Effect of dehydroleucodine on intestinal transit: structural basis of the interaction with the α(2)-adrenergic receptor.

The activity of dehydroleucodine, a sesquiterpene lactone obtained from Artemisia douglasiana, was studied in mice small intestinal transit. Its mechanism was evaluated in the presence of several adrenergic and cholinergic antagonist drugs and one opioid antagonist. Docking of dehydroleucodine into the homology model of the α2-adrenergic receptor allowed us to analyze the structural basis of their interactions. The experiments showed that dehydroleucodine delayed intestinal transit. The docking of dehydroleucodine showed a unique binding site, equivalent to the binding site of carozolol in the ß-adrenergic receptor. The results suggested that dehydroleucodine produced an inhibitory effect on intestinal transit. Its action could be mediated, at least in part, through the α2-adrenergic receptor.

The classical competitive antagonism of phentolamine on smooth muscle preparations, investigated by two procedures.

1. In isolated smooth muscle tissues taken from rats, rabbits and guinea-pigs, all at 37.5 degrees C, the equilibrium dissociation constant (K(beta)) of the competitive, reversible alpha-adrenoceptor antagonist phentolamine varied between 4 and 28 nm. 2. The concentration of the antagonist required to inhibit contractions to direct- or indirect-acting alpha-adrenenoceptor agonists by 50% (IC50) also varied between 5 and 30 nm. 3. From one tissue to another, the IC50/K(beta) ratio of the blocker varied from 1 to 2.5, the values being close to those predicted by classical receptor theory based on the law of mass action. 4. At 27.5 degrees C, using phenylephrine as the spasmogen in rat aorta, the IC50/K(beta) ratio for phentolamine was 3.1. 5. A significantly higher IC50 compared with K(beta) for phentolamine indicates that the procedures for estimating affinity constants for a competitive antagonist are not equivalent.

Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; P<0.05; n = 8) and ODQ (100 microM) (62.7+/-5.1 vs 26.8+/-3.9%; P<0.05; n = 8). Charybdotoxin and apamin (K(Ca)-channel blockers) did not affect the phentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; P<0.05; n = 8) of the phentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; P<0.05; n = 8). The results suggest that phentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

[Effect of nifedipine on perfusion pressure changes produced by alpha 1 adrenergic agonists and depolarizing solution in rat hindlimbs]. / Effecto de la nifedipina sobre los cambios en la presión de perfusión producidos por agonistas alpha 1 adrenérgicos y solución despolarizante en el tren posterior de la rata.

The calcium channels blocker, nifedipine (NIF) inhibited in a partial non competitive manner the changes in perfusion pressure (delta P) caused by noradrenaline (NA) in the rat hindquarters, being the maximum inhibition of 31.0 +/- 8.3% (n = 5) for NIF 1.10(-5) M. The vasoconstrictor response of K+ 80 mM - phentolamine 3.10(-6)M was inhibited with lower concentrations of NIF than the one produced by phenylephrine (F) 1.10(-4)M. When the hindquarters were perfused with Krebs OCa-EGTA 2 mM NA developed contractile response. The administration of Ca 2.5 mM after the intracellular calcium store had been depleted, generated vasoconstriction in the presence of F 1.10(-4)M and K+ 80 mM - phentolamine 3.10(-6)M which were blocked in a 60.4 +/- 5.7 (n = 12) and 91.1 +/- 2.3% (n = 10) respectively by NIF 1.10(-5)M. The results suggest that the activation of the adrenergic receptor by NA in the perfused rat hindquarters, probably releases calcium from intracellular stores and promotes calcium influx through pathway scarcely sensitive to NIF. Both mechanisms would be responsible for the partial blockade found in our results.

Efecto de la nifedipina sobre los cambios en la presion de perfusion producidos por agonistas alpha 1 adrenergicos y solucion despolarizante en el tren posterior de la rata / Effect of nifedipine on perfusion pressure changes produced by alpha 1 adrenergic agonists and depolarizing solution in rat hindlimbs

El bloqueante de los canales de calcio nifedipina (NIF), inhibió en forma no competitiva incompleta, los cambios en la presión de perfusión (delta P) provocados por noradrenalina (NA) en el tren posterior de la rata, siendo la máxima inhibición de 31.0 ñ 8.3 (n=5) con NIF 1.10**5 M. La respuesta vasoconstrictora de K+ 80 mM - fentolamina 3.10**-6 M, fue inhibida con menores concentraciones de NIF que aquella producida por fenilefrina (F) 1.10**-4 M. Cuando el tren posterior fue perfundido con krebs OCa-EGTA 2 mM NA desarrolló respuesta contráctil. La administración de Ca**2+ 2.5mM luego de agotados los depósitos intracelulares, generó vasoconstricción en presencia de F 1.10**-4 M y de K+ 80 mM - fentolamina 3.10**-6 M. Estas respuestas fueron bloqueadas en un 60.4 ñ 5.7% (n=12) y 91.1 ñ 2.3% (n=10) respectivamente por NIF 1.10**-5M. Los resultados sugieren que la activación del receptor adrenérgico por NA y F en el tren posterior de la rata, liberaría calcio de depósitos intracelulares y promovería el influjo de calcio a través de vías escasamente sensibles a NIF. Estos mecanismos serían los responsables del bloqueo parcial observado (AU)

Efecto de la nifedipina sobre los cambios en la presion de perfusion producidos por agonistas alpha 1 adrenergicos y solucion despolarizante en el tren posterior de la rata / Effect of nifedipine on perfusion pressure changes produced by alpha 1 adrenergic agonists and depolarizing solution in rat hindlimbs

El bloqueante de los canales de calcio nifedipina (NIF), inhibió en forma no competitiva incompleta, los cambios en la presión de perfusión (delta P) provocados por noradrenalina (NA) en el tren posterior de la rata, siendo la máxima inhibición de 31.0 ñ 8.3 (n=5) con NIF 1.10**5 M. La respuesta vasoconstrictora de K+ 80 mM - fentolamina 3.10**-6 M, fue inhibida con menores concentraciones de NIF que aquella producida por fenilefrina (F) 1.10**-4 M. Cuando el tren posterior fue perfundido con krebs OCa-EGTA 2 mM NA desarrolló respuesta contráctil. La administración de Ca**2+ 2.5mM luego de agotados los depósitos intracelulares, generó vasoconstricción en presencia de F 1.10**-4 M y de K+ 80 mM - fentolamina 3.10**-6 M. Estas respuestas fueron bloqueadas en un 60.4 ñ 5.7% (n=12) y 91.1 ñ 2.3% (n=10) respectivamente por NIF 1.10**-5M. Los resultados sugieren que la activación del receptor adrenérgico por NA y F en el tren posterior de la rata, liberaría calcio de depósitos intracelulares y promovería el influjo de calcio a través de vías escasamente sensibles a NIF. Estos mecanismos serían los responsables del bloqueo parcial observado

Pharmacological properties of contraction caused by sodium removal in muscle strips isolated from canine coronary artery.

Contractions produced by Na+ removal were studied in muscle strips isolated from canine coronary artery. In the presence of 20 mM K+ and 0.5 mM Ca2+, rapid contractions were observed repeatedly on complete replacement of NaCl with sucrose. This contraction in the absence of Na+ (0-Na) was not affected by phentolamine but was strongly inhibited by verapamil. Ouabain slowly potentiated the O-Na contraction and markedly reduced the inhibition due to verapamil. The O-Na contraction was dependent on external Ca2+ both with and without ouabain. Bepridil had effects very similar to those of verapamil. Amiloride and excess Mg2+ reduced the O-Na contraction and the degree of their inhibition was similar after ouabain treatment. The decrease in verapamil susceptibility could suggest that the O-Na contraction has verapamil-sensitive and -insensitive components. The former is probably due to Ca2+ influx through voltage-dependent channels and the latter to Ca2+ influx through an Na(+)-Ca2+ exchange process. Ouabain is considered to increase the contribution of Na(+)-Ca2+ exchange to the O-Na contraction. Mg2+ may inhibit both verapamil-sensitive and -insensitive pathways. Amiloride probably exerts its inhibitory effect on the contractile machinery.

Antagonism by methysergide of neurogenic vasoconstriction in the dog forelimb.

In the flow-regulated dog forelimb, electrical stimulation of the efferent median nerve produced frequency-dependent increases in perfusion pressure. These vasoconstrictor effects were attenuated by a large dose of phentolamine, an alpha 1 and alpha 2 blocking drug. Administration of methysergide after phentolamine completely reversed the vasoconstrictor responses to vasodilation at most frequencies of stimulation. In the absence of phentolamine pretreatment, even a lower dose of methysergide reversed or caused biphasic responses (attenuated constriction followed by dilatation) during the nerve stimulation at the lower frequencies (0.5-4.0 Hz). This lower dose of methysergide completely abolished vascular effects of exogenous 5-hydroxytryptamine (5-HT) and potentiated those of norepinephrine; hence, the antagonism by methysergide of neurally mediated vasoconstriction is not caused by an action on alpha-adrenergic receptors. Unlike methysergide, selective 5-HT2 antagonists ketanserin and ritanserin have no modifying effect on exogenous 5-HT responses. These studies have provided pharmacological evidence that suggests that 5-HT may be the neurotransmitter mediating neurogenic vasoconstriction in the dog forelimb, and that this effect does not involve activation of 5-HT2 receptors.

Coronary blood flow during exercise following nonselective and selective alpha 1-adrenergic blockade with indoramin.

Studies were performed to compare the effects of alpha 1-adrenergic blockade with indoramin and nonselective alpha-adrenergic blockade with phentolamine on coronary blood flow and myocardial oxygen consumption during exercise. Nine dogs trained to run on a motor-driven treadmill were instrumented with electromagnetic flowmeter probes on the left circumflex coronary artery, and aortic and coronary sinus catheters for determination of myocardial arteriovenous oxygen difference. During control conditions, myocardial oxygen consumption and coronary blood flow increased as a direct function of heart rate during exercise. Phentolamine caused a significant decrease in blood pressure, while heart rate, coronary blood flow, and myocardial oxygen consumption were significantly increased at rest and during all exercise stages. Although alpha 1-adrenergic blockade with indoramin caused a similar reduction of arterial pressure, heart rate was unaltered both at rest and during exercise. Coronary blood flow and myocardial oxygen consumption were unchanged by alpha 1-adrenergic blockade at rest and during light exercise: however, during the heaviest exercise stages alpha 1-blockade caused a significant decrease in myocardial oxygen consumption. These findings are in agreement with the concept that phentolamine, by blocking presynaptic alpha 2-adrenoceptors which normally modulate norepinephrine release, increases sympathetic activity during exercise while indoramin, by acting as a selective alpha 1-adrenergic blocker, does not produce this effect.

Reduction of centrally-stimulated gastric acid secretion by tizanidine, a new imidazoline derivative, in anesthetized rats.

The effect of a novel imidazoline derivative (tizanidine) on stimulated gastric acid secretion was studied in the perfused stomach of anesthetized rats. Tizanidine, which did not prevent peripherally-stimulated gastric acid secretion, inhibited 2DG- or TRH-stimulated gastric acid secretion. Yohimbine and phentolamine reduced the inhibition of TRH-stimulated acid secretion by tizanidine. Clonidine was found to have similar effects to tizanidine at a lower dose. These results indicate that tizanidine may inhibit gastric acid secretion via the central alpha-adrenergic system similar to clonidine in anesthetized rats.

Contribution of extracellular and intracellular calcium to the enhanced effect of an alpha-adrenergic agonist on amylase release from dispersed rat parotid cells.

The role of calcium in the potentiation of amylase release by the alpha-adrenergic agonist methoxamine (MTX) was examined using dispersed rat parotid acinar cells. The stimulatory effect of MTX (10 microM) on the beta-adrenergic agonist isoproterenol (ISP, 1 microM)-induced amylase release was blocked by the alpha-adrenergic antagonist phentolamine (10 microM). In Ca-free (1 mM EGTA) medium, the increment of amylase release by MTX in normal medium was decreased by about 70%, but MTX still potentiated ISP-induced amylase release. MTX did not affect the cyclic AMP accumulation activated by ISP in either normal or Ca-free medium. MTX enhanced the ISP-stimulated uptake and efflux of 45Ca2+. These results suggest that both extracellular Ca2+ and intracellular stored Ca2+ may play an important role in the potentiation of amylase release from rat parotid acinar cells.

Phentolamine--an unexpected agonist in the rabbit.

Phentolamine (0.1-10 microM) caused an anomalous rightward shift of the relationship between the number of electrical field pulses and tachycardia in the rabbit isolated right atrium. Phentolamine was apparently acting as a presynaptic agonist on sympathetic nerve endings to inhibit transmitter release. The effect was prevented by benextramine treatment and antagonized 10 fold by yohimbine (1 microM) but not by prazosin (0.1 microM). In ganglion-blocked (mecamylamine) conscious or anaesthetized rabbits, phentolamine (3-1000 micrograms kg-1) caused a dose-related rise in blood pressure that was antagonized by yohimbine (1 mg kg-1). These pressor and inhibitory cardiac sympathetic nerve effects of phentolamine are not found in similar preparations from the guinea-pig or rat. Therefore, these rabbit-specific agonist effects of phentolamine at sites similar to alpha 2-adrenoceptors make this drug unsuitable as an alpha-adrenoceptor antagonist in rabbits.

Nadolol: evidence for sympathetic nerve inhibition by a beta blocker in essential hypertension.

Although beta blockers' antihypertensive mechanisms have not been clearly delineated, their long-term effects may involve chronic reduction in systemic vascular resistance, which may be the result of sympathetic outflow inhibition. Although a central site of action has been advocated, we sought to identify a peripheral non-cardiac sympatholytic mechanism by studying autonomic function in a small group of nine hypertensive males during treatment with placebo and chronic oral nadolol, a noncardioselective hydrophilic beta blocker with little predicted brain penetration. Nadolol reduced blood pressure and heart rate (both P less than 0.005) while suppressing the blood pressure response to cold stimulus only after parasympathetic inhibition (P less than 0.05); the blunted response to cold stimulus did not correlate with the drug's overall blood pressure lowering effect. Baroreceptor sensitivities to phenylephrine and amyl nitrate stimuli were not enhanced. Several biochemical measures of sympathetic nervous system activity were not influenced by nadolol. Thus, nadolol, while not enhancing baroreflex sensitivity, does seem to have a peripheral non-cardiac sympatholytic effect, but this effect does not account entirely for the long term reduction in blood pressure observed in patients on the drug.

the role of differential blockade of alpha-adrenergic agonists in chlorpromazine-induced hypotension.

We studied the effects of chlorpromazine HCl (CPZ) on the pressor responses of norepinephrine and phenylephrine in the isolated, perfused hindlimbs of anesthetized dogs. Our results show that, in contrast to phentolamine, CPZ produced a much greater degree of blockade of the pressor responses of phenylephrine than of norepinephrine. Pre-treatment with cocaine prevented the differential blockade. On the other hand, CPZ acted as a classical competitive antagonist in the isolated femoral artery strip preparation. We conclude that differences in affinity for the Uptake system of the adrenergic nerve endings provide the basis for the differential alpha-adrenergic blocking effect of CPZ. These results explain the finding that although a significant degree of alpha-adrenergic receptor blockade may be present after hypotensive doses of CPZ, responses to the naturally occurring neurotransmitter, norepinephrine, are not decreased.

Antagonism by chlorisondamine and propranolol, but not by atenolol, of the circadian phase-dependent phentolamine-induced changes in the cardiac noradrenaline turnover in the rat.

The effects of phentolamine alone or in combination with propranolol, atenolol and chlorisondamine were studied on the concentration and turnover of noradrenaline in the heart of light-dark (L:D = 12:12 h) synchronized rats. In order to detect possible circadian phase-dependent variations in the drug effects, the same experiments were performed in the light-period and dark-period, respectively. The parameters of the turnover were calculated from the exponential decline of i.v. injected 3H-(-)-noradrenaline. Phentolamine significantly decreased the noradrenaline concentration during L, but not during D. Reduction in 3H-noradrenaline accumulation by phentolamine was 42.3% during L and 22.2% during D. Phentolamine increased the turnover rate of cardiac noradrenaline more than 3-fold in either photoperiod. Chlorisondamine reversed all the effects of phentolamine studied. Propranolol, but not atenolol, antagonized the effects of phentolamine in a dose-dependent and stereospecific way, being more effective when applied during D. Thus, the chronopharmacological studies in unrestrained rats show a circadian phase-dependency of the effects of adrenoceptor blocking drugs. It is concluded that a central site of action is responsible for the antagonism by propranolol of the phentolamine-induced increase in the turnover of the cardiac noradrenaline in vivo.

Pre- and postsynaptic alpha-adrenoceptor antagonists: differentiated cardiovascular effects in the rat.

Intravenous (i.v.) injections of yohimbine, phentolamine, prazosine and phenoxybenzamine lowered blood pressure and increased heart rate in conscious rats. Intracerebroventricular (i.c.v.) injections of yohimbine and phentolamine increased blood pressure and heart rate; this was antogonized by pretreatment with clonidine. Phenoxybenzamine and prozosine had no effect or gave hypotension and tachycardia on i.c.v. injection. Pentobarbitone anaesthesia partly antagonized the cardiovascular effects of all alpha-adrenoceptor antagonist. Synthesis and utilization of central noradrenaline was increased by i.v. or i.c.v. yohimbine; anaesthesia partly antagonized this effect. In peripheral tissues others have found that yohimbine, tolazoline, piperoxan and phentolamine are potent blockers of the presynaptic alpha-adrenoceptors while phenoxybenzamine and prazosine act preferentially on postsynaptic alpha-adrenoceptors. The differentiated cardiovascular response to i.c.v. injection of these blockers may reflect their different affinity to central pre- and postsynaptic alpha-adrenoceptors.

Comparative study of propranolol and dicyclohexylamine protection of alpha-receptor blockade.

During the course of a study into protection against alpha-receptor blockade, it was observed that the dicyclohexylamine (DCHA) salt of 5-hydroxyindole acetic acid exhibited protection, whilst the free acid did not protect against alpha-receptor blockade. A study has been made into the protection of alpha-receptor blockade by DCHA and this compared with the beta-blocker propranolol, with which DCHA may share some structural similarity. It was also observed that at higher concentrations DCHA itself produced spiked contractions of the vas deferens. These properties of DCHA and their interrelationships with those of propranolol are discussed.

Positive inotropic effect of ethylephrine on the isolated rat atria.

Ethylephrine, a sympathomimetic amine which belongs to the phenolamine group, was assayed on the driven left rat atrium. The frequency response curve was performed for norepinephrine and ethylephrine. The maxima was attained for both compounds at 1 Hz. The agonist under study has an inotropic action less potent than the classical catecholamines. Propranolol (10(-8) and 10(-7) M) produced a parallel shift to the right in the log dose-response curves of ethylephrine with no decrease in the maximal response, indicating that the antagonism was competitive. In the presence of cocaine or with reserpine-pretreatment the sensitivity of the preparation to the amine did not vary. The alpha-blocker, phentolamine (10(-8) to 3.10(-5) M) did not possess an inotropic effect per se. In contrast, phentolamine, delivered to the bath beforehand, did not block the agonist. However at 10(-8) and 10(-7) M increase the maximal response both in normal and reserpinized preparations. It is suggested that ethylephrine is a direct inotropic preparation. It is suggested that ethylephrine is a direct inotropic agent on the driven left rat atrium and its effects are mediated by beta-receptors. The results also indicate the lack of evidence that ethylephrine has any action on the alpha-receptors.