Further characterization of antagonism by alpha 2-adrenoceptor agonists of contractions induced by alpha 1-adrenoceptor agonists.

The population of alpha 1-adrenoceptor subtypes functionally mediating contraction in response to phenylephrine was examined in rat thoracic aorta and tail arteries. In thoracic aorta, chloroethylclonidine (CEC), which alkylates the alpha 1B and alpha 1D subtypes, shifted the concentration-response curve for phenylephrine substantially to the right without reduction in maximum contraction. The pA2 value (7.8) for 5-methylurapidil was consistent with the values reported for alpha 1D subtype, but was higher than those in tissues in which the alpha 1B subtype is dominant. In tail arteries, CEC did not shift the concentration-response curve for phenylephrine, but somewhat inhibited the maximum contraction. Schild analysis for 5-methylurapidil yielded a straight line with a slope significantly less than unity. Prazosin antagonized phenylephrine-induced contraction of tail arteries in a competitive manner with a pA2 value of 8.5, consistent with values for alpha 1L subtype. Clonidine relaxed the active tone induced by phenylephrine in both thoracic aorta and tail arteries, but quite different responses to clonidine by the two tissues were observed. After pretreatment with CEC, the relaxation induced by clonidine was abolished in thoracic aorta, but not in tail arteries. These results suggest that alpha 1D-and alpha 1L-adrenoceptors are mainly present in thoracic aorta and tail arteries, respectively. This difference in the populations of alpha 1-adrenoceptor subtypes may be related to regional differences in the modes of relaxant action of clonidine.

Characteristics of depression by clonidine of the pressor response to alpha 1-adrenoceptor agonists in pithed rats.

Effects of clonidine on the elevated arterial diastolic blood pressure induced by infusion of alpha 1-adrenoceptor agonists were studied in pithed rats. Intravenous injection of clonidine resulted in a dose-dependent pressor response at a resting state and a further increase in the moderately elevated diastolic blood pressure induced by infusion of lower doses of phenylephrine and methoxamine. However, clonidine produced a depressor response when diastolic blood pressure was elevated by around 100 mmHg during infusion of higher doses of alpha 1-adrenoceptor agonists. The depressor response to clonidine was dose-dependent at a dose range of 10 to 1000 micrograms/kg. On the other hand, clonidine failed to cause the depressor response while diastolic pressure was elevated by infusion of vasopressin by around 100 mmHg. NG-methyl-L-arginine, a nitric oxide synthase inhibitor, and propranolol, a beta-adrenoceptor antagonist, did not affect the depressor response to clonidine. After pretreatment with yohimbine, an alpha 2-adrenoceptor antagonist, the depressor response to clonidine was inhibited, but prazosin, an alpha 1-adrenoceptor antagonist, did not suppress the depressor response. These results demonstrate that clonidine specifically depresses the pressor response to alpha 1-adrenoceptor agonists in pithed rats via a mechanism which is not mediated by beta-adrenoceptors and independent of endothelium-dependent relaxing factor, and suggest that a yohimbine-sensitive mechanism may be related to the clonidine effect.

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists.

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.

Serotonin relaxes porcine pial veins.

The effect of serotonin [5-hydroxytryptamine (5-HT)] on pial venous tone of the pig was examined using in vitro tissue bath techniques. Isolated pial venous rings exhibited spontaneous rhythmic contractions (SRC) on mechanical stretching and/or applications of several vasoactive substances, including norepinephrine. On the other hand, KCl induced sustained active muscle tone (SAT) without SRC. The SRC induced by mechanical stretching were not affected by tetrodotoxin, nitro-L-arginine, alpha- and beta-adrenergic, histaminergic, and muscarinic receptor antagonists, indicating that the SRC in porcine pial veins are of myogenic origin. The SRC induced by stretching or applications of vasoactive substances and SAT induced by KCl were inhibited by 5-HT in a concentration-dependent manner. The inhibition was prevented by methysergide and methiothepin but not by ketanserin, propranolol, 3 alpha-tropanyl-1H-indole-3-carboxylic acid ester, hemoglobin, or nitro-L-arginine. The SRC and SAT were inhibited by 5-carboxamidotryptamine (5-CT), 8-hydroxy-2-di-N-propylaminotetralin HBr (8-OHDPAT), 1-[3-(trifluoromethyl)phenyl]piperazine (TFMPP), and 5-methoxytryptamine (5-MT), but not by sumatriptan, alpha-methylserotonin, or 2-methylserotonin. On the other hand, 5-CT, 8-OHDPAT, TFMPP, 5-MT, and sumatriptan constricted the porcine pial arteries exclusively. In 15% of pial venous preparations examined, 5-HT at low concentrations induced ketanserin-sensitive constrictions. These results indicate that the porcine pial venous smooth muscle contains multiple subtypes of 5-HT receptors. The 5-HT inhibition of SRC and SAT is predominant and is mediated by 5-HT1-like receptors, which, however, do not seem to correspond to 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, 5-HT1E, or 5-HT1F receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible mechanisms of action of the antispasmodic agent tiropramide in the isolated detrusor from rats.

The effects of tiropramide hydrochloride on Ca(2+)-induced contraction, cytoplasmic free Ca2+ levels and tissue cyclic AMP concentrations were investigated to elucidate the mechanisms of its antispasmodic action in the isolated detrusor from rats. Tiropramide inhibited the Ca2+ (3 mM)-induced contractions of the isolated urinary bladder depolarized in a Ca(2+)-free medium, and the IC50 value was 3.3 x 10(-6) M. When tiropramide was added during the sustained phase of the K+ (60 mM)-contracture, IC50 values of tiropramide for the contraction and the increased fluorescence were 1.9 x 10(-5) M and 16.4 x 10(-5) M, respectively. On the other hand, the IC50 values for the K(+)-induced contraction and fluorescence after pretreatment of the isolated urinary bladder with tiropramide were 2.1 x 10(-5) M and 2.6 x 10(-5) M, respectively. Tissue cyclic AMP levels at 1 min after addition of 10(-5) M tiropramide were significantly increased. Papaverine, IBMX or forskolin potentiated the inhibitory effect of tiropramide on carbachol-induced contraction and its cyclic AMP-elevating effect. However, a good correlation between the degrees of potentiation of the inhibitory effect and the increase in cyclic AMP levels was not observed. The present results suggest that the smooth muscle relaxant activity of tiropramide in the isolated detrusor from rats may be intimately associated with predominant inhibition of Ca2+ influx and, to a lesser extent, an increase in intracellular cyclic AMP levels.

[Effects of tiropramide hydrochloride on the isolated detrusor and intravesical pressure of the bladder in situ in rats].

The effects of tiropramide on the isolated detrusor and intravesical pressure of the bladder in situ in rats were compared with those of flavoxate, oxybutynin and terodiline. The IC50 values (x 10(-5) M) of tiropramide for carbachol (CCh)-, K+ (60 mM)-, Ba2+ (10 mM)-, and electrical stimulation-induced contractions were 3.6, 4.2, 5.8, and 2.9, respectively. The four antispasmodics used (2 and 4 mg/kg, i.v., each) abolished the rhythmic bladder contractions in situ in anesthesized rats. Of the four compounds, oxybutynin was most potent and no significant differences were observed between the inhibitory effects of tiropramide, flavoxate and terodiline. The administration of flavoxate (30 and 60 mg/kg) into the duodenum little influenced the rhythmic bladder contractions. Tiropramide, flavoxate, oxybutynin and terodiline (8 and 12 mg/kg, i.v., each) dose-dependently prolonged the time to the volume-evoked micturition reflex, and the activity of tiropramide was not statistically different from those of the other three antispasmodics. Under unilateral pelvic and bilateral hypogastric nerve transection, both of the contractions induced by electrical stimulation of the peripheral and central cut ends of the pelvic nerve were dose-dependently inhibited to the same extent by tiropramide and terodiline. These results suggest that the effects of tiropramide on the function of urinary bladder in rats may be mainly due to direct actions on the smooth muscle, and that tiropramide is more potent than flavoxate and less potent than oxybutynin and terodiline.

[Effects of lactulose on intestinal functions in mice and rats].

Effects of lactulose on transit of charcoal meals and the luminal water and insoluble contents in the intestinal tract of rats and mice were investigated. The ED50 value for lactulose to induce diarrhea was 3.8 g/kg (p.o.). The sufficient dose (5.4 and 8.6 g/kg, p.o.) needed to produce diarrhea increased the luminal water content of the small intestine and the caecum in rats and mice and sped transit of the intestinal charcoal in mice. In addition, the administration of lactulose at 5.4 g/kg significantly decreased the luminal insoluble contents of the rat small intestine. These results indicate the cathartic effect of lactulose in smaller animals such as rats as well as humans and suggest the possible application of full doses of lactulose to flush the luminal contents from the small intestine.

[Effects of lactulose on blood ammonia levels in beagles with end-to-side portacaval shunt].

The effects of lactulose on blood ammonia and fecal pH in beagles with an end-to-side portacaval shunt were investigated. Concentrations of blood ammonia before and two weeks after the operation were 4.65 +/- 0.34 micrograms/ml (N = 12) and 8.66 +/- 0.60 micrograms/ml (N = 6), respectively (P less than 0.01), while there was no significant difference in fecal pH values before and after the operation. The blood ammonia concentrations in the control and lactulose-treated (2.1 g/kg, p.o.) groups after administration of meat were 12.65 +/- 1.64 micrograms/ml (N = 8) and 8.45 +/- 0.90 micrograms/ml (N = 8), respectively (P less than 0.05). The fecal pH values in the control and lactulose-treated (2.1 g/kg, p.o.) beagles were 6.24 +/- 0.09 (N = 8) and 5.58 +/- 0.08 (N = 8), respectively (P less than 0.01). The lowering effect of lactulose on the blood ammonia levels could not be ascribed to its laxative action alone, because the oral administration of lactulose at doses less than 2.1 g/kg induced no fluid evacuation. The present studies in the beagle suggest that the action of lactulose produce a lowering of fecal pH, causing an increase in the concentration of less absorbable ammonium ions and a decrease in the production of toxic nitrogenous compounds such as ammonia, resulting in a reduction in blood ammonia levels.

Possible mechanisms of spasmolytic action of bile salts on the isolated guinea-pig gallbladder.

The spasmolytic action of bile salts on gallbladder smooth muscle could explain the alleged relief of biliary colic seen during bile acid therapy. The mechanisms of spasmolytic action of bile salts, ursodeoxycholate and deoxycholate were studied in the isolated gallbladder of guinea-pigs. The bile salts accelerated the 45Ca-efflux from the gallbladder with synchronous relaxation and inhibited the cellular 45Ca-uptake by the depolarized muscle preparation. Further, they sensitively inhibited CaCl2-induced contraction of the depolarized muscle. The tissue cyclic AMP content of the gallbladder was significantly elevated by the bile salts. Dibutyryl cyclic AMP mimicked the effects of bile salts on the Ca-efflux and the muscle relaxation, but showed no effect on the cellular Ca-uptake. From these results, it is suggested that the bile salts produce the relaxant action through accelerating Ca-efflux, which is probably coupled with the elevation of the cellular cyclic AMP level, and through suppressing the Ca-influx across the cell membrane.

Inhibition by beta-CCM of cholecystokinin-induced release of acetylcholine from the longitudinal muscle-myenteric plexus preparation in the guinea-pig ileum.

The antagonism between cholecystokinin (CCK) and methyl beta-carboline-3-carboxylate (beta-CCM) in the nervous system was studied by measuring the release of acetylcholine (ACh) from the longitudinal muscle-myenteric plexus preparation of guinea-pig. The ACh release was assessed by measuring [3H] output from the preparation preincubated with [3H] choline. Thirty mM of KCl caused a pronounced release of [3H] ACh from the preparation. Sulfated cholecystokinin octapeptide (CCK8) also increased the release of [3H] ACh in a dose-dependent manner at concentrations ranging from 10(-10)M to 10(-8)M. CCK8 at a concentration of 10(-8)M released [3H] ACh by about 300% of the 30 mM KCl-induced [3H] ACh release. In the presence of beta-CCM (10(-8)M to 10(-4)M), the release of [3H] ACh by KCl was not affected, but that by CCK8 was significantly inhibited depending on the concentrations of beta-CCM. These results show that the action of CCK8 to stimulate neurons in the myenteric plexus can be selectively antagonized by beta-CCM.

Effects of sucrose- or Tris-substitution for Na+ on responses to D 600, papaverine and KCN in the K+-depolarized taenia coli of guinea pigs.

Effects of D 600, papaverine and KCN on sustained contracture induced by 20 mM KCl in a Na+-free sucrose- or Tris-substituted solution were investigated in the taenia coli of guinea pigs. D 600 (10(-5) M) and papaverine (10(-4) M), added after 40 min incubation in the 20 mM KCl sucrose solution, relaxed the muscle by 88.2 +/- 8.0% (mean +/- S.E., n = 4) and 78.8 +/- 6.3% (n = 4), respectively. In contrast to this, KCN (10(-3) M) relaxed the muscle by 11.0 +/- 4.7% (n = 4) after a 20 min incubation in the sucrose solution. The concentration-relaxation curves for these relaxants were slightly shifted to the right in the Na+-free 20 mM KCl Tris solution. The cellular Na+ content of the taenia coli was greatly reduced by exposure to the sucrose or Tris solution, while the Ca2+ contents in both solutions were gradually increased with the exposure time. When a glucose-free solution was applied 60 min after the KCl-induced sustained contracture, gradual relaxation in the normal solution was observed. This relaxation was completely inhibited in the Na+-free sucrose solution. These results suggest that the relaxing activities of the different inhibitors are changed to different degrees by the replacement of Na+ with sucrose in the guinea-pig taenia coli and that sustained KCl-contracture in a Na+-free sucrose solution may be less energy-dependent as compared with that in a Na+-free Tris medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of Ca2+-release from the isolated smooth muscle cells in suspension from the guinea-pig ileum.

Collagenase-dispersed cells from the guinea-pig ileum were prepared and Ca2+ release into Ca2+-depleted solution from the isolated single cells obtained by the centrifugation of the dispersed cells on isotonic sucrose solution was determined with a Ca2+-selective electrode. A technique employing an isotonic sucrose solution for washing isolated cells permitted removal of contaminating extracellular fluids and obtaining the isolated cells with minimum loss of cellular Ca2+. The release of Ca2+ from the dispersed cells consisted of at least two phases. The Ca2+ release into an isotonic sucrose-Tris solution (ISTS) was significantly reduced and the early phase of the Ca2+ release disappeared. At 16 degrees C, Ca2+ release depended on the composition of the bathing solution in a Ca2+-free salt solution, the later phase of Ca2+-release was abolished whereas in ISTS both phases disappeared. Furthermore, Ca2+ release was significantly reduced after the treatment of the dispersed cells with disperse (1,500 units/ml) for 10 min. These results show that Ca2+ release into Ca2+-depleted solution from the isolated ileal cells with minimum loss of cellular Ca2+ show a biphasic curve and suggest that Ca2+ sources responsible for these phases may be of distinct origin.

Mechanisms of inhibitory action of phenylephrine in guinea-pig taenia coli.

The effect of phenylephrine, an alpha-agonist, on the Ca movements and the influence of removal of external Na+ on the relaxant activity of phenylephrine were examined in the taenia coli of guinea pigs. Phenylephrine (10(-7)-10(-5)M) caused dose-dependent relaxation of the taenia coli contracted by 20 mM KCl in Locke-Ringer solution. Phenylephrine (10(-5) M) suppressed the spike discharges of the taenia coli evoked by 20 mM KCl without affecting the membrane potential, and this was accompanied by the muscle relaxation. Phenylephrine also inhibited the cellular 45Ca-uptake in the taenia coli, but had no discernible effect on the 45Ca-efflux from the smooth muscle. These effects of phenylephrine were not observed in a Na-free solution or in the highly depolarized smooth muscle. These findings suggest that the inhibition of Ca-influx in the taenia coli may be involved in the phenylephrine-induced relaxation in the partly depolarized tissue. Reasons for reduction of phenylephrine action encountered under the Na-free condition were also discussed.

Mechanisms of spasmolytic action of bile salts in depolarized guinea-pig taenia coli.

The effects of bile salts on the calcium movements and the electrical activity of the guinea-pig taenia coli were investigated and compared with those of papaverine in order to explore the mechanisms of their spasmolytic action. Four bile salts, deoxycholate, chenodeoxycholate, ursodeoxycholate and cholate, as well as papaverine, dose-dependently relaxed the depolarized taenia coli. The bile salts and papaverine caused the acceleration of 45Ca-efflux with the synchronous muscle relaxation and inhibited the cellular 45Ca-uptake by the depolarized muscle preparation. The bile salts also inhibited the increased spike frequency and the developed tension in the depolarized taenia coli. Furthermore, the dose-relaxation curves for bile salts were shifted to the right as the external calcium ion was increased. These findings suggest that the bile salts, like papaverine, may exert their spasmolytic action through accelerating the Ca-efflux and inhibiting the Ca-influx of the smooth muscle cells.

Reversal of antinociceptive effect of caerulein by benzodiazepine.

Benzodiazepines, chlordiazepoxide and diazepam reversed the antinociceptive action of caerulein in mice. Benzodiazepines (1-5 mg/kg) were administered intraperitoneally and 100 ng of caerulein was injected intracisternally to mice. Benzodiazepines did not change the basal pain threshold of mice but significantly antagonized the antinociceptive effect of caerulein. Proglumide (200 mg/kg, i.p.), which has been claimed to be a specific cholecystokinin receptor antagonist, could also antagonize the antinociceptive effects of caerulein. Naloxone (5 mg/kg) partially but significantly antagonized the antinociceptive effect of caerulein, suggesting that one of the mechanisms of antinociceptive action of caerulein is related to endogenous opioid peptides since benzodiazepines do not act on opioid receptors. Benzodiazepines may decrease the antinociceptive effect of caerulein through acting on cholecystokinin receptors in the central nervous system.

Inhibition of cholecystokinin response in the gallbladder by dibenamine and its protection by benzodiazepines.

The contractile response of the guinea-pig gallbladder to cholecystokinin (CCK) and acetylcholine (ACh) was irreversibly inhibited by 5 X 10(-5) M dibenamine, and the dibenamine-induced inhibition in the CCK response was prevented by 10(-4) M chlordiazepoxide (CDP) and diazepam (DZP), but not by 10(-2) M proglumide or 10(-6) M atropine. The dibenamine-induced inhibition in the ACh response was prevented by 10(-6) M atropine, but not by 10(-4) M CDP. These findings suggest that the binding of CCK to the CCK receptor can be inhibited by benzodiazepines.

Possible mechanisms of inhibitory action of protamine on contractile activity of rat aorta.

Experiments were performed to determine possible mechanisms of inhibitory action of protamine chloride on noradrenaline (10 microM)-, KCl (40 mM)-, BaCl2 (1 mM)- and CaCl2 (10 mM)-induced contractions in rat aorta. Protamine, La3+ and gallopamil (D600), inhibited the K+-induced contractions more effectively than the noradrenaline-induced responses on the basis of the concentrations giving 40% inhibition. Lanthanum (1-5 mM) reduced tissue Ca content in both normal and Ca2+-depleted Tris-buffered solutions and produced an increase in 45Ca efflux from the aortic strip into the Ca2+-depleted Tris solution. Protamine (1-5 mg ml-1) reduced tissue Ca content in normal Tris solution, but to a lesser extent than La3+ in the Ca2+-depleted solution. Furthermore, protamine (3 mg ml-1) produced no increase in 45Ca efflux from aorta. These results suggest that protamine chloride may preferentially inhibit the Ca2+ influx stimulated by K+ depolarization and that its inhibitory action on rat aorta may be due to non-specific displacement of the superficially located bound Ca2+ of the cell membrane, which can also be readily removed by treatment with Ca2+-depleted solution.

Mechanism of relaxant action of papaverine. VI. Sodium ion dependence of its effect on 45Ca-efflux in guinea-pig taenia coli.

The present study was undertaken to investigate the roles of sodium ion and the cyclic AMP system in the relaxant effect of papaverine. The effects of papaverine on the 45Ca-efflux and the mechanical activity of guinea-pig taenia coli were tested in solutions in which the concentration of sodium ion was varied and compared with those of dibutyryl cyclic AMP. Papaverine dose-dependently caused an acceleration of 45Ca-efflux and the synchronous relaxation of a depolarized preparation in normal bathing solution. Dibutyryl cyclic AMP mimicked these effects of papaverine. In Na-free solution, papaverine lost its ability to accelerate the 45Ca-efflux, and its relaxant activity was markedly reduced, while dibutyryl cyclic AMP had neither an effect on the 45Ca-efflux nor an effect on the muscle relaxation in Na-free solution. Reintroduction of a small amount of sodium ion to the solution, however, recovered these effects of papaverine and dibutyryl cyclic AMP on the 45Ca-efflux and the muscle relaxation. These findings indicate that the relaxant effect of papaverine may be in part due to an increase in cyclic AMP-mediated Ca-efflux which requires the presence of external sodium ion. The sodium ion dependence of this Ca-efflux process was also discussed.