Adenosine and selective A(2A) receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation.

To examine whether adenosine reduces ischemia/reperfusion (I/R)-induced liver injury by inhibiting leukocyte activation via A(2) receptor (A(2)R) stimulation, we investigated the effects of adenosine and selective A(2A) receptor (A(2A)R) agonists (YT-146 and CGS21680C) on I/R-induced liver injury in rats. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited neutrophil elastase release by about 30 to 40% and increased intracellular Ca(2+) concentrations in isolated neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited tumor necrosis factor (TNF)-alpha production by monocytes stimulated with endotoxin by about 50%. Although ZM241385, a selective A(2A)R antagonist, significantly enhanced the increase in neutrophil elastase release and intracellular Ca(2+) concentrations in neutrophils stimulated with fMLP, this agent did not affect the endotoxin-induced TNF-alpha production by monocytes. Rats were subjected to liver ischemia for 60 min. Serum levels of transaminases increased after hepatic I/R, peaking at 12 h after reperfusion. The i.v. infusion of adenosine (1 and 10 mg/kg/h), YT-146 (0.1 and 1 mg/kg/h), and CGS21680C (0.1 and 1 mg/kg/h) significantly inhibited the I/R-induced increase in serum transaminase levels 12 h after reperfusion. The I/R-induced decrease in hepatic tissue blood flow was significantly prevented by adenosine and YT-146. Hepatic levels of TNF-alpha, cytokine-induced neutrophil chemoattractant (equivalent to human interleukin-8), and myeloperoxidase were significantly increased after I/R. These increases were significantly inhibited by the administration of adenosine, YT-146, and CGS21680C. Although the histological neutrophil accumulation in the liver was significantly increased after I/R as evaluated by the naphthol AS-D chloroacetate technique, the administration of adenosine, YT-146, and CGS21680C significantly inhibited this increase. These findings suggest that adenosine reduces I/R-induced liver injury both by inhibiting the synthesis of inflammatory mediators and by inhibiting neutrophil degranulation directly, probably through A(2A)R stimulation.

Effects of SC-52458, a new nonpeptide angiotensin II receptor antagonist, on increase in cytoplasmic Ca2+ concentrations and contraction induced by angiotensin II and K(+)-depolarization in guinea-pig taenia coli.

1. The effects of angiotensin (Ang) II receptor antagonist, SC-52458, on peak and plateau components of Ang II-induced contraction were evaluated in the guinea-pig taenia coli. 2. SC-52458 suppressed both the components of and increases in cytoplasmic Ca2+ concentrations, [Ca2+]i, coupled with the contraction by Ang II; tetrodotoxin and atropine did not affect the contractions. 3. SC-52458 inhibited a plateau component of the contraction induced by K(+)-depolarization to some extent, without affecting a peak component. 4. SC-52458 suppressed both the contraction and increase of [Ca2+]i by antagonizing AT1 receptors in the smooth muscle.

Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and alpha, beta-methylene ATP in guinea-pig urinary bladder.

1. The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea-pig urinary bladder and compared with those evoked by ATP and alpha, beta-methylene ATP (a P2-purinoceptor agonist). 2. AP4A, ATP and alpha, beta-methylene ATP produced concentration-dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate-6-azophenyl- 2'-4'-disulphonic acid), 0.3- 30 microM, a P2x-purinoceptor antagonist, and suramin, 1-300 microM, a P2-purinoceptor antagonist in a concentration-dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and alpha, beta-methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3. AP4A (10 microM) and ATP (100 microM) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of alpha, beta-methylene ATP (1 microM). 4. In fluorescence experiments with fura-2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L-type Ca2+ channel blocker. 5. These findings suggest that P2x-purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea-pig urinary bladder, and that the AP4A-evoked contraction results from Ca2+ influx.

Discrimination by nimodipine, but not by nifedipine, between phasic and tonic contractions of guinea-pig taenia coli induced by K+ depolarization.

Effects of Ca2+ channel blockers, such as nifedipine, nimodipine, gallopamil, verapamil, diltiazem, loperamide, Mn2+ and Ni2+, and papaverine, on contractile responses to K+ depolarization were evaluated in longitudinal muscles of taenia coli isolated from guinea-pig. Depolarization with high K+ solution (K+, 40 mM) produced a biphasic (phasic and tonic) contraction, which was inhibited by the above blockers in a concentration-dependent manner. Ratios of IC50 for the phasic contraction to IC50 for the tonic contraction of nimodipine, verapamil, gallopamil, nifedipine, loperamide, diltiazem, papaverine, Ni2+, and Mn2+ were 516.1, 73.7, 22.0, 6.4, 5.3, 4.9, 1.2, 0.7, and 0.1, respectively, indicating that nimodipine suppressed the tonic contraction more effectively than the phasic contraction. In a fluorescence study with fura 2, K+ depolarization elicited an increase in intracellular free Ca2+, [Ca2+]i, which was coupled with the phasic and tonic contraction. The increases in [Ca2+]i coupled with both types of the contraction were abolished by exposure to Ca(2+)-free solution. In addition, the increase of [Ca2+]i coupled with the phasic contraction was abolished by nifedipine, 10(-7) M, but not by nimodipine, 10(-7) M, whereas the increase with the tonic contraction was suppressed by both nifedipine and nimodipine. These findings suggest that the phasic and tonic contractions evoked by K+ depolarization are due to increases in [Ca2+]i via activation of respective nimodipine-resistant and nimodipine-sensitive Ca2+ channels in the longitudinal muscles of the taenia coli. Accordingly, nimodipine, but not nifedipine, appears to be a useful tool for distinguishing between the phasic and tonic contractions.

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.

Inhibition by ouabain and veratridine of acetylcholine-evoked phasic contraction in the guinea-pig taenia coli.

Inhibitory action of ouabain and veratridine on acetylcholine (ACh)-evoked phasic contraction was examined in the guinea-pig taenia coli. ACh (5 x 10(-4)M) produced a biphasic (phasic and tonic) contraction. As the phasic contraction, but not the tonic contraction, was resistant to gallopamil (D600, 2 x 10(-7)M), a blocker of voltage-dependent Ca2+ channels, all experiments were carried out in the presence of gallopamil at this concentration. Addition of high Ca2+ (30 mM) to the solution containing ACh induced a sustained contraction, which increased in the presence of ouabain (10(-5)M), an inhibitor of the Na(+)-K+ exchange system. On the other hand, the ACh-evoked phasic contraction was suppressed by ouabain (10(-7)-10(-5) M) in a time- and dose-dependent manner. The suppression by ouabain (10(-6)M) of the phasic contraction was transiently potentiated by the addition of veratridine (10(-6)M), an activator of Na+ channel. In contrast, the greater suppression by ouabain (10(-5)M) of the contraction was antagonized by amiloride (10(-4)M), a blocker of Na+ channel. This antagonism by amiloride was transiently inhibited in the presence of veratridine. In the absence of ouabain, the amplitude of the phasic contraction was transiently reduced by adding veratridine but was increased by amiloride. In addition, the phasic contraction by ACh increased 80 min after exposure to Na(+)-free isotonic high-K+ solution (K+, 143 mM), which elicited greater depolarization of the cell membrane. In a fluorescence study with Fura-2, an intracellular free-Ca2+ indicator, ACh increased the fluorescence intensity from the tissue by excitative light at 340 nm, which coupled with the phasic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism by nifedipine of contraction and Ca2(+)-influx evoked by ATP in guinea-pig urinary bladder.

1. The effects of Ca2(+)-antagonists, especially nifedipine, on contraction and increase of intracellular Ca2+ (Fura-2/AM method) evoked by ATP were evaluated in a thin outer layer segment of guinea-pig urinary bladder. 2. The ATP-evoked contraction was markedly inhibited by dihydropyridine-type Ca2(+)-antagonists, such as nifedipine and nitrendipine, but not by D-600, omega-conotoxin and tetramethrin. 3. This antagonism by nifedipine of ATP-evoked contractions was competitive from the Schild plot analysis, the pA2 value being 8.23. The reduction of ATP-evoked contraction by nifedipine (0.1 microM) was fully reversed by administration of Bay K 8644 (0.1 microM). 4. ATP (100 microM) caused an increase of fluorescence brightness after loading Fura-2/AM, which was coupled with a contraction of the bladder. Both the contraction and the elevation of intracellular Ca2+ evoked evoked by the nucleotide were completely antagonized by nifedipine. by the nucleotide were completely antagonized by nifedipine. 5. These results suggest that ATP may activate the dihydropyridine-sensitive, voltage-dependent Ca2(+)-channels in a direct or indirect fashion and, thereby, elicit a contraction of the bladder.

A possible coupling of postjunctional ATP release and transmitters' receptor stimulation in smooth muscles.

The nature of ATP release from mainly smooth muscles of guinea-pig was evaluated with KCl and agonists for different kinds of receptors. In ileal longitudinal muscles, amounts of net ATP release by ACh and bethanechol (1-10 microM) were much larger (about 10 fold) than that by other drugs, e.g., histamine, 5-hydroxytryptamine, prostaglandin-F2 alpha, substance P and bradykinin, including KC1, although differences between contractions of the tissue evoked by test drugs were approximately 1.5 times at most. The ATP release, as well as the contraction, evoked by ACh or bethanechol was markedly reduced by atropine (0.3 microM), thus, indicating primarily postjunctional release of ATP. The remarkable ATP release from vas deferens by norepinephrine (NE), but not by substance P, was abolished almost completely by prazosin (0.3 microM). Increases in intracellular Ca2+ and subsequent contraction in the ileal tissue were produced by ATP and these responses were fully antagonized by nifedipine (0.1 microM). These findings provide evidence that the drugs-stimulated ATP release from smooth muscles does not result from contractility of muscles, but is substantially elicited only by stimulation of neurotransmitter (NE or ACh) receptors, suggesting the existence of the receptor-stimulus-postjunctional ATP release coupling. The released ATP may contribute, in part, to the muscle contractility via increase of Ca2(+)-influx, presumably, in a manner related to the voltage-gated Ca2(+)-channels.

Two phases of the prostaglandin F2 alpha-induced contraction in guinea-pig taenia coli involve different Ca2+ channels.

Properties of the contraction produced by PGF2 alpha in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F2 alpha (3 x 10(-7) M) and acetylcholine (ACh, 10(-5)M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF2 alpha or ACh were abolished in Ca2+ -free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF2 alpha and ACh were markedly suppressed by alpha-[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]-methylamino]-propyl]- 3,4,5-trimethoxy-alpha-(1-methylethyl)benzeneacetonitrile hydrochloride (D600, greater than 10(-7)M) as well as nifedipine (5 x 10(-9)M), a Ca2+-antagonist. However, the phasic contraction produced by PGF2 alpha, but not by ACh, was greatly inhibited by Mn2+ (greater than 10(-4)M). Furthermore, the phasic contraction caused by PGF2 alpha was abolished in 18 mM K+ Krebs solution with D600 (2 x 10(-7)M), whereas that induced by ACh and the tonic contractions produced by PGF2 alpha as well as by ACh were unaffected in this high K+ solution without D600. Membrane potentials of the tissue in normal (K+, 5.9 mM) and 18 mM K+ Krebs solution containing D600 were about -55 mV and -43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca2+ indicator in the presence of D600, PGF2 alpha and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between Bay-K-8644 and various relaxant agents on K+-depolarized contracture of guinea pig taenia coli.

1. Interaction between Bay-K-8644 and nifedipine, D600, diltiazem, papaverine or W-7 on a K+-contracture of the guinea pig taenia coli have been investigated. 2. These relaxing drugs except Bay-K-8644 inhibited the K+-contracture in a concentration-dependent manner. 3. Complete inhibition by nifedipine (1 x 10(-7) M) of the contracture was absolutely antagonized by Bay-K-8644 (1 x 10(-6) M), a Ca2+-agonist, an apparent pA2 value of Bay-K-8644 as an antagonist for nifedipine obtained from Schild plot being 7.94 and its slope 0.99. 4. Marked inhibitions by D600 (1 x 10(-5) M), diltiazem (1 x 10(-5) M), papaverine (1 x 10(-4) M) and W-7 (1 x 10(-4) M) were unaffected but mild inhibitions by around IC50 of these relaxing agents, except W-7, were fully antagonized by Bay-K-8644. 5. These results suggest that Bay-K-8644 is a competitive antagonist for nifedipine, but not for other agents, on common Ca2+ channel sites.

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens.

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.

Mechanism of extraneuronal adrenaline release induced by K+-free medium and ouabain, but not by high KCl in vas deferens of guinea-pig and rat.

The effects of K+-free medium, ouabain or high KCl on the contractile response and spontaneous efflux of adrenaline were analyzed in denervated guinea-pig or rat vasa deferentia preloaded with 3 x 10(-5) M (+/-)-adrenaline. The K+-free medium-induced contraction of guinea-pig tissues was related to the concentration of adrenaline used during preloading and was suppressed by the addition of 2.5 mM Rb+ but not by 2.5 mM Cs+. The contractile effects of K+-free medium and 10(-5) M ouabain, but not the response to high KCl, were prevented by 5 x 10(-6) M phentolamine or 3 x 10(-5) M deoxycorticosterone. These contractions, therefore, appear to be mediated by adrenaline released from the extraneuronal compartment. The K+-free medium- or ouabain-induced contractions of denervated rat vasa deferentia were small in comparison with those of the guinea-pig tissue. The amount of spontaneous efflux of adrenaline from the guinea-pig preparation was significantly enhanced by omission of K+ or by 10(-4) M ouabain but not by 80 mM KCl. K+-free medium and 3 x 10(-5) M ouabain merely elicited a small membrane depolarization accompanied by a slight decrease of membrane resistance in the guinea-pig vas deferens, whereas 30 mM KCl markedly altered these electrical parameters. From these findings, it is suggested that the rapid increase in extraneuronal amine release following inhibition of Na+-K+-ATPase may be due to some direct effect of K+ deficiency or of ouabain on the binding between the enzyme and the catecholamine rather than to alterations of membrane properties as a consequence of enzyme inhibition.