Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium

The effects of forskolin and isoproterenol on total cyclic AMP accumulation and relaxation were compared in uterine segments from estrogen-treated rabbits and term-pregnant rats. In the rabbit, forskolin (0.2-5.0 microM) inhibited spontaneous and acetylcholine-induced contractions and increased cyclic AMP levels. At lower doses forskolin significantly inhibited contractions but had little effect on cyclic AMP (cAMP) levels. Isoproterenol (0.005-0.5 microM), doses equi-effective to those of forskolin for inhibition of contractions, did not alter cyclic AMP levels. In the rat, forskolin (0.5 microM) and isoproterenol (0.05 microM) inhibited spontaneous and potassium-induced contractions by 80%. This reduction was accompanied by an increase in cyclic AMP levels, but the increase was significantly greater with forskolin than with isoproterenol. We conclude that: (1) cAMP may mediate relaxation under certain conditions but it is not an obligatory mediator, (2) some form of compartmentalization of cyclic AMP might exist in the myometrium.     

Rate-limiting steps in oral absorption of a leucotriene D4 antagonist in the beagle dog.

Oral administration of a leucotriene D4 antagonist drug (1) in the Beagle dog at doses of 2, 20, 100, 300, and 800 mg/kg resulted in dose-dependent bioavailability values (4-50%). To understand the dose dependence of the absorption of 1 in the dog, initial rates of absorption of 1, which were estimated from Loo-Riegelman analysis of the concentration in blood data, were analyzed in terms of dissolution and absorption rates. From the Loo-Riegelman plots, the initial rates of absorption of 1 were estimated as 8.2, 41.9, 41.1, 76.1, and 72.8 micrograms.mL-1.h-1, respectively, for the doses given earlier. These data, which indicate leveling of the initial absorption rates at high doses, were consistent with an absorption model in which the dissolution rate is the rate-controlling step in the intestinal absorption of 1 at doses less than 100 mg/kg. The powder dissolution rate of 1 in 17 mM bile salt solution was estimated as 14 and 700 micrograms.mL-1.h-1 for amounts of 1 equivalent to the amounts given to dogs at 2- and 100-mg/kg doses, respectively. After consideration of the volume of distribution and the volume of intestinal fluid in the dog, the value of the initial dissolution rate was much lower than the initial absorption rates at the 2-mg/kg dose. Oral administration of 1 at 2 mg/kg in a 3% polysorbate 80 solution enhanced both the rate and the extent of absorption of the compound. These results confirm the validity of the conclusion that the intestinal absorption of 1 is limited by dissolution rate at low doses.     

Cardiac inotropic responses to calcium and forskolin are not altered by prolonged isoproterenol infusion.

Effects of prolonged isoproterenol infusion upon the density of cardiac calcium channels, calcium-mediated contractile responses, and the ability of forskolin to enhance tension development and cyclic AMP accumulation were studied in ventricular muscle preparations from Sprague-Dawley rats. Isoproterenol infusion (400 micrograms/kg per h s.c., 4 days) significantly decreased calcium channel density (Bmax) in cardiac microsomal membranes as quantified by a 32% decrease in specific [3H]nitrendipine binding sites; binding affinity (KD) was unchanged. A 57% decrease of beta-adrenoceptors confirmed homologous down regulation. To examine functional effects of decreased [3H]nitrendipine binding sites, responses to calcium, BAY K8644 and nifedipine were determined in isolated right ventricular strips. Significant decreases in basal developed tension were observed in muscles from isoproterenol-infused rats. However, concentration-dependent increases in contractility in response to CaCl2 or BAY K8644 were comparable, and the negative inotropic effect of nifedipine was unchanged. Whereas isoproterenol infusion was associated with significantly decreased basal cardiac cyclic AMP concentrations, exposure of ventricular strips from either vehicle- or isoproterenol-infused rats to 10 microM forskolin resulted in comparable increases in cyclic AMP and in developed tension. Cumulative, submaximal concentrations of forskolin also produced similar increases in contractility with maximum responses in ventricular strips from vehicle-infused animals attained at 4.4 microM forskolin. Higher concentrations resulted in automaticity. By contrast, ventricle from isoproterenol-infused animals responded to 14.4 microM forskolin with maximal increases in force of contraction.     

Rate-limiting steps in cytochrome P-450-catalysed reactions: Studies on isotope effects in the O-de-ethylation of 7-ethoxycoumarin

1. Primary deuterium isotope effects ranging from 2 to 6 were measured for the O-de-ethylation of 7-ethoxycoumarin catalysed by microsomal and purified cytochrome P-450 isozymes.

2. Interpretation of the observed deuterium isotope effect in terms of the contribution of the C-H bond-cleavage step to the overall rate requires the determination of the intrinsic isotope effect.

3. Evidence is presented that at least one irreversible step occurs before C-H bond cleavage which kinetically divides the cytochrome P-450 reaction cycle into two portions. Intermolecular primary isotope effects are shown to provide kinetic information only about steps following the irreversible step. Consequently, the isotope effect may be a measure of the rate limitation imposed by the C-H bond-cleavage step in the sequence of events following the irreversible step but be totally independent of other slow steps preceding the irreversible step. These results indicate that multiple rate-limiting steps may exist in cytochrome P-450-catalysed reactions.     

Effect of vasoactive intestinal peptide on myocardial contractility and coronary blood flow in the dog: comparison with isoproterenol and forskolin

The effects of intracoronary injections of vasoactive intestinal peptide (VIP) on left ventricular (LV) dp/dt, coronary blood flow (CBF), and myocardial oxygen consumption (MVO2) were compared with isoproterenol (ISO) and forskolin in 18 dogs using a preparation in which cardiac output, mean systemic arterial pressure, and heart rate were fixed. In eight dogs in which the effects of VIP and ISO were compared using doses ranging from 2 x 10(-12) to 2 x 10(-8) mol, both agents significantly increased LV dp/dt at doses greater than or equal to 6.6 x 10(-10) mol. At maximal doses (2 x 10(-9) to 2 x 10(-8) mol) the effect of ISO was significantly greater than VIP. Both VIP and ISO significantly increased CBF at all doses, but at maximal doses the effect of VIP on CBF was significantly greater than ISO. The increase in CBF relative to the increase in MVO2, an index of direct coronary vasodilation, was significantly greater for VIP compared with ISO. In 10 additional dogs the effects of VIP and ISO were compared with forskolin given in doses ranging from 2 x 10(-9) to 2 x 10(-7) mol. At maximal doses (2 x 10(-7) mol) the increase in LV dp/dt was similar to VIP but significantly less than ISO, whereas the increase in CBF was similar to ISO but significantly less than VIP. The increase in CBF relative to the increase in MVO2 was significantly greater for forskolin compared with ISO, indicating a direct vasodilator effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of isoproterenol and forskolin on amylase release from parotid tissue after chronic pilocarpine administration in rats following ligation removal

The influence of chronic pilocarpine administration on the recovery of the parotid gland from an obstruction was investigated by studying the amylase release induced by secretagogues in rats. Treatment with pilocarpine (5 mg/kg, daily for 7 days) increased amylase release induced by isoproterenol (10(-5) M) or forskolin (10(-5) M) in the parotid tissue of rats after the removal of a 7-day-duct ligation. However, there was no significant difference in the amylase activity of the parotid tissue between pilocarpine-treated rats and control rats. These results suggest that the accelerated recovery of amylase release from parotid tissue in rats chronically treated with pilocarpine may be due to the increased response of amylase release, rather than the increased accumulation of secretory materials in the cells; and furthermore, cyclic AMP-mediated events may be involved in the increased response of amylase release.     

Effect of alkaline cations on cyclic 3' 5'-AMP stimulated lipolysis in rat adipocytes

In Krebs-Ringer phosphate medium, cyclic AMP had little effect on production of free fatty acids by fat cells in vitro, whereas dibutyryl cyclic AMP or epinephrine stimulated the production of free fatty acids. On the other hand, although under non-physiological condition, cyclic AMP was found to stimulate the lipolysis in simple KCl-Tris medium. The stimulation level was similar to that elicited by dibutyryl cyclic AMP. Cyclic AMP also stimulated lipolysis in LiCl-Tris or RbCl-Tris medium, but not in NaCl-Tris medium. In KCl-Tris medium, addition of divalent alkaline cations (Mg2+, Ca2+ and Sr2+, at 40 mM) completely inhibited the stimulation by cyclic AMP, but not those by dibutyryl cyclic AMP and epinephrine. No cyclic AMP-induced lipolysis was observed in homogenized or freeze-thawed cells.     

In vitro effects of glucidamine on adrenergic stimulated lipolysis and on lipoprotein lipase activity in human adipose tissue

Glucidamine, a purified glycoprotein-mucopolysaccharide complex displays dose-dependent lipolytic effect on human adipose tissue in vitro. On adipose tissue lipoprotein lipase activity glucidamine exhibits an heparin-like effect in eluting the enzyme from the tissue stores to the medium of incubation. No activating effects of glucidamine on lipoprotein lipase eluted from human adipose tissue was observed.     

Bronchodilator and antiallergy activity of forskolin

Forskolin is a diterpene from the roots of Coleus forskohli which directly activates the adenylate cyclase and raises cyclic AMP levels in a variety of tissues. Forskolin was studied for its effects on the tone of airway smooth muscle and the immunologic release of leukotrienes and histamine. The bronchospasm induced by inhaled antigen in sensitized guinea pigs was prevented in a dose-related fashion by the intravenous (i.v.) or intratracheal administration of forskolin. Forskolin was more potent than aminophylline and less potent than salbutamol. There was no evidence that forskolin would potentiate the in vivo bronchodilator effects of either salbutamol or aminophylline. Forskolin was approximately 100 times more potent than aminophylline by the i.v. and intratracheal routes to reverse an established allergic bronchospasm. Forskolin given intratracheally also inhibited the bronchospasm to i.v. histamine, with a short duration of action. In vitro forskolin (less than 1 microM) inhibited contractions of lung parenchyma provoked by histamine, LTC4 or antigen. Forskolin (1 microM) also inhibited the immunologically stimulated release of LTD4 and histamine from sensitized guinea pig lung. These studies show that forskolin shares with other agents that elevate cyclic AMP levels the ability to relax airway smooth muscle and inhibit mediator release in vitro and elicit a bronchodilation in vivo.     

Profile of cardiovascular effects of NKH477, a novel forskolin derivative, assessed in isolated, blood-perfused dog heart preparations: comparison with isoproterenol.

Cardiac and coronary vasodilator effects of NKH477, a novel water-soluble forskolin derivative, and isoproterenol, a nonselective beta-adrenoceptor full agonist, were compared in isolated, blood-perfused papillary muscle, sinoatrial (SA) node, and atrioventricular (AV) node preparations of dogs. Both agents were injected intraarterially. The two agents increased the force of contraction of the paced papillary muscle and of the unpaced muscle, and the rate of automaticity of the latter. They increased sinus rate and accelerated AV nodal conduction. In producing these cardiac effects, both agents were similar, although NKH477 was 120-350 times less potent than isoproterenol; however, NKH477 differed distinctly from isoproterenol in that the former increased coronary blood flow more greatly than the latter. Thus, NKH477 is more coronary vasodilatory than positive inotropic, and more positive inotropic than positive chronotropic. Such a cardiovascular profile of NKH477 was similar to that of forskolin, except for the duration of actions; NKH477 was longer-acting than forskolin.     

Bronchodilating activity of forskolin in vitro and in vivo

The effect of forskolin, a receptor-independent adenylate cyclase activator on acetylcholine (ACh)-, histamine- and leukotriene D4 (LTD4)-induced contractions in isolated tracheal spirals was investigated. Forskolin reduced the contraction due to these agonists (IC50S: ACh 5.8 X 10(-6) M; histamine 4 X 10(-7) M; LTD4 3.1 X 10(-6) M). Forskolin was also effective against ACh- and histamine-induced contraction when added during contraction. In ovalbumin-sensitized guinea pigs, intravenously and intraduodenally administered forskolin significantly reduced the bronchospasm due to aerosol administration of ovalbumin. These results suggest that forskolin could prove useful in delineating the role of cyclic nucleotides in pulmonary smooth muscle responses.     

Rate-limiting step in the metabolism of polar and non-polar monoamines in lung and liver

1 Uptake of the non-hydroxylated amines, [14C]-tryptamine and [14C]-benzylamine in rat lung, infused through the pulmonary circulation, was not saturable over the concentration range 2.5-1,000 microM. 2 The kinetic constants for deamination of a variety of hydroxylated and non-hydroxylated monoamines in liver, perfused via the portal circulation, with monoamine oxidase activity in homogenates of liver were similar. 3 In lung, uptake of both [14C]-tryptamine and [14C]-benzylamine was inhibited by the monoamine oxidase inhibitor deprenyl and competition occurred between tryptamine, benzylamine and beta-phenylethylamine for uptake. 4 These results indicate that tryptamine and benzylamine metabolism in lung is not limited by uptake, unlike that of the hydroxylated amines 5-hydroxytryptamine and noradrenaline and that uptake resembles that of beta-phenylethylamine in lung. 5 the selectivity of the lung in handling monoamines is not shown by the liver, suggesting that lung has a specific role in clearing certain biogenic monoamines.     

Prostacyclin and lipolysis in rat fat cells

In rat fat cells incubated for 15 min at 37 degrees and pH 8.5, glycerol release was highly stimulated both by norepinephrine and by theophylline. Prostacyclin (PGI2) (10(-8)-10(-7)M) did not alter the basal rate of glycerol release but potentiated the lipolytic effect of 2 X 10(-6)M norepinephrine. The rate of norepinephrine-induced glycerol release was increased by PGI2 during 10 min of incubation and then maintained for the next 5 min. Lipolysis induced by concentrations of norepinephrine which produced maximal effects was not altered by PGI2. PGI2 (10(-7)-10(-6)M) also potentiated the effect of 5 X 10(-4)M theophylline on glycerol release, but antagonized the stimulation induced by a maximally effective concentration of the methylxanthine (2 X 10(-3)M). Incubation of the cells with norepinephrine in the presence of 2 X 10(-4) or 5 X 10(-4)M theophylline caused a loss of the potentiating effect of PGI2 on norepinephrine-induced lipolysis. In the presence of 10(-3)M theophylline, the lipolytic action of norepinephrine was inhibited by PGI2. In fat cells incubated with adenosine deaminase (0.5 U/ml), 2.5 X 10(-7)M PGI2 did not alter the response to 5 X 10(-4)M theophylline and inhibited the effect of norepinephrine both in the absence and in the presence of theophylline. The present results show that, under appropriate experimental conditions, PGI2 may act as a lipolytic agent in isolated fat cells and that some kind of interaction exists between stimulation of methylxanthine-sensitive adenosine receptors and stimulation of PGI2 receptors.     

Relaxant effects of forskolin in smooth muscle

Forskolin was found to cause concentration dependent, reversible relaxations of isolated smooth muscle preparations including rat aorta, bovine coronary artery, canine coronary artery, guinea pig taenia caeci and rabbit small intestine. The relaxant effects of forskolin in guinea pig taenia caeci and rabbit small intestine were potentiated by cyclic nucleotide phosphodiesterase inhibitors Ro 20-1724 and MIX. In rabbit small intestine, Ro 20-1724 also potentiated the relaxant effects of isoproterenol but not those of verapamil or 2-chloroadenosine. However, in bovine coronary arteries the phosphodiesterase inhibitors had to be used at concentrations of 100 nM or below because of relaxant effects and did not alter forskolin-induced relaxations. Forskolin caused a concentration dependent activation of adenylate cyclase in broken cell preparations from guinea pig taenia caeci and rabbit small intestine, exceeding the stimulatory effect of 10 mM NaF. These results indicate that relaxations of smooth muscle by forskolin are mediated by cyclic AMP and, in turn, that cyclic AMP may well serve as an intracellular mediator of physiological relaxant stimuli.