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We examined the effect of diphenylamine-2-carboxylic acid (DPC), which has been shown to inhibit the efflux of cyclic nucleotides from vascular smooth-muscle cells, on the relaxant responses to forskolin, an adenylyl cyclase activator, and sodium nitroprusside (SNP), an NO donor, in the porcine coronary arteries. DPC (100 microM), which caused only a minor effect by itself, significantly augmented the relaxant responses to forskolin and SNP in the preparations contracted with 30 mM KCl. On the other hand, DPC did not affect the relaxant responses to nifedipine and cromakalim. Forskolin (10 microM) induced an accumulation of adenosine 3', 5'-cyclic monophosphate (cAMP) in the porcine coronary arteries, which was associated with an accumulation of cAMP in the incubation media. The intracellular cAMP response to forskolin was enhanced by DPC, whereas the extracellular cAMP response was reduced. The effects of SNP on guanosine 3', 5'-cyclic monophosphate (cGMP) accumulation were examined in the presence of 3-isobutyl-l-methylxanthine (500 microM) because cGMP was not found in the tissue and the incubation medium in the absence of the phosphodiesterase inhibitor. DPC significantly decreased the SNP-induced release of cGMP to the extracellular space, whereas it did not affect the accumulation of cGMP in the tissue. These results suggest that DPC inhibits the efflux of cyclic nucleotides. It is likely that the inhibitory effect of DPC on cAMP efflux contributes to the enhancement of tissue cAMP accumulation and relaxation produced by the agents that activate adenylyl cyclase. Thus, the transport system(s) of cyclic nucleotides may be a novel target for the prevention and/or treatment of various cardiovascular diseases.

We examined how lidocaine affects muscarinic receptor-mediated inhibition of adenylyl cyclase in bovine tracheal smooth muscles. Lidocaine (100 microM) augmented the relaxant responses to forskolin in the bovine tracheal smooth muscle contracted with methacholine (0.3 microM). On the other hand, lidocaine failed to affect the relaxant effects of forskolin on the histamine (100 microM)- and KCl (40 mM)-contracted preparations. Lidocaine (100 microM) enhanced both basal and forskolin-stimulated cAMP accumulation in the presence of methacholine (0.3 microM). However, in the absence of methacholine, neither basal nor forskolin-stimulated cAMP accumulation was affected by lidocaine. Similar phenomenon was observed when the bovine tracheal smooth muscles were treated with methoctramine (0.03 microM). In radioligand binding experiments, lidocaine inhibited [3H]N-methyl scopolamine binding to cloned human muscarinic receptors (M(1)-M(5)) expressed in Chinese hamster ovary cells. These results suggest that lidocaine prevents muscarinic receptor-mediated signaling pathway and thereby reverses inhibition of adenylyl cyclase by methacholine in bovine tracheal smooth muscle.

We examined how diabetes affects the beta-adrenoceptor subtypes mediating relaxation of rat urinary bladder smooth muscle contracted with carbachol. The relaxant responses to isoproterenol were larger in muscles from rats 8 to 10 weeks after induction of diabetes with streptozotocin (80 mg/kg, i.p.) as compared to the control muscles. In contrast, forskolin-induced relaxations did not differ significantly in the control and diabetes groups. Propranolol (1 microM) abolished the diabetes-induced augmentation of relaxant responses to isoproterenol. The relaxant responses to T-0509 ((-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)-amino]ethanol hydrochloride), a beta(1)-adrenoceptor agonist, were small but significantly augmented by diabetes. On the other hand, diabetes did not change the relaxations produced by clenbuterol, a beta(2)-adrenoceptor agonist, and BRL37344 ((+/-)-(R*,R*)-(4-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]phenoxy)acetic acid), a beta(3)-adrenoceptor agonist. These results suggest that diabetes selectively augments the beta(1)-adrenoceptor-mediated relaxation of the rat urinary bladder smooth muscle.

Guanosine 3',5'-cyclic monophosphate (cGMP) has an important role in regulating vascular smooth muscle tone. We examined whether mRNA for multidrug resistance protein (MRP) 4 and MRP5, which were recently identified as ATP-dependent export pumps for cyclic nucleotides, is expressed in the porcine coronary and pulmonary arteries. The results showed that both arteries express mRNA for MRP4 and MRP5, and thus these proteins may be novel targets for the prevention and/or treatment of various cardiovascular diseases.