Simultaneous measurement of intracellular calcium and tension in vascular smooth muscle: validation of the everted ring preparation.

This study details and validates a method that facilitates the eversion of vascular smooth muscle, a preparation employed in in vitro Ca2+ fluorometric assays. Vascular segments of porcine coronary artery, approximately 2 cm in length, were sutured to portions of polyethylene tubing inserted into the lumen of the vessel. After being secured and stabilized by the tubing, the vessel was easily everted while immersed in physiological buffer. Intracellular calcium concentrations ([Ca2+]i) and tension were measured simultaneously in everted rings denuded of the endothelium. In these preparations, increases in tension generated by KCl and prostaglandin F2 alpha (PGF2 alpha) were accompanied by increases in [Ca2+]i, as measured by fura-2 fluorescence. Isoproterenol (ISO) and sodium nitroprusside (SNP) elicited reductions in muscle tension as well as [Ca2+]i in both KCl- and PGF2 alpha-contracted rings. Comparison of the responsiveness of everted and uneverted coronary artery rings demonstrated that, while fura-2 fluorescence in uneverted rings was negligible, the magnitudes of contraction of both preparations to KCl or PGF2 alpha were similar. The relaxant responses to ISO and SNP were also similar in the everted and uneverted rings contracted with KCl or PGF2 alpha. The data suggest that the procedure employed in everting vascular segments maintains the integrity of the smooth muscle, thus making it a suitable model for the simultaneous measurement of [Ca2+]i and tension.

Evidence against KATP channel involvement in adenosine receptor-mediated dilation of epicardial vessels.

The purpose of this study was to determine whether ATP-glyburide-sensitive K+ (KATP-glyburide) channels are involved in the adenosine-induced vasorelaxation of porcine and canine epicardial vessels in vitro. Adenosine and its analogues, 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), R-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclopentyladenosine (CPA), N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)]adenosine (DPMA), 2-phenylaminoadenosine (CV-1808), 2-[m-(carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS-22988), 2-[(2-cyclohexylethyl)amino]adenosine (CGS-22492), 2-[(p-amino)phenethylamino]adenosine (APE), and 2-(1-octynyl)adenosine (YT-146) (10 nM-100 microM), produced concentration-dependent relaxations in endothelium-intact and -denuded arterial ring segments contracted with 30 mM KCl, 10 nM endothelin-1, or 10 microM prostaglandin F2 alpha. Sodium nitroprusside (SNP; 1 nM-10 microM) and KATP-channel activator, pinacidil (10 nM-10 microM), also produced similar vasodilatory responses. Glyburide, a KATP-channel blocker, caused a rightward shift of the concentration-response curve to pinacidil but did not alter the responses elicited by SNP or adenosine and its analogues. The data suggest that KATP-glyburide channels are not involved in the mechanism whereby adenosine and its analogues elicit their vasorelaxant response in isolated porcine or canine epicardial vessels.

Adenosine receptor-mediated relaxation of porcine coronary artery in presence and absence of endothelium.

This study was designed to investigate the effects of a series of adenosine analogues on porcine coronary artery in vitro. In both endothelium-intact and -denuded rings, 5'-(N-ethylcarboxamido) adenosine (NECA), 2-[p-(2-carboxyethyl)]phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), 2-chloroadenosine (CAD), N6-R-phenylisopropyladenosine (R-PIA), 2-phenylaminoadenosine (PAA), N6-cyclohexyladenosine (CHA), N6-cyclopentyladenosine (CPA), and N6-S-phenylisopropyladenosine (S-PIA) produced concentration-dependent relaxations. The rank order of potency was consistent with A2-adenosine receptor identification. The xanthine adenosine antagonist, 8-(sulfophenyl) theophylline (8-SPT), attenuated the relaxant responses to all the agonists in the endothelium-intact rings and to only CAD, R-PIA, PAA, CHA, CPA, and S-PIA in the denuded preparations. Except for NECA and CGS-21680, the slopes of the relaxation curves and the dissociation constant (Kb) values for 8-SPT were similar for all agonists. In addition, endothelium removal selectively reduced the responses to NECA and CGS-21680. The adenosine receptor agonist, CGS-22988, also relaxed the denuded rings in a manner insensitive to blockade by 8-SPT. The data suggest that multiple A2-adenosine receptors exist on the smooth muscle and endothelium of porcine coronary artery, mediating relaxation. Whereas the smooth muscle contains both xanthine-sensitive and -insensitive A2-receptors, which can be activated by a wide range of adenosine agonists, the endothelium possesses xanthine-sensitive receptors that can be stimulated selectively by certain adenosine agonists, including 5'-uronamide derivatives, such as NECA and CGS-21680. The smooth muscle also appears to contain xanthine-insensitive A4-receptors activated by CGS-22988.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity relationship of 2-(ar) alkoxyadenosines at the adenosine A2 receptor in coronary artery.

We examined the ability of four 2-(ar)alkoxyadenosines (2-(2-phenylethoxy)adenosine, PEA; 2-[2-(2-naphthyl)ethoxy]adenosine, NEA; 2-[2-(4-methylphenyl)ethoxy]adenosine, mPEA; 2-(1-hexyloxy)adenosine, HOA) to relax porcine coronary artery in vitro. All four compounds produced concentration-dependent relaxations in rings contracted with 30 mM KCl. The EC25 values are as follows (x 10(-9) mol/l): CGS21680, (2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosi ne) (32.7) approximately NECA, 5'-N-ethylcarboxamidoadenosine (51.4) approximately mPEA (74.3) approximately NEA (160.7) > HOA (855.1) approximately PEA (1259) approximately 2-chloroadenosine (1871) > adenosine (9705). However, EC75 values for all the compounds except adenosine and 2-chloroadenosine converged to a range of 8.16 to 22.86 microM, suggesting a biphasic response. Furthermore, the responses were found to be independent of endothelial integrity. The unselective adenosine receptor antagonist 8-p-sulphophenyltheophylline (100 microM) attenuated the relaxant response to NEA (EC25 = 1172 nM), suggesting that adenosine receptors mediated relaxation. Structure-activity correlations suggest that the adenosine A2 receptor in porcine coronary artery contains a region of limited bulk tolerance juxtaposed to the region occupied by adenine C-2 and distal to that a large hydrophobic region.

Adenosine-5'-uronamides rapidly desensitize the adenosine A2 receptor in coronary artery.

This study examined the structure-activity-relationship (SAR) of adenosine analogs and their ability to induce tachyphylaxis in vascular smooth muscle. Adenosine-5'-uronamides,5'-N-ethylcarboxamidoadenosine (NECA), 5'-N-cyclopropylcarboxamidoadenosine (CPCA), and 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680), evidenced rapid desensitization of the A2 vasorelaxant response in porcine coronary artery in vitro whereas adenosine, 2-chloroadenosine (CAD), or 2-[(2-cyclohexylethyl)-amino]adenosine (CGS 22492) failed to do so. Tissues with prior exposure to NECA exhibited mitigated relaxation responses to adenosine, CAD, and NECA but not to isoproterenol, forskolin, pinacidil, or sodium nitroprusside (SNP). The data suggest that adenosine-5'-uronamides homologously desensitize the A2 receptor in porcine coronary artery smooth muscle.

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.

Protein kinase C and phospholipase C in adenosine receptor-mediated relaxation in coronary artery.

The effect of adenosine, 2-chloroadenosine (CAD), and 5'-(N-ethylcarboxamido)-adenosine (NECA) on the contraction produced by phorbol 12,13-dibutyrate (PDB) was investigated in porcine coronary artery in vitro to determine whether adenosine receptor-mediated relaxation was linked to protein kinase C. Also, the coronary relaxation produced by adenosine and NECA in KCl-contracted coronary rings was investigated before and after treatment with the phospholipase C inhibitor neomycin to examine a possible link between phospholipase C and adenosine receptor-mediated relaxation. Ring segments of coronary artery were suspended in organ baths for measurement of isometric force. PDB (10 nM-1 microM) caused concentration-dependent contraction, and this response was significantly attenuated by pretreatment with the protein kinase C inhibitor staurosporine (200 nM) but not 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (10 microM). Treatment of rings with either adenosine, CAD, or NECA (100 microM) significantly attenuated the PDB-induced contraction, whereas treatment with either sodium nitroprusside (SNP; 1 microM) or isoproterenol (Isop; 1 microM) did not affect the contraction produced by PDB. The attenuation of the PDB-induced contraction by adenosine and its analogues was blocked by prior treatment of the coronary rings with 8-phenyltheophylline (10 microM). In a separate series of experiments, pretreatment of rings with the phospholipase C inhibitor neomycin (1 mM) resulted in a significant attenuation of the relaxing response to both adenosine and NECA while having no significant effect on the relaxation-response to SNP or Isop. These results provide indirect evidence that adenosine receptor-mediated relaxation in porcine coronary artery may be linked to modulation of protein kinase C and phospholipase C.

Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.

This study was designed to evaluate whether the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) would attenuate the relaxation produced by adenosine analogs in order to provide functional evidence in support of the working hypothesis that adenosine receptor-mediated relaxation of coronary artery involves adenylate cyclase. Rings from porcine left anterior descending coronary artery were mounted in organ chambers for measurement of isometric force. Rings contracted with KCl (30 mM) relaxed in a concentration-dependent manner to 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), isoproterenol, sodium nitroprusside (SNP) and forskolin. Treatment of coronary rings with DDA (50 microM) significantly attenuated the relaxation produced by CAD, NECA, forskolin and isoproterenol, but had no effect on the relaxation response to SNP. The nucleoside transport inhibitor dilazep (10 microM) completely reversed the inhibitory effect of DDA on the relaxation produced by forskolin and CAD, whereas dilazep only partially reversed the DDA inhibition of NECA-induced relaxation. In a membrane preparation from porcine coronary artery CAD, but not NECA, increased cyclic AMP production in a GTP-dependent manner. DDA significantly decreased basal cyclic AMP production and also decreased CAD-, forskolin-, GTP- and NaF-stimulated cyclic AMP production. These results provide functional and biochemical evidence in support of the working hypothesis that adenosine receptor-mediated coronary relaxation involves adenylate cyclase. Furthermore, the results from this study suggest that the signaling mechanisms responsible for adenosine receptor-mediated coronary relaxation are more complicated than a single receptor coupled with adenylate cyclase because 1) dilazep completely reversed the inhibitory effect of DDA on the CAD relaxation but not the NECA relaxation, and 2) NECA did not increase cyclic AMP production.