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Transient receptor potential-vanilloid type-1 (TRPV1) is a ligand-gated cation channel with preference for divalent cations, especially Ca(2+) (sequence of conductances: Ca(2+)>Mg(2+)>Na(+) approximately/= K(+) approximately/= Cs(+)). In the present study, the two-electrode voltage-clamp technique was used on oocytes of Xenopus laevis expressing TRPV1 to evaluate whether human TRPV1 also conducts protons. In medium devoid of K(+), Na(+), Mg(2+), and Ca(2+), capsaicin 1 microM induced a significant inward current (62% of the current in physiological medium). The effects of capsaicin were abolished in the presence of capsazepine 3 microM. The capsaicin-induced currents in medium devoid of Na(+), K(+), Mg(2+), and Ca(2+) were dependent on pH, causing larger inward currents and less negative reversal potentials at low pH and vice versa. The same current was also demonstrated in Chinese hamster ovary cells expressing human TRPV1. We conclude that TRPV1 conducts protons, in addition to Na(+), K(+), Mg(2+), and Ca(2+). The proton conductance may help to initiate action potentials and to translocate H(+) dependent on TRPV1 activation and membrane potential.

Heat transduction mechanisms in primary nociceptive afferents have been suggested to involve a vanilloid receptor channel with high calcium permeability. To characterize the changes in free cytosolic calcium evoked by noxious heat stimuli (< or =51 degrees C, 10s), we performed microfluorometric measurements in acutely dissociated small dorsal root ganglion neurons (< or =32.5 microm) of adult rats using the dye FURA-2. Only neurons that responded with a reversible increase in intracellular calcium to high potassium were evaluated. Heat-induced calcium transients (exceeding mean + 3S.D. of the temperature dependence of the dye) were found in 66 of 105 neurons. These transients increased non-linearly with temperature. In contrast, heat-insensitive neurons showed a small linear increase of intracellular calcium throughout the range of 12-49 degrees C, similar to cardiac muscle cells. The vanilloid receptor agonist capsaicin induced calcium transients in 72 of 99 neurons. Capsaicin sensitivity and heat sensitivity were significantly associated (P<0.001, chi(2)-test), but 16 of 34 heat-insensitive cells responded to capsaicin and four of 49 heat-sensitive cells were capsaicin insensitive. The competitive vanilloid receptor antagonist capsazepine (10 microM) reversibly reduced the heat-induced calcium transients by 47+/-13%. In contrast, high potassium-induced calcium transients were not affected by pre-incubation with capsazepine. In calcium-free extracellular solution, the heat-induced rise in intracellular calcium was reduced by 76+/-5%. Heat-induced calcium transients were also reversibly reduced by 75+/-6% in sodium-free solution and by 62+/-7% with the L-type calcium channel blocker nifedipine (5 microM). These results indicate that noxious heat rapidly increases intracellular calcium in nociceptive primary sensory neurons. Heat-sensitive vanilloid receptors are involved in the induction of calcium transients, and calcium is also released from intracellular stores, but the main fraction of calcium passes through voltage-operated calcium channels.

The effects of a nitric oxide-donor, S-nitroso-N-acetylpenicillamine, and a direct activator of soluble guanylyl cyclase, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), on force of contraction (F(c)) and L-type Ca(2+) currents (I(Ca(L))) were investigated in myocardial preparations from neonatal and adult rats. Since hearts from adult and neonatal animals contained 160 and 47 mg/100 g wet weight myoglobin, respectively, its possible interaction with both drugs was also investigated. Both S-nitroso-N-acetylpenicillamine (100 microM) and YC-1 (30 microM) were ineffective in myocardial preparations from adult rats but reduced the magnitude of I(Ca(L)) and F(c) in preparations from neonatal rats. The latter effects were antagonised by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 50 microM) and attenuated by myoglobin (30-300 microM), which also attenuated the effects of both drugs on pre-contracted aortic rings. The differential effects of S-nitroso-N-acetylpenicillamine and YC-1 in the myocardium from adult and neonatal rats may result from developmental changes in the content of myoglobin and/or in the NO/soluble guanylyl cyclase signal pathway.

This study addresses the question whether K(+) channels are involved in the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1 ). In rat aorta, guinea pig aorta, and guinea pig a. carotis, YC-1 inhibited contractions induced by phenylephrine (3 microM) more potently than those induced by K(+)(48 mM). In rat aorta, tetraethylammonium (10 mM), charybdotoxin (0.2 microM), and iberiotoxin (0.1 microM), but not glibenclamide (10 microM), attenuated the relaxant effects of YC-1. In guinea pig a. carotis, YC-1 (30 microM) induced a hyperpolarisation which was antagonised by 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 50 microM). In rat aorta, YC-1 (30 microM) increased the rate constant of 86Rb-efflux. The effect of YC-1 was potentiated by zaprinast (10 microM), but inhibited by ODQ (50 microM) or charybdotoxin (0.2 microM). In smooth muscle cells from rat aorta, YC-1 (10 microM) increased BK(Ca) channel activity. It is suggested that YC-1-induced vasorelaxation is partially mediated by the activation of K(+) channels.

The effects of isoliquiritigenin on force of contraction (Fc), L-type Ca2+ current (I(Ca)) and intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat ventricular heart muscle. Isoliquiritigenin increased Fc and I(Ca) and, after longer exposure times, resting tension and [Ca2+]i. The effect of isoliquiritigenin (100 microM) on I(Ca) was diminished by Rp-cAMPS (30 microM). 1H-[1,2,4]oxa- diazolo[4,3-a]quinoxalin-1-one (50 microM) did not influence the effects of isoliquiritigenin on Fc and I(Ca). The positive inotropic effects of isoprenaline and forskolin, but not of 3-isobutyl-1-methylxanthine, were potentiated by isoliquiritigenin (100 microM). In the presence of milrinone (10 microM), no further effects of isoliquiritigenin (100 microM) on Fc and I(Ca) were observed. It is suggested that the increase in Fc and I(Ca) by isoliquiritigenin is due to an accumulation of cyclic AMP. These effects are probably unrelated to an effect of the drug on soluble guanylyl cyclase, as reported for smooth muscle, but rather due to a direct inhibition of phosphodiesterase III activity.

We investigated the effects of isoliquiritigenin and YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole) on tension in endothelial-free rat aortic rings precontracted with phenylephrine (3 microM). Both compounds induced a concentration-dependent relaxation (EC50 of YC-1 1.9 microM and of isoliquiritigenin 9.4 microM). The effects developed faster with YC-1 than with isoliquiritigenin, and the effects of YC-1 were potentiated by isoliquiritigenin (10 microM). 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (30 microM) inhibited the effect of YC-1, but not of isoliquiritigenin. These results suggest that the effects of YC-1 are due to stimulation of soluble guanylyl cyclase activity, whereas the effects of isoliquiritigenin are rather related to inhibition of phosphodiesterase activity.

The effect of verapamil on L-type Ca2+ current (ICa) was compared at external pH 7.4 and 8.5 in rat ventricular myocytes. Alkalinisation increases the fraction of uncharged molecules of verapamil (pK 8.75) and thereby facilitates membrane permeation of the drug. Verapamil (1 microM) reduced the amplitude of ICa (ICa(peak)) by 36 +/- 4% at pH 7.4 and by 40 +/- 6% at pH 8.5, whereas alkalinisation from pH 7.4 to 8.5, without drug, increased ICa(peak) by 12 +/- 3%. It is suggested that the efficiency of verapamil is not influenced by the amount of protonation or membrane permeation.

Several activators of cGMP-dependent protein kinase (protein kinase G) such as 8-Br-cGMP reduced force of contraction in rat left atria. Inhibitors of protein kinase G antagonized the negative inotropic effect of 8-Br-cGMP but not of acetylcholine in atria. However, the acetylcholine-induced relaxation in aortic rings was significantly inhibited by protein kinase G inhibition. It is concluded that the reduction by 8-Br-cGMP of force of contraction in atria is related to activation of protein kinase G. In response to acetylcholine, activation of protein kinase G is probably a major step in smooth muscle relaxation but is not involved in the reduction of force of contraction in atria.

The effects of cromakalim on spontaneous and induced mechanical activity of human detrusor muscle were investigated in vitro. Cromakalim produces a concentration-related decrease of spontaneous as well as carbachol- and K(+)-evoked contractions. This is the first study to utilize the patch clamp technique to elucidate the mechanism of action of cromakalim on human detrusor cells. Cromakalim hyperpolarizes the detrusor cells by increasing the net outward current which is most likely carried by potassium ions. In the human urinary bladder, this effect is mediated by a glibenclamide-sensitive potassium channel, as glibenclamide is able to diminish the relaxant effect of cromakalim and to prevent the drug-induced hyperpolarization. Our results show that the effectiveness of cromakalim in relaxing the detrusor muscle is about an order of magnitude higher in the rat and guinea pig than in humans. It is therefore deemed unlikely that cromakalim or drugs with the same mode of action and similar tissue specificity will be of value in the clinical treatment of bladder instability and hyperreflexia. Moreover, it is suggested that when studies are undertaken to elucidate the effects and clinical potential of new drugs acting upon the bladder, human detrusor tissue should be used. The rat and guinea pig are unsuitable for this purpose.