 microM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC50 values 7.57+/-0.54 and 8.08+/-0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis. 6. These studies are consistent with the action of stable adenosine analogues at prejunctional A1 and postjunctional A1-like adenosine receptors. The prejunctional A1 adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of alpha1-adrenoceptor-, but not of ATP-induced contractility.     

Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat

1. The receptors mediating the vasodilator responses to adenosine in the isolated mesenteric arterial bed of the rat were identified by use of selective agonists and antagonists and the involvement of the endothelium was examined. 2. Adenosine-mediated dilatation of the mesentery was potentiated by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but in contrast, removal of the endothelium substantially reduced the responses to adenosine. 3. The order of potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-p-(-2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > 2-chloro-N6-cyclopentyl-adenosine (CCPA) > or = adenosine, suggesting the presence of A2A receptors. 4. Adenosine-mediated dilatation was inhibited by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (3 microM) and by the A2A receptor antagonist 8-(3-chlorostyryl)caffeine (500 nM), but was unaffected by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM). 5. Reducing the pH of the perfusate to 6.8 potentiated the actions of both CGS 21680 and adenosine, but the vasodilator effects of carbachol were the same at both pH values. The adenosine response at the lower pH as at pH 7.4, was unaffected by DPCPX. The actions of the nitrovasodilator, sodium nitroprusside, were also potentiated at pH 6.8 relative to those at the higher pH value but smaller responses were obtained at the lower pH value with forskolin, a stimulator of adenylyl cyclase, than at pH 7.4. 6. It is concluded that the adenosine receptor mediating dilatation of the rat mesenteric arterial bed is of the A2A subtype, that the response, under the conditions used, is apparently partly dependent on the endothelium (but not due to the release of nitric oxide), and that the response to activation of this receptor is potentiated by a reduction in pH which is similar to that seen in ischaemic conditions.     

Photoaging and tretinoin

BACKGROUND: Adenosine is a potent vasodilator of vascular smooth muscle. Endothelium-derived nitric oxide (NO) elicits vasodilation. We have previously reported that adenosine stimulates the production of NO from porcine carotid arterial endothelial cells (PCAEC) via a receptor-mediated mechanism. This study was to determine whether adenosine also enhances NO production from human arterial endothelium and to define the involvement of adenosine A1 and A2 receptors. MATERIALS AND METHODS: Human iliac arterial endothelial cells (HIAEC) and PCAEC were harvested and cultured in dishes. NO production was evaluated with a NO electrode sensor which measured continuously real-time NO production. RESULTS: NO content of the medium bathing HIAEC and PCAEC was significantly increased with adenosine (100 micromol/L). Ethylcarboxamidoadenosine (NECA), a nonselective adenosine receptor agonist, and carboxyethyl-phenethylamino-ethylcarboxamidoadenosine (CGS-21680), a selective adenosine A2a receptor agonist, increased NO production by HIAEC and PCAEC with respective EC50 values of 3.32 and 6.96 nmol/L for NECA and 30.97 and 29.47 nmol/L for CGS-21680. Chlorofuryl-triazolo-quinazolinamine (CGS-15943; 1 micromol/L), an adenosine A1 and A2 receptor antagonist, and aminofuryltriazolotriazinyl-aminoethylphenol (ZM-241385; 1 micromol/L), a selective adenosine A2a receptor antagonist, inhibited the effect of CGS-21680. Chlorocyclopentyl-adenosine (CCPA; 1 micromol/L), an adenosine A1 receptor agonist, significantly depressed NO production by both HIAEC and PCAEC: This effect was inhibited by cyclopentyl-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist. CONCLUSIONS: The results demonstrate that adenosine A2a receptors increase, and adenosine A1 receptors decrease, the production of NO by human and porcine arterial endothelial cells.     

Differential role of vasoactive prostanoids in porcine and human isolated pulmonary arteries in response to endothelium-dependent relaxants

The pig is increasingly being used in medical research, both as a model of the human cardiovascular system, and as a possible source of organs for xenotransplantation. However, little is known about the comparative functions of the vascular endothelium between porcine and human arteries. We have therefore compared the effects of two endothelium-dependent vasorelaxants, acetylcholine (ACh) and the Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) on the porcine and human isolated pulmonary artery using isometric tension recording. ACh and CPA produced endothelium-dependent relaxations of both the human and porcine pulmonary arteries. In the porcine pulmonary artery, the cyclo-oxygenase inhibitor, flurbiprofen had no effect on relaxations to ACh (Emax: control 67.8+/-8.8% versus 72.4+/-9.5% (n=11)) or CPA (Emax: control 79.6+/-5.0% versus 94.0+/-10.6% (n=7)). The nitric oxide synthase inhibitor, L-NAME converted relaxations to both ACh and CPA into contractile responses (maximum response: ACh 30.0+/-11.1% (n = 10); CPA 80.4+/-26.2% (n = 8) of U46619-induced tone). These contractile responses in the presence of L-NAME were abolished by flurbiprofen. In the human pulmonary artery, L-NAME and flurbiprofen partly attenuated relaxations to ACh (Emax: control: 45.1+/-12.1%; flurbiprofen: 33.4+/-13.5%; L-NAME: 10.1+/-7.2%) and CPA (Emax: control: 78.1+/-5.5%; flurbiprofen: 69.6+/-7.2%; L-NAME 37.9+/-10.7% of U46619-induced tone). These responses were abolished by the combination of both inhibitors. We have demonstrated that while the release of nitric oxide is important in responses to endothelium-dependent vasorelaxants in both human and porcine pulmonary arteries, in the human arteries, there is an important role for vasorelaxant prostanoids whilst in the porcine arteries, vasoconstrictor prostanoids are released.     

Vasodilatory effects of adenosine A2 receptor agonists CGS 21680 and CGS 22492 in human vasculature

The vasodilatory effects of the adenosine analogs, 5'-N-ethylcarboxamidoadenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) and 2-[(2-cyclohexylethyl)amino]adenosine (CGS 22492) in human coronary, internal mammary artery and saphenous vein were examined in vitro. All produced concentration-dependent relaxations in arterial as well as venous rings contracted with 35 mM KCl. The concentration-response curves for NECA and CGS 21680 were parallel in the coronary. The adenosine A2 receptor antagonist, 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943A) significantly attenuated the relaxing response to the adenosine analogs in coronary artery. Although NECA and CGS 22492 were equally as effective at the highest concentration administered (both achieving approximately 70% relaxation at 10(-4) M) NECA (EC50 = 1.25 +/- 0.11 microM) induced greater vasodilation at lower concentrations than CGS 22492 (EC50 = 11.27 +/- 1.53 microM). CGS 21680 was the least potent of the agents tested achieving only 44% relaxation at 10(-4) M (EC50 = 4.71 +/- 0.46 microM). Coronary artery appeared to be more responsive than internal mammary artery or saphenous vein which displayed only marginal relaxation to these agents.     

Skin ulceration from tibial cement extrusion: case report and literature review

BACKGROUND/AIMS: Endothelium dependent vasodilatation is an important regulator of blood flow to the eye but its role has not been investigated in vessels supplying the ciliary body. This study assessed the role of the endothelium in modulating vasoconstrictor responses of the intraocular bovine anterior ciliary artery. METHODS: Bovine anterior ciliary arteries (n = 33) were mounted in a myograph, containing physiological salt solution at 37 degrees C, for isometric force measurement. Cumulative concentration-response curves were obtained to the constrictor agonists 5-hydroxytryptamine (5-HT), noradrenaline, phenylephrine, prostaglandin, F2 alpha, endothelin-1, and KCl in both endothelium intact and denuded arteries. RESULTS: All vasoconstrictors produced sustained contractile responses which were unaffected by the removal of the endothelium. Responses to 5-HT were also unaffected by inhibition of nitric oxide synthase. CONCLUSION: These results indicate that neither agonist stimulated nor basal release of nitric oxide from the endothelium modulates responses to vasoconstrictor agonists in the isolated bovine anterior ciliary artery when measured in a no flow isometric system.     

Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat

1. The aim of this study was to characterize adenosine receptors located in the nucleus tractus solitarius (NTS) that mediate decreases in blood pressure in the anaesthetized rat. To determine the adenosine receptor subtype involved, a range of selective agonists and antagonists were studied and their relative potencies evaluated. 2. The rank order of agonist potency in inducing decreases in diastolic blood pressure was N6-cyclopentyladenosine (CPA) > N6-cyclohexyladenosine (CHA) > N-ethyl-carboxamidoadenosine (NECA) > or = 2-phenylaminoadenosine (CV1808) > 2-p-(carboxyethyl)phenethylamino-5' N-ethylcarboxamidoadenosine (CGS 21680) > N6-(2-(4-aminophenyl)ethyl)-adenosine (APNEA). 3. The hypotensive action of CPA following microinjection into the NTS was antagonized by i.v. infusions (50 micrograms kg-1 min-1) of adenosine receptor antagonists, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), 8-phenyltheophylline (8-PT), 8-(p-sulphophenyl)theophylline (8-SPT), and 1,3-dipropyl-8-N-(2-diethylamino)ethyl)-N methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo) benzenesulphonamidexanthine (PD 115199). The antagonist potency order was DPCPX > PD115199 > or = 8-PT. Intravenous infusion of 8-SPT had no effect on blood pressure responses to microinjection of CPA into the NTS. 4. The results suggest that adenosine A1 receptors in the NTS mediate hypotensive responses in the anaesthetized rat preparation.     

Increased production of nitric oxide in coronary arteries during congestive heart failure

Experiments were designed to determine whether a heterogeneity of endothelium-dependent relaxations in arteries from different vascular beds exists in experimental congestive heart failure (CHF) and to determine the mediators of those responses. CHF was produced in dogs by rapid ventricular pacing for 15 d. Rings of coronary, femoral, and renal arteries with and without endothelium from control and CHF dogs were suspended in organ chambers for measurement of isometric force. In arteries contracted with prostaglandin F2 alpha, endothelium-dependent relaxations to BHT 920 (an alpha 2-adrenergic agonist) were increased in coronary arteries from dogs with CHF (maximal relaxation: control -15 +/- 9% vs CHF -92 +/- 5%; n = 5-6; P < 0.05), with a modest enhancement in renal arteries. Relaxations to adenosine diphosphate and the calcium ionophore were unchanged. Relaxations to BHT 920 in CHF were reduced by NG monomethyl-L-arginine (L-NMMA) and pertussis toxin but not by indomethacin. These data suggest that endothelium-dependent relaxations are affected heterogeneously in CHF. The enhanced response to alpha 2-adrenergic agonists in the coronary artery is mediated by nitric oxide through a mechanism sensitive to inhibition by pertussis toxin. This selective increase in endothelium-dependent relaxations in the coronary artery may contribute to preserving coronary blood flow during CHF.     

Regulation of purinoceptors in guinea pig pulmonary artery: functional evidences

In isolated guinea pig pulmonary arteries (precontracted with 1 microM noradrenaline) N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist, exerted a concentration-dependent contraction, whereas 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective A1/A2 receptor agonist, in the presence of DPCPX (a highly selective A1 receptor antagonist), produced a concentration-related rapid relaxation. Pulmonary arteries obtained from guinea pigs treated with aminophylline (APH) or 8-phenyltheophylline (8-PT) for 10 consecutive days, displayed more pronounced contraction in response to CPA compared to those of solvent-treated animals. Relaxant action of NECA was, however, attenuated in arteries prepared from methylxanthine-treated guinea pigs. Opposite changes were found in vascular tissues excised from chronically dipyridamole(DP)-treated guinea pigs.     

Impairment of vasodilator function in basilar arteries from aged rats

BACKGROUND AND PURPOSE: Aging is associated with a reduction in cerebral perfusion. Impaired vasodilatation in large brain arteries could be implicated. This study sought age-related changes in vasodilator responses to norepinephrine in rat basilar artery and investigated which aspects of norepinephrine's action are responsible. To study the effect of aging per se, we used the rat, an animal with resistance to development of age-related pathologies. METHODS: Vascular responses were studied in basilar arteries from young (3 to 4 months old) and old (20 to 22 months old) normotensive Sprague-Dawley rats with wire myography. Endothelial structure was assessed with confocal microscopy. RESULTS: There was no age-related difference in blood pressure and in KCl or 5-hydroxytryptamine (5-HT) contractions. Relaxation to bradykinin or its absence predicted an intact or denuded endothelium, confirmed by confocal microscopy. Norepinephrine produced concentration-dependent relaxation that was significantly smaller in old rats, with or without endothelium. This response was significantly smaller in endothelium-denuded vessels, or after preincubation with NG-nitro-L-arginine methyl ester or propranolol, but not with rauwolscine. CONCLUSIONS: In old and young rats the vasodilator action of norepinephrine in basilar artery is dependent on beta-adrenoceptors and nitric oxide. The impaired vasodilatation to norepinephrine found in the basilar artery from old rats might be caused by (1) a reduction in nitric oxide production and/or release or (2) beta-adrenoceptor alteration at the endothelium and/or the vascular smooth muscle. This impairment of vasodilator function can be ascribed to the aging process per se and not to other age-related alterations, such as hypertension.     

Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different roles of endothelium-derived substances on inhibiting platelet aggregation in whole blood

1. The inhibitory effects of adenosine, nitroprusside (a nitric oxide donor) and prostacyclin on collagen-induced rabbit platelet aggregation were studied under two different conditions: in whole blood with an impedance method and in platelet-rich plasma (PRP) with a turbidimetric method. 2. All substances tested were less potent in whole blood than in PRP, and the differences in IC50 value between whole blood and PRP were not of the same order of magnitude; adenosine (669-fold), nitroprusside (54-fold) and prostacyclin (2-fold). 3. These results imply that (a) some other, as yet unknown, factors in blood modulate the platelet aggregation; (b) adenosine and nitric oxide act close to the endothelium, and (c) prostacyclin acts as a relatively long lasting circulating hormone.     

Endothelial calcium-calmodulin dependent nitric oxide synthase in the in vitro vascular hyporeactivity of portal hypertensive rats

BACKGROUND/AIMS: Increased nitric oxide production has been implicated in impaired vascular responsiveness to vasoconstrictors in portal hypertension. However, there is no firm evidence concerning the involved nitric oxide synthase isoform. The present study investigated the possible contribution of one nitric oxide synthase isoform, the endothelial constitutive Ca2+-calmodulin dependent, in the overproduction of nitric oxide in portal hypertension. METHODS: Vascular responses to norepinephrine and acetylcholine were evaluated in isolated thoracic aortic rings from normal and portal vein stenosed rats. RESULTS: An impaired concentration-dependent contraction to norepinephrine was observed in intact rings from portal hypertensive rats compared to controls. The hyporeactivity to norepinephrine was reversed after endothelium denudation, the inhibition of nitric oxide synthase with L-NOARG or the inhibition of calmodulin with W-7, but not after pre-incubation with indomethacin. Stimulation of intact rings with norepinephrine after the inhibition of calmodulin with calmidazolium was followed by a decreased vascular response in vessels from normal rats but not in those from portal hypertensive rats. Stimulation of intact rings with norepinephrine in a Ca2+-free medium was followed by a decreased vascular response in vessels from both portal hypertensive and normal rats. No difference in vasoconstrictive responses was observed between the two groups after calmidazolium or in a Ca2+-free medium. Relaxation induced by acetylcholine in norepinephrine-precontracted rings was more marked in rings from portal hypertensive rats than in controls. No differences in the vasodilator responses were observed after relaxations had been inhibited by the removal of the endothelium, pre-incubation with L-NOARG, indomethacin, W-7 or calmidazolium and in a Ca2+-free medium. CONCLUSIONS: This study demonstrates the involvement of the endothelial constitutive Ca2+-calmodulin dependent nitric oxide synthase isoform in the overproduction of nitric oxide in portal hypertension.     

The Trypanosoma cruzi ribosomal RNA-encoding gene: analysis of promoter and upstream intergenic spacer sequences

The present study was designed to investigate the influence of endothelium-derived nitric oxide on the contractile responses of isolated human omental arteries to electrical field stimulation and noradrenaline. We measured isometric tension in artery rings obtained from portions of human omentum during the course of abdominal operations (32 patients). Electrical field stimulation induced frequency-dependent contractions which were abolished by tetrodotoxin (10(-6) M) and prazosin (10(-6) M), thus indicating that this effect was due to noradrenaline released from adrenergic nerves acting on alpha 1-adrenoceptors. The increases in tension induced by electrical field stimulation were of greater magnitude in arteries denuded of endothelium. NG-Nitro-L-arginine (L-NAME, 10(-4) M) potentiated the contractile response to electrical field stimulation in artery rings with endothelium but did not influence the contractile responses of endothelium-denuded arteries. The potentiation induced by L-NAME was completely reversed by L-arginine (10(-4) M), but not by D-arginine (10(-4) M). Contractile responses to noradrenaline were similar in arteries with and without endothelium. L-NAME (10(-4) M) had no significant effect on the contractile responses to noradrenaline. Our results suggest that electrical field stimulation releases endothelium-derived nitric oxide which inhibits the contractile responses of human omental arteries. The constrictor responses to noradrenaline are not modulated by the endothelium.     

Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors

1. The role of endothelial factors and potassium channels in the action of the pineal hormone melatonin to potentiate vasoconstrictor responses was investigated in the isolated perfused tail artery of the rat. 2. Melatonin (100 nM) potentiated contractile responses to both adrenergic nerve stimulation and alpha1-adrenoceptor stimulation by phenylephrine. After removal of the endothelium, melatonin no longer caused potentiation. 3. The potentiating effect of melatonin was also lost when nitric oxide synthase was inhibited with L-NAME (10 nM). Thus potentiating effects depend on the presence of nitric oxide released by the endothelium. However, melatonin did not affect relaxation responses to acetylcholine in endothelium-intact arteries, nor did melatonin modulate relaxing responses to sodium nitroprusside in endothelium-denuded arteries. While melatonin does not appear to modulate agonist-induced release of nitric oxide nor its effect, melatonin may modulate nitric oxide production induced by flow and shear stress. 4. When the Ca2+-activated K+ channel opener, NS 1619 (10 microM), was present, potentiating effects of melatonin were restored in endothelium-denuded vessels. However, addition of the opener of ATP-sensitive K+ channels, cromakalim (3 microM), did not have the same restorative effect. Furthermore, addition of a blocker of Ca2+-activated K+ channels, tetraethylammonium (1 mM), significantly attenuated potentiating effects of melatonin. These findings support the hypothesis that melatonin inhibits the activity of large conductance Ca2+-activated K+ channels to produce its potentiating effects. 5. Thus in the rat perfused tail artery, potentiation of constriction by melatonin depends on the activity of both endothelial factors and Ca2+-activated K+ channels. Our findings suggest that melatonin inhibits endothelial K+ channels to decrease flow-induced release of nitric oxide as well as block smooth muscle K+ channels to enhance vascular tone.     

Intraspinal injection of adenosine agonists protect against L-NAME induced neuronal loss in the rat

Intraspinal injection of the nonspecific inhibitor of nitric oxide synthase N-nitro-L-arginine methyl ester (L-NAME) results in a dose-dependent loss of neurons in the rat spinal cord. This effect is thought to result from a reduction in basal levels of nitric oxide (NO), thereby producing an ischemic reaction secondary to vasoconstriction and reduced spinal cord blood flow (SCBF). An important component of this ischemic reaction is the release of excitatory amino acids and the initiation of an excitotoxic cascade. In the present study, microinjections of adenosine A1 and A2 receptor agonists were made in the spinal cord to evaluate the neuroprotective effects of these drugs against neuronal loss produced by L-NAME. Animals were divided into six groups based on the composition of injected solutions: (a) L-NAME; (b) L-NAME + N6-cyclopentyladenosine (CPA, A1 agonist); (c) L-NAME + 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA, A2 agonist); (d) L-NAME + CPA + CPCA; (e) N-methyl D-aspartate (NMDA); and (f) NMDA + CPA. Injections of L-NAME or NMDA produced a unilateral loss of spinal neurons, a local inflammatory response, and darkly stained pyknotic nuclei surrounding the area of neuronal loss. CPA and CPCA significantly reduced the area of L-NAME-induced neuronal loss, and a synergistic effect was observed when ineffective doses of these agonists were co-injected with L-NAME. The excitotoxic effects of NMDA were not affected by CPA. The results have shown that A1 and A2 receptor agonists provide significant neuroprotection against L-NAME induced neuronal loss, presumably by inhibiting ischemia induced release of excitatory amino acids (A1 agonist), or by restoring SCBF secondary to vasodilation (A2 agonist). It is suggested by these results that the intraspinal injection of L-NAME is an effective model to study the pathological consequences of vasoconstriction, reduced SCBF, and ischemia secondary to decreased NO production in the rat spinal cord. Finally, the results provide support for the continued investigation of specific adenosine agonists as therapeutic agents directed against the ischemic and excitotoxic components of spinal injury.     

Further investigations into adenosine A1 receptor-mediated contraction in rat colonic muscularis mucosae and its augmentation by certain alkylxanthine antagonists

1. The alkylxanthine antagonists, 8-phenyltheophylline (8-PT), 8-p-sulphophenyltheophylline (8-SPT) and 1,3,7-trimethylxanthine (caffeine) produced rightward displacements of contractile concentration-effect curves to 5'-N-ethylcarboxamidoadenosine (NECA) in rat isolated colonic muscularis mucosae (RCMM) with concentration-ratios consistent with adenosine receptor blockade. The non-xanthine antagonist, 9 fluro-2-(2-furyl)-5,6-dihydro [1,2,4] triazo to [1,5-c]-quinazin-imine (CGS15943A) also antagonized contractions to NECA with an affinity (pKB8.1-8.5) consistent with adenosine A1 receptor blockade. 2. In addition to producing rightward shifts of the concentration-response curves, the maximum contractions to 5'-N-ethylcarboxamidoadenosine (NECA) were also markedly increased in the presence of 8-PT (by 83 +/- 16% at 1 microM), 8-SPT (by 37 +/- 7% at 10 microM) and caffeine (by 45 +/- 5% at 100 microM) but were unaffected by CGS15943A (at 0.01 and 0.03 microM). 3. As with NECA, the maximum contractions to the adenosine A1 receptor agonists R-phenylisopropyladenosine (R-PIA) and N-[(1S, trans)-2-hydroxyclopentyl] adenosine (GR79236) were both antagonized and augmented by 8-PT. In addition, the contractions to NECA in the presence of 8-PT (1 microM) were inhibited by nanomolar concentrations of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 4. The non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (1 microM) produced a marked increase in the NECA maximum without producing a rightward shift in the NECA curve, whereas a higher concentration (10 microM) virtually abolished responses. The PDE type III inhibitor,milrinone (1 microM), the type IV inhibitor, rolipram (10 microM), and the type V PDE inhibitor, zaprinast(3 microM), were all without effect on NECA responses in RCMM.5. Partial inhibitions of contractions to NECA were produced by indomethacin (at 3 or 10 micro M) or piroxicam (at 3 microM). Responses to GR79236 were also partially inhibited by indomethacin. In the presence of indomethacin, 8-PT was still able to enhance markedly the maximum contractions obtained to NECA in RCMM.6. The present study has shown that certain alkylxanthine antagonists (but not the non-xanthineCGS15943A) produced a marked augmentation of adenosine Al receptor-mediated contractions inRCMM. The mechanism of this augmentation is, as yet, not known but is unlikely to result from inhibition of PDE. This study has also shown that adenosine Al receptor-induced contractions inRCMM are mediated, in part, via products of the cyclo-oxygenase pathway.     

Impaired endothelium-dependent relaxation after coronary reperfusion injury: evidence for G-protein dysfunction

This study was done to determine whether abnormal receptor-dependent release of endothelium-derived relaxing factor (EDRF) might be caused by G-protein dysfunction. Dogs were exposed to global myocardial ischemia (45 minutes, induced by aortic cross-clamping) followed by reperfusion (60 minutes) while on cardiopulmonary bypass, and coronary arteries were then studied in vitro in organ chamber experiments. After reperfusion, endothelium-dependent relaxation to the receptor-dependent agonists adenosine diphosphate and acetyl-choline was significantly impaired as well as to sodium fluoride, which acts on a pertussis toxin-sensitive G-protein. In contrast, endothelium-dependent relaxations to the receptor-independent agonists A23187 and phospholipase C were normal. Furthermore, endothelium-dependent relaxation to poly-L-arginine (molecular weight, 139,200), which appears to induce endothelium-dependent relaxation of the canine coronary artery by a nonnitric oxide pathway, was unaffected by ischemia and reperfusion. These experiments suggest that global myocardial ischemia and reperfusion selectively impair receptor-mediated release of EDRF (nitric oxide) but that the ability of the endothelial cell to produce EDRF or generate endothelium-dependent relaxation to nonnitric oxide-dependent agonists remains intact. We hypothesize that coronary reperfusion injury leads to G-protein dysfunction in the endothelium.     

Influence of endothelial nitric oxide on adrenergic contractile responses of human cerebral arteries

The present study was designed to investigate the influence of the endothelium and that of the L-arginine pathway on the contractile responses of isolated human cerebral arteries to electrical field stimulation (EFS) and norepinephrine. Rings of human middle cerebral artery were obtained during autopsy of 19 patients who had died 3-8 h before. EFS (1-8 Hz) induced frequency-dependent contractions that were abolished by tetrodotoxin, prazosin, and guanethidine (all at 10(-6) M). The increases in tension were of greater magnitude in arteries denuded of endothelium. N(G)-monomethyl L-arginine (L-NMMA 10(-4) M) potentiated the contractile response to EFS in artery rings with endothelium but did not influence responses of endothelium-denuded arteries. L-arginine (10(-4) M) reversed the potentiating effects of L-NMMA on EFS-induced contractions. Norepinephrine induced concentration-dependent contractions, which were similar in arteries with and without endothelium or in arteries treated with L-NMMA. Indomethacin (3 x 10(-6) M) had no significant effect on the contractile response to EFS or on the inhibition by L-NMMA of acetylcholine-induced relaxation. These results suggest that the contractile response of human cerebral arteries to EFS is modulated by nitric oxide mainly derived from endothelial cells; although adrenergic nerves appear to be responsible for the contraction, the transmitter involved in the release of nitric oxide does not appear to be norepinephrine. The effects of L-NMMA in this preparation appear to be due to inhibition of nitric oxide formation rather than caused by cyclooxygenase activation.