We examined the inhibitory mechanism of BRL37344, a beta-adrenoceptor agonist that is considered to be specific to beta(3)-subtype, on muscarinic receptor-mediated contraction of the rat urinary bladder smooth muscle. BRL37344 produced apparently biphasic concentration-relaxation curves in the urinary bladder smooth muscle contracted with carbachol (0.6 microM). The first and second phases had estimated p D(2) (-logEC(50)) values of 7.80+/-0.34 and 4.62+/-0.18, respectively ( n=6). The first component of the BRL37344 concentration-response curve was not affected by propranolol (1 microM), whereas it was inhibited by higher concentrations of the drug (10 microM or 30 microM). The second component was completely resistant to propranolol. On the other hand, BRL37344 produced monophasic concentration-relaxation of 30 mM KCl-precontracted urinary bladder smooth muscle with a p D(2) value of 8.34+/-0.18 ( n=6). Pretreatment of the urinary bladder smooth muscles with BRL37344 (30, 100 and 300 microM) significantly ( P<0.05) shifted the concentration-response curves for carbachol-induced contractions. In radioligand binding experiments, BRL37344 concentration-dependently displaced the specific binding of [(3)H] N-methyl scopolamine to muscarinic receptors on rat urinary bladder smooth muscle membranes. Additionally, BRL37344 inhibited [(3)H] N-methyl scopolamine binding to cloned human muscarinic receptors (M(1)-M(5)) expressed in Chinese hamster ovary cells. These results suggest that BRL37344 attenuates muscarinic receptor-mediated contractions through prevention of the agonists binding to their receptors, in addition to stimulation of beta(3)-adrenoceptors, in rat urinary bladder.

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The effects of ibudilast, an inhibitor of phosphodiesterases (PDEs), on tension, levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) were investigated in bovine tracheal smooth muscle. We especially examined the combined effect of ibudilast with the cGMP-elevating agents on these parameters. Ibudilast was equipotent to attenuate the precontractions induced by both 0.3 microM methacholine and 40 mM K(+). By contrast, the relaxant effects of sodium nitroprusside and salbutamol on 40 mM K(+)-contracted preparations were smaller than those on 0.3 microM methacholine-contracted ones. Neither N(omega)-nitro-L-arginine (100 microM), an inhibitor of nitric oxide synthase, nor ODQ (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 5 microM), an inhibitor of soluble guanylyl cyclase, affected the ibudilast-induced relaxation. The relaxations induced by ibudilast and diltiazem on 40 mM K(+)-contracted preparations were significantly attenuated when extracellular CaCl(2) was increased from 2.54 mM to 10 mM. Ibudilast (10 microM), which caused only minor effect by itself, significantly ( P<0.05) shifted the concentration-response curves for the relaxant responses to sodium nitroprusside (SNP), atrial natriuretic peptide (ANP) and salbutamol to the left. On the other hand, ibudilast did not change the relaxant responses to diltiazem. Unlike ibudilast, diltiazem (3 microM) failed to affect the SNP- and salbutamol-induced relaxations. Ibudilast significantly ( P<0.05) increased basal levels of cGMP and cAMP. Furthermore, ibudilast enhanced SNP (0.3 microM)- and ANP (0.3 microM)-induced cGMP accumulation and salbutamol (10 nM)-induced cAMP accumulation. Zaprinast (10 microM), a type 5 PDE inhibitor, enhanced both relaxation and cGMP accumulation induced by SNP and ANP without changing salbutamol-induced responses. These findings suggest that blockade of voltage-gated Ca(2+) channels is involved in the relaxing action of ibudilast in bovine tracheal smooth muscle. However, ibudilast potentiates relaxation responses to ANP and SNP by inhibition of PDE 5, not by blockade of Ca(2+) channels. The enhancement of cGMP-mediated response may contribute to the therapeutic effects of ibudilast.