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Effect of isoprenaline and phenylephrine on the adenosine 3',5' monophosphate content and mechanical activity of cold stored and fresh taenia caecum from the guinea pig
British Journal of Pharmacology
Abstract
1 Cold storage treatment of the guinea‐pig taenia caecum had a greater inhibitory effect on the isoprenaline‐induced relaxation than that induced by phenylephrine. Prolonged cold storage (12‐14 days) almost abolished the effect of isoprenaline but only reduced the phenylephrine effect. The ED 50 of cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) that elicited muscle relaxation was not altered by the prolonged cold storage.
2 After cold storage treatment, tissue cyclic AMP content was decreased; however, isoprenaline still caused a dose‐dependent increase in the cyclic AMP level. The threshold dose of isoprenaline for cyclic AMP accumulation was the same in fresh and cold‐stored preparations.
3 In the fresh preparation, the onset of the isoprenaline (10 ⁻⁶ M)‐induced relaxation preceded the increase in tissue cyclic AMP.
4 Isoprenaline, phenylephrine, adrenaline and noradrenaline at doses (ED 50 ) sufficient to induce muscle relaxation did not always increase the cyclic AMP level.
5 Similarly, the responses to papaverine and nitroglycerine were not accompanied by an increase in cyclic AMP.
6 The adenylate cyclase and phosphodiesterase (low and high Km ) activities of taenia caecum were not attenuated by the prolonged cold storage.
7 Propranolol inhibited both the isoprenaline‐induced relaxation and cyclic AMP accumulation; however, the pA 2 values were significantly different for the two events.
8 Based on these results, both the relaxation and cyclic AMP accumulation caused by isoprenaline are mediated by activation of β‐adrenoceptors but are independent phenomena.
... Activation of beta-adrenoceptors is generally known to stimulate adenylate cyclase (Sutherland and Rail, 1960;Stiles, Carm, and Lefkowitz, 1984). In taenia coli myocytes of guinea pig, it also increases the intracellular cAMP concentration (Honda, Katsuki, Miyahara, and Shibata, 1970). However, it is not clear whether the increase of K + conductance by ISO in smooth muscle cells could be mainly or exclusively accounted for by the activation of adenylate cyclase. ...
In freshly dispersed guinea pig taenia coli myocytes the activity of the large conductance Ca(2+)-activated K+ channel (maxi-K+ channel) predominates. The open probability (Po) of this channel is increased by micromolar concentrations of the beta-adrenergic agonist isoproterenol (ISO). Low concentrations of cholera toxin (CTX, 1 pM) and guanosine 5'-O-2-thiodiphosphate (GDP beta S, 0.5 mM) suppress the ISO-induced increase of Po. Higher concentrations of CTX (e.g., 0.5 nM) as well as forskolin and dibutyryl cAMP increase the Po. 1,9-Dideoxyforskolin, the forskolin analogue, which lacks the adenylate cyclase-stimulating effect, does not. A specific protein kinase A inhibitor (Wiptide), applied intracellularly via diffusion from the patch electrode, suppresses the ISO-induced increase of whole-cell outward K+ current during step depolarization. In contrast, intracellularly applied protein kinase C (19-36), a specific protein kinase C inhibitor, has no effect on the whole-cell current. TMB-8, an inhibitor of intracellular calcium mobilization, does not affect either the whole-cell outward K+ current during step depolarization or the Po. These observations show that ISO increases the Po of the maxi-K+ channels in the guinea pig taenia coli myocytes through the G protein-adenylate cyclase-protein kinase A system.
. In order to study the effects of local stimulation of β-adrenoceptors on limb blood flow during exercise and on muscle metabolism, the non-selective β-adrenoceptor agonist, isoprenaline, was infused intra-arterially into the forearm of six healthy, young subjects. Isoprenaline caused a marked increase in forearm blood flow not only at rest but also during dynamic forearm exercise. The forearm release of lactate increased considerably during drug infusion whereas glucose uptake decreased. The net uptake of free fatty acids during exercise decreased. There were no systemic effects of isoprenaline. These findings suggest that the resistance vessels are responsive to p-adrenergic vaso-dilatation also during muscle exercise. The increase in blood flow through the exercising forearm may be secondary to an increased muscle metabolism. The increased lactate release from the exercising muscles indicates an increased muscle glycogen breakdown during P-adrenoceptor stimulation.
We examined the relationships between the relaxation mediated by β-adrenoceptor and either the associated cyclic AMP production or the activation of cyclic AMP-dependent protein kinase (A-PK) in canine saphenous and portal veins. In the saphenous vein constricted with methoxamine, isoproterenol caused concentration-dependent relaxation (maximum relaxation 92.7%), and concomitant increases in cyclic AMP and A-PK activity ratio from (52.8 to 73.5%). The portal vein was only slightly relaxed by isoproterenol (14.7% in the longitudinal strips) after constriction with methoxamine, while cyclic AMP and A-PK activation increased significantly. Isoproterenol markedly activated A-PK of the portal vein after KCl construction (from 52.6 to 74.6%) but the maximum relaxation was only slight (13.3%). The portal vein also showed a smaller relaxation response to either forskolin or dibutyryl cyclic AMP than the saphenous vein. These results indicated that the relationship between the relaxation response to isoproterenol and either cyclic AMP production or A-PK activation was different in the saphenous and portal veins.
Both parameters of isometric contraction, Td and dT/dt max, of the isolated hemidiaphragm of the rat, as measured during the action of isoprenaline in the course of direct electrical stimulation, were significantly affected by lowering the temperature of the bath to 18°C. The action of isoprenaline was only depressed, whereas the effect of aminophylline was completely blocked by this procedure. The effect of calcium chloride in antagonizing the blocking action of di-Na-EDTA was even stronger at 18°C than at 36°C. Calcium ionophores Ro-2-2985 and A 23187 produced an increase in Td and dT/dt max when used in lower concentrations, but higher concentrations regularly produced depression or block of the isometric contraction. The increasing effect of Ro-2-2985 on Td and dT/dt max was completely reversed by lowering the temperature to 18°C. It was shown that at 18°C neither calcium nor isoprenaline antagonized the blocking action of verapamil on Td and dT/dt max, thus indicating the alteration of calcium channels at low temperatures. A 23187 potentiated the action of isoprenaline on Td and dT/dt max, whereas Ro-2-2985 blocked it. Similarly, the inrcreasing effect of db-cAMP on Td and dT/dt max was reversed by Ro-2-2985. It is concluded that both lowering the temperature of the medium and the calcium ionophores produce changes in the response to isoprenaline and aminophylline, during direct electrical stimulation, in a way which is compatible with the view that the interaction between calcium and cAMP system is essential to the isometric contraction of skeletal muscle.
2‐2′‐Pyridylisatogen tosylate (PIT) slowly relaxed taenia caeci preparations of the guinea‐pig in a concentration‐dependent manner (threshold 2.5 μ m ). The relaxant effect did not show tachyphylaxis.
The relaxation was not affected by tetrodotoxin (0.3 μ m ), guanethidine (17 μ m ) nor by a combination of phentolamine (36 μ m ) and propranolol (4 μ m )
In taenia caeci preparations suspended in K ⁺ ‐depolarizing, Ca ²⁺ ‐free Ringer, addition of Ca ²⁺ (0.1 to 30 m m ) resulted in a slow contraction. PIT (50 μ m ) and papaverine (15 μ m ) antagonized these contractions, whereas indomethacin (28 μ m ) was ineffective.
Although PIT (50 μ m for 30 min) caused a relaxation of the taenia, and, when the tone of the preparations was restored with carbachol, antagonized adenosine 5′‐triphosphate (ATP)‐induced relaxations, relaxation of the taenia with papaverine (30 μ m for 5 min) did not antagonize ATP‐induced relaxations. It is concluded that the relaxant and ATP‐receptor blocking actions of PIT are independent properties of the compound.
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.
The effects of beta 1- and beta 2-selective and nonselective adrenergic agonists and antagonists were studied in the cat and rabbit superior cervical ganglion (SCG) in situ and in vitro. In experiments on the cat SCG in situ beta-adrenergic agonists induced an increase in positive afterpotential (PAP) of compound ganglionic action potential evoked by preganglionic stimulation in the following order of potency: isoprenaline (ISO) greater than salbutamol much greater than tazolol. Beta-adrenergic antagonists inhibited the ISO response in the following order of potency: propranolol greater than exaprolol much greater than H 35/25. Practolol did not exhibit beta-adrenergic blocking action; on the contrary, in higher doses it enhanced the response to ISO. In higher doses all beta-adrenergic antagonists tested depressed ganglionic transmission in an approximately similar range of doses. In experiments in vitro, using sucrose-gap technique. ISO induced depolarization of the cat SCG and this effect was inhibited in a dose-dependent manner by propranolol. Beta-adrenergic agonists did not induce depolarization of the rabbit SCG in experiments in situ and in vitro. The level of noradrenaline and dopamine in the cat and rabbit SCG did not differ significantly. Low level of adrenaline was found in the cat SCG; however, no adrenaline was detected in the rabbit SCG. No statistically significant change of cyclic AMP content was observed in the cat and rabbit SCG or in the incubation medium under conditions similar to those in which ISO induced depolarization of the cat SCG. It is concluded that the beta-adrenergic receptors present in the cat SCG resemble the beta 2-subtype and that the presence of an electrophysiologic response to beta-adrenergic agonists appears to be species-dependent. The depolarization of the cat SCG due to beta-adrenergic receptor stimulation is not coupled with cyclic AMP increase.
Inhibition by isoprenaline of the aggregatory response of human and rat platelets induced by various excitatory agonists is blocked by beta 2-adrenoceptor antagonists. beta 1-Adrenoceptor antagonists are ineffective. beta 2-Adrenoceptor agonists cause inhibition of the response of human platelets to various excitatory agonists. The maximal extent of inhibition is less than that observed for isoprenaline. beta 1-Adrenoceptor agonists fail to cause detectable inhibition of this response. Neither beta 1 nor beta 2-adrenoceptor agonists cause inhibition of the response of rat platelets to excitatory agonists. Only beta 2-adrenoceptor agonists block the inhibitory response to isoprenaline. The extent of inhibition by isoprenaline is a function of the excitatory agonist used and in human platelets is correlated with the ability of that agonist to suppress elevated platelet cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. Inhibition by isoprenaline is prevented in the presence of an inhibitor of adenylate cyclase. Isoprenaline increases platelet cyclic AMP levels with an EC50 similar to that required to observe inhibition of the aggregatory response. These data indicate that human platelets carry beta 2-adrenoceptors whose occupancy causes inhibition of the response to excitatory agonists as a consequence of elevation of platelet cyclic AMP. The beta-adrenoceptor present on rat platelets also appears to be of the beta 2-subtype.
In order to study the effects of local stimulation of beta-adrenoceptors on limb blood flow during exercise and on muscle metabolism, the non-selective beta-adrenoceptor agonist, isoprenaline, was infused intra-arterially into the forearm of six healthy, young subjects. Isoprenaline caused a marked increase in forearm blood flow not only at rest but also during dynamic forearm exercise. The forearm release of lactate increased considerably during drug infusion whereas glucose uptake decreased. The net uptake of free fatty acids during exercise decreased. There were no systemic effects of isoprenaline. These findings suggest that the resistance vessels are responsive to beta-adrenergic vasodilatation also during muscle exercise. The increase in blood flow through the exercising forearm may be secondary to an increased muscle metabolism. The increased lactate release from the exercising muscles indicates an increased muscle glycogen breakdown during beta-adrenoceptor stimulation.
Even after 21 days of cold storage at 2 degrees C, rabbit atria still showed a detectable response only to isoproterenol while other cardiostimulants (norepinephrine, ouabain, anthopleurin-A and nicotine) failed to activate tissues stored up to 14 days. The specific fluorescence of catecholamines disappeared from the tissue after 5 days of cold storage. The maximum decrease in oxygen consumption and tissue catecholamine content was observed after 14 days and a concomitant decrease in tissue ATP content, Na+-K+, Ca2+ and Mg2+-ATPases was nearly maximal after 7 days of cold storage. The cold storage had no apparent effect on the ultrastructures of myocardial cells except a disappearance in glycogen granules.
In freshly dispersed guinea pig taenia coli myocytes the activity of the large conductance Ca(2+)-activated K+ channel (maxi-K+ channel) predominates. The open probability (Po) of this channel is increased by micromolar concentrations of the beta-adrenergic agonist isoproterenol (ISO). Low concentrations of cholera toxin (CTX, 1 pM) and guanosine 5'-O-2-thiodiphosphate (GDP beta S, 0.5 mM) suppress the ISO-induced increase of Po. Higher concentrations of CTX (e.g., 0.5 nM) as well as forskolin and dibutyryl cAMP increase the Po. 1,9-Dideoxyforskolin, the forskolin analogue, which lacks the adenylate cyclase-stimulating effect, does not. A specific protein kinase A inhibitor (Wiptide), applied intracellularly via diffusion from the patch electrode, suppresses the ISO-induced increase of whole-cell outward K+ current during step depolarization. In contrast, intracellularly applied protein kinase C (19-36), a specific protein kinase C inhibitor, has no effect on the whole-cell current. TMB-8, an inhibitor of intracellular calcium mobilization, does not affect either the whole-cell outward K+ current during step depolarization or the Po. These observations show that ISO increases the Po of the maxi-K+ channels in the guinea pig taenia coli myocytes through the G protein-adenylate cyclase-protein kinase A system.
Cyclic nucleotide phosphodiesterase has been prepared from the liver, skeletal muscle, heart muscle, kidney, epididymal fat pad, and brain cortex of the rat. Kinetic analysis indicated that all of these tissues, with the exception of liver exhibit complex behavior indicative of multiple enzymatic activities. The phosphodiesterase activities of brain cortex, kidney, and adipose tissue have been resolved on Bio-Gel A-1.5m gel filtration columns into three fractions. These are an excluded fraction, a high molecular weight fraction in which the enzymatic affinity for cyclic guanosine 3',5'-monophosphate is greater than that for cyclic adenosine 3',5'-monophosphate, and a lower molecular weight fraction with a high affinity for cyclic adenosine 3',5'-monophosphate. The lower molecular weight enzyme appears to be negatively cooperative. The muscle preparations have little, if any, of the excluded fraction which in kidney, adipose tissue, and brain has kinetic behavior similar to that of the lower molecular weight enzyme. Liver has no detectable phosphodiesterase activity in any except the intermediate fraction which has a molecular weight of approximately 400,000. This enzymatic activity has been purified 88-fold by centrifugation, ammonium sulfate fractionation, and gel filtration. The potential for metabolic control through regulation of cyclic nucleotide phosphodiesterase is discussed in view of the existence of at least two distinct enzymatic activities in all of tissues studied except the liver.
Publisher Summary The ATPase activity which requires Na + , K + , and Mg ++ together and is inhibited by cardiac glycosides is a part of the enzyme system for the stoichiometric transport of Na + outward and K + inward across cell membranes. It is widely distributed in animal tissues and species. Organs, which transport Na+ and K+ to energize electrical activity or secretion, show much activity. The chapter examines the preparation, purification, and properties of sodium and potassium-stimulated ATPase. From the rate of release of inorganic phosphate from ATP in the presence of Na + , K ++ , and Mg ++ is subtracted the rate of release in the presence of Mg ++ and a cardiac glycoside inhibitor, such as ouabain. Released inorganic phosphate may be measured simply by adding 2.5 ml of 0.48 M HC104, mixing, filtering, and taking a 2.0-ml aliquot for assay. The method is described that separates phosphomolybdate from ATP by extraction into butyl acetate. The general principle of purification procedure described in the chapter is to obtain a microsomal fraction by differential centrifugation of a sucrose homogenate and to form dispersed and activated particles with a detergent, urea, or a concentrated iodide solution. Aging, freezing and thawing, and mild detergents may improve the activity or sensitivity of fresh homogenates or microsomal fractions. The K m for ATP is about 0.3 mM with Mg ++ in slight excess. As the amount of Mg ++ is reduced with maximal ATP, the activity decreases but the sensitivity increases. The Km for Na + is about 1.5 mM and for K + about 0.4 mM. The apparent affinity of the Na + -site for K + is about 7-fold less than for Na + whereas the apparent affinity of the K + -site for Na + is about 160-fold less than for K + .
: Papaverine and nitroglycerine relaxed rabbit colon muscle. The relaxation was preceded by an increase of the cyclic AMP content of the muscle. There was a correlation between the degree of relaxation and the increase of cyclic AMP. A reduction of ATP content was obtained with these drugs; this effect was not present in the Ca++-poor preparation in spite of the fact that the cyclic AMP level was still increased. Papaverine but not nitroglycerine reduced the phosphodiesterase activity of the intact rabbit colon. In a homogenate of colon muscle a marked inhibition of phosphodiesterase activity was obtained. The phosphodiesterase activity from different cell fractions was also studied. Phosphodiesterase from the cytoplasmic fraction was activated by papaverine and nitroglycerine, while that of a Ca++-binding microsomal fraction like that of mitochondria was inhibited. Diazoxide and hydralazine relaxed bovine mesenteric artery but not rabbit colon. The relaxation was associated and correlated with an increase in the cyclic AMP content. A reduction in the ATP content and an activation of phosphorylase a activity was also obtained; the ATP reduction was Ca++-dependent. A weak inhibitory effect on phosphodiesterase activity was observed in hornogenate of the artery after diazoxide. Hydralazine activated phosphodiesterase in a homogenate of mesenteric artery in a low concentration but inhibited it in a high concentration. It is suggested that cyclic AMP mediates, at least partly, the relaxing action of these four drugs probably by stimulating a Ca++-binding process in the smooth muscle cell.
In colonic muscle from the rabbit a relaxing effect was induced by stimulation both of adrenergic α-receptors with phenylephrine and of β-receptors with isoprenaline. The α-receptor induced relaxation was not accompanied initially by any metabolic effects. After a latency period of 3 min did a decrease in the phosphorylase α activity, a decrease in the concentrations of hexose phosphates and lactate and an increase in the concentration of high-energy phosphate compounds (ATP and CrP) become evident. There was a decrease of the cyclic AMP content. These effects were inhibited by dibenamine. The relaxation induced via adrenergic β-receptors was preceded by an increase in the cyclic AMP content and the phosphorylase α activity, an increase in the concentration of hexose phosphates and lactate and an initial decrease of the ATP and CrP concentrations, which after 10 min was succeeded by an increase. The magnitudes of the relaxing and metabolic responses following stimulation of preceptors were correlated with regard to time and dose. These responses were blocked by an adrenergic β-receptor blocking agent (sotalol) and were also eliminated in carbohydrate-poor muscle. The relaxing and metabolic effects of isoprenaline could be reproduced completely by cyclic AMP, only incompletely by 5'AMP and not at all by other tested cyclic nucleotides.
In experiments on taenia coli from rabbits, a study was made of the mechanisms whereby catecholamines induce a relaxing effect. In a low concentration (≤2.1 × 10–6 g/ml), l-phenylephrine selectively stimulated adrenergic a-receptors; this effect was blocked completely by the adrenergic α-blocking agent dibenamine. In a low concentration (≤ 3.7 × 10–8 g/ml), 1-isoprenaline selectively stimulated β-receptors; this effect was blocked completely by the adrenergic β-receptor blocking agent sotalol. In higher concentrations, phenylephrine and isoprenaline stimulated both adrenergic α-and β-receptors; their relaxing effect could only be blocked completely by a combination of dibenamine and sotalol. In taenia coli from rabbits, the threshold concentration for β-receptor-mediated relaxation was lower than that for relaxation induced via a-receptors; in taenia coli from guinea pigs the reverse was found. The relaxant effect induced via adrenergic a-receptors was blocked selectively by factors such as cold treatment, digitalis glycosides, desoxycorticosterone, which could be assumed to decrease the ionic gradients of K+ and Na+ across the cell membrane or reduce its ionic permeability. — On successive reduction of the carbohydrate reserves of the muscle, the relaxation induced via adrenergic β-receptors was inhibited more rapidly than that induced via α-receptors. Theophylline and puromycin, which inhibit the enzymatic hydrolysis of cyclic AMP, potentiated selectively the β-receptors mediated relaxation.
Physiologically active concentrations of epinephrine produce an increase in the concentrations of cyclic adenosine 3′,5′-phosphate in isolated intestinal smooth muscle preparations from the taenia coli of the guinea-pig. This effect is not associated with a change in the concentrations of hexose phosphate esters in this tissue.
The intracellular level of cyclic AMP in the taenia from the guinea pig caecum was significantly increased by isoprenaline in a dose-dependent manner. Papaverine also increased the intracellular cyclic AMP level, but a synthetic antispasmodic agent, Aspaminol (1,1-diphenyl-3-piperidinobutanol hydrochloride) did not modify it. The results suggest that there are two mechanisms for the so-called papaverine-like actions. The increase in the intracellular cyclic AMP concentration produced by isoprenaline was prevented by propranolol. In taenia depolarized by KCl, papaverine caused relaxation but isoprenaline did not. This difference can be explained by the observation that the intracellular cyclic AMP level in the KCl-depolarized taenia is increased by papaverine but not by isoprenaline.
Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low Km cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μm) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μm cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP (Ka ∼- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP (Ki ∼- 450 μM) and cyclic AMP (Ki ∼- 150 μM) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 mm) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.
Studies on the role of cyclic nucleotides, that is cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in smooth muscle relaxation began in the mid 1960s. If the role of cAMP (cyclic nucleotides) in smooth muscle relaxation appears to be relatively straight-forward, the role of cGMP (cyclic nucleotides) was less clear. The initial studies suggested a role for cGMP in mediating smooth muscle contraction. These conclusions were based on correlations between cGMP content and contraction in response to contractile agonists such as acetylcholine. A more thorough analysis, however, revealed that the elevations in cGMP observed with acetylcholine actually occurred after the onset of contraction, suggesting that cGMP was produced in response to intracellular Ca2+ elevations. It was not until the late 1970s that several investigators demonstrated that nitrogen oxide containing vasodilators such as nitroprusside and nitroglycerine elevated cGMP in smooth muscle tissues. These findings suggested that cGMP mediated relaxation of smooth muscle, a finding that was eventually supported by the discovery of the potent smooth muscle relaxing effects of cGMP analogs. Nevertheless, there have been dissociations reported between cyclic nucleotide elevating compounds or analogs on one hand, and the relaxation of different smooth muscle preparations on the other. Uterine smooth muscle relaxation, for example, is unaffected by elevations in cGMP. Vascular and bronchial smooth muscle, however, are highly responsive to the relaxing effects of cGMP. The diversity of responses to cyclic nucleotides in the various smooth muscle types may reflect different mechanisms that lead to the decrease in tone in the different types of smooth muscle.
An adenosine 3',5'-monophosphate-dependent protein kinase, which catalyzes the phosphorylation of histone by ATP, has been
purified from bovine brain and some of its properties have been studied. Under appropriate conditions, the activity of the
enzyme was stimulated more than 20-fold by adenosine 3',5'-monophosphate, with an apparent Km for the cyclic nucleotide of about 2.5 x 10-7 m. The activation of the enzyme by adenosine 3',5'-monophosphate was associated with a decrease in Km of the enzyme for ATP that occurred in the presence of the cyclic nucleotide. In contrast, adenosine 3',5'-monophosphate
had no significant effect on the Km of the enzyme for the histone.
The optimum pH for the activity of the enzyme was 6.5. The purified enzyme had an absolute requirement for a divalent metal.
The stimulation by cyclic AMP was greatest in the presence of Mg++, Mn++, or Co++. Cyclic AMP strongly inhibited enzyme activity in the presence of Ca++. The activation observed in the presence of adenosine 3',5'-monophosphate was also observed with the 3',5'-monophosphate
derivatives of inosine, guanosine, uridine, and cytidine as well as with dibutyryl adenosine 3',5'-monophosphate, but much
higher concentrations of these other cyclic nucleotides were required. Deoxythymidine 3',5'-monophosphate, the only cyclic
deoxyribonucleotide studied, was completely inactive. Adenosine and ADP were inhibitors of the enzyme.
A simple and sensitive assay for adenosine 3':5'-cyclic monophosphate (cAMP) has been developed that is based on competition for protein binding of the nucleotide, presumably to a cAMP-dependent protein kinase. The nucleotide-protein complex is adsorbed on a cellulose ester filter. Assay conditions are such that a binding constant approaching 10(-9) M is obtained, and the assay is thus sensitive to 0.05-0.10 pmol of cAMP.
Theophylline, diazoxide, and papaverine in low concentrations relaxed the uterus with minimal or no elevation of cyclic AMP (cAMP) levels. In higher concentrations, theophylline relaxed the uterus and increased its cAMP levels, but imidazole reversed the increase in cAMP without causing recontraction. Imidazole and NaF caused uterine contractures but did not detectably decrease cAMP levels until several minutes after the onset of contractures. The uterine relaxations produced by theophylline and/or dibutyryl cAMP in amounts which increased uterine cAMP were not reversed by propranolol. These results eliminate the possibility that propranolol interfered with a relaxant action of cAMP. Along with previous data, these results also show that uterine contractile activity was not determined primarily by the general levels of cAMP and that phosphodiesterase activity in the uterus was insufficient to rapidly affect these cAMP levels. Also, substances like theophylline, diazoxide, and papaverine, postulated to inhibit phosphodiesterase activity, did not bring about their relaxant effects by this mechanism.
In rabbit colon muscle there was a quantitative correlation and a co-ordination in time between relaxation and an increase in cyclic AMP on stimulation of adrenergic β-receptors. The cyclic AMP content had increased before the muscle had started to relax. There was an increase in the phosphorylase a activity and reduction of the ATP content. The adrenergic β-receptor blocking agent sotalol inhibited the relaxation and the increase in cyclic AMP content. Reduction of the Ca++ content of the muscle decreased the cyclic AMP content and the phosphorylase a activity. These effects were restored on addition of Ca++ ions. In a Ca-poor muscle adrenergic β-receptor stimulation still produced an increase in cyclic AMP content and phosphorylase a activity but there was an increase in the ATP content instead. The reduction of cyclic AMP which followed an adrenergic a-receptor stimulation was eliminated in the Ca-poor muscle. The existence of a Ca++-accumulating ATP-utilizing mechanism, stimulated by cyclic AMP, might explain the relation between relaxation, cyclic AMP and Ca++ and adrenergic β-receptor stimulation.
The effects of prolonged cold storage on the mechanical and membranal responses to stimulation of α‐ and β‐adrenoceptors by phenylephrine and isoprenaline, respectively, were studied on the guinea‐pig taenia caecum.
Cold storage invariably caused a decrease in the resting membrane potential, and this effect was enhanced as the duration of treatment was prolonged.
After cold storage (18 days) the tissue potassium ion content (89·7 ± 1·7 mmol/kg wet wt.) was decreased to 30·5 ± 1·9 mmol/kg wet wt. whereas that for sodium (69·2 ± 1·4 mmol/kg wet wt.) increased to 134·0 ± 2·3 mmol/kg wet wt.
In the fresh preparations, phenylephrine (1 and 2 μ m ) caused a cessation of spontaneous action potentials, accompanied by hyperpolarization of the membrane and relaxation of the muscle. These effects were markedly diminished after 18 days of cold storage. Isoprenaline (1 and 2 μ m ) also blocked the action potentials and caused a concomitant muscle relaxation, but in most cases the hyperpolarization was not observed. After 14 days of cold storage these mechanical and membranal changes associated with isoprenaline treatment were not demonstrable in most preparations.
Nicotine (5 μ m and 50 μ m ) produced a biphasic effect, cessation of the action potential, hyperpolarization and subsequent relaxation followed by a long lasting depolarization, an accelerated discharge of action potentials and an increase in muscle tension. After a few days of cold storage the hyperpolarization effect disappeared but the intensity of the long‐lasting depolarization as well as the contractile effects were increased. After cold storage for more than 7 days, nicotine did not affect mechanical or electrical activity.
Dibutyryl 3′5′ cyclic AMP (1 μ m to 500 μ m ) failed to affect the mechanical and electrical activities of taenia caecum.
Phenylephrine and isoprenaline had no effect on the high potassium‐depolarized taenia.
These observations suggest that the electro‐mechanical effect of an α‐adrenoceptor stimulant on the guinea‐pig taenia caecum is more resistant to cold treatment than that of a β‐adrenoceptor stimulant. This inhibitory system of stimulation of both α‐ and β‐receptors of guinea‐pig taenia caecum may react by different mechanisms. The results also demonstrate that cold storage itself changes the membrane permeability to ions and the tissue ion content (Na ⁺ and K ⁺ ) of smooth muscle of guinea‐pig taenia caecum.
In the following investigation we have found that an influence on the c-AMP metabolism is of significance both during relaxation and contraction of bovine mesenteric artery. All tests were done with isometrically contracted muscle. Cyclic AMP was analyzed according to KAKIUCHI and RALL (Mol. Pharmacol. 4, 367–378 (1968)).
Cold storage (2° C) treatment progressively reduced noradrenaline uptake by the taenia caecum of the guinea‐pig. After 15 days of cold treatment, [ ³ H]‐noradrenaline uptake by tissue was reduced to about 10% of control. On the other hand, prolonged cold storage failed to decrease [ ³ H]‐adenosine uptake by the taenia caecum.
Cocaine (10 μ m ) inhibited noradrenaline uptake by about 82% but nucleoside uptake was inhibited by 20%. Cocaine treatment failed to decrease the residual noradrenaline uptake in the cold stored strips (more than 10 days). Phenoxybenzamine (1 μ m ) or oligomycin (1 μg/ml) treatment decreased the tissue adenosine uptake to about 34% and 28% of the respective controls.
Based on thin layer chromatography, it was estimated that approximately 68% of [ ³ H]‐adenosine was converted into and retained as [ ³ H]‐ATP in the fresh tissues, a small fraction was accountable as [ ³ H]‐adenosine (18%) but virtually no [ ³ H]‐AMP, [ ³ H]‐cyclic AMP or [ ³ H]‐ADP was detected. Similar distribution of radioactivity of nucleotides was observed in tissues cold stored for 8 days.
The inhibition of the mechanical activity of taenia by noradrenaline (10 μ m ), papaverine (100 μ m ) and nitroglycerine (100 μ m ) was accompanied by [ ³ H]‐adenine nucleotide but not [ ³ H]‐noradrenaline release. Treatment with phentolamine and propranolol (both 1 μ m ) had no effect on the adenine nucleotide release elicited by nicotine and electrical field stimulation, whereas such treatment reduced the inhibitory action of both stimuli.
These results suggest that the nucleotide release after application of electrical and chemical stimulation may be from an extraneurogenic source. Thus, we conclude that ATP or a related nucleotide is not the chemical transmitter of the non‐adrenergic inhibition in the taenia caecum of the guinea‐pig.
Relaxation produced by phenylephrine, epinephrine, norepinephrine and isoproterenol on guinea-pig taenia coli was not modified after cold storage of up to 6 days. These responses of the cold stored strip to phenylephrine, epinephrine and norepinephrine were prevented by pretreatment with alpha-blocking agents (phenoxybenzamine and phentolamine) but not by treatment with beta-blocking agents (propranolol and MJ 1999). The opposite was observed for the response to isoproterenol.On the other hand, the contraction produced by methacholine, (10−7 M), physostigmine (10−7 M), KCl (10 mM), and Ba (1 mM) was decreased after 3–6 days cold storage. Similarly, the relaxation produced by ATP (10−6 - 10−8 M) and 3',5'-cyclic AMP (10−4 - 10−5 was also decreased after 3−6 days cold storage; the response of fresh preparations to these agents was not prevented by either alpha- or beta-blocking drugs. In low Ca media (0.6 mM) catecholamine-induced relaxation of fresh strip was not changed, but that of ATP and 3',5'-cyclic AMP was decreased.These results indicate that cold storage does not interfere with the inhibitory system related to catecholamine but does with ATP and 3',5'-AMP. Also, prolonged cold storage does affect the membrane system involved in methacholine, KCl, Ba and physostigmine-induced contractions. This data suggests that the relaxation mechanisms of catecholamines may be different from that of exogenous high energy substances.
An adenylate cyclase that is activated specifically by low concentrations of dopamine has been demonstrated in homogenates of caudate nucleus of rat brain. A half-maximal increase in the activity of the enzyme occurred in the presence of 4 muM dopamine. Concentrations of dopamine as low as 0.3 muM stimulated the activity of the enzyme. The adenylate cyclase activity of the homogenates was also stimulated by low concentrations of apomorphine, a substance known to mimic the physiological and pharmacological effects of dopamine. The stimulatory effect of dopamine was blocked by low concentrations of either haloperidol or chlorpromazine, agents known to block the actions of dopamine in mammalian brain. The results suggest that dopamine-sensitive adenylate cyclase may be the receptor for dopamine in mammalian brain. The isolation of this enzyme from caudate nucleus should facilitate the search for new therapeutic agents useful in the treatment of extrapyramidal diseases.
In the guinea‐pig taenia caecum, fluorescent adrenergic fibres terminate in both muscle layers. The density of these fibres is greater in the taenia than in the underlying circular muscle layer. The myenteric plexus and individual ganglion cells are also densely innervated by intensely fluorescent adrenergic nerve fibres.
After three days of cold storage, the specific fluorescence disappeared from all tissue layers of the taenia caecum and smooth muscle fibres. In contrast, cholinesterase active substances were still demonstrable in all tissue layers even after seven days of cold storage but the density of these substances was decreased.
Cold storage (3–7 days) decreased the tissue noradrenaline content and did not modify the cholinesterase enzyme activity (4 days).
In cold stored strips, the inhibitory response to nicotine, 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP) or electrical transmural stimulation was abolished and enhancement of the contractile response occurred. Cold storage also inhibited the inhibitory action of tyramine. Similar results were observed after reserpine treatment.
In fresh taenia, the relaxation produced by nicotine, DMPP and electrical transmural stimulation was inhibited by adrenoceptor blocking agents and bretylium. In cold storage preparations, contraction produced by these stimuli was blocked by parasympathetic blocking agents and potentiated by anticholinesterase. These results indicate that the inhibitory response to these stimulants is mediated by stimulation of the adrenergic nerve system more than by non‐adrenergic nerves; the excitatory effect is probably due to stimulation of cholinergic nerves.
These results suggest that the adrenergic mechanisms of the taenia caecum are more labile in cold storage than the cholinergic mechanisms. Thus, the inhibitory action of cold storage on the relaxation produced by nicotine, DMPP, and transmural stimulation is probably explained by selective physical degeneration of the adrenergic nerve terminal. Also, enhancement of the contractile response to these stimulants in cold stored preparations is explained by the lack of adrenergic inhibitory mechanisms.
Physiologically active concentrations of epinephrine produce an increase in the concentrations of cyclic adenosine 3′,5′-phosphate in isolated intestinal smooth muscle preparations from the taenia coli of the guinea-pig. This effect is not associated with a change in the concentrations of hexose phosphate esters in this tissue.
Serum protein determination
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Contrasting effect on cyclic 3′,5′‐AMP and its dibutyryl derivatives on hepatic glycogenolysis and ideal relaxation
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LEVINE, R.A. (1958). Contrasting effect on cyclic 3',5'-AMP and its dibutyryl derivatives on hepatic glycogenolysis and ideal relaxation. Fed. Proc., 27, 354 (Abstract).
Cyclic AMP Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Archs int
- G A Butcher
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New York: Academic Press. ROBISON, G.A., BUTCHER, R.W. & SUTHERLAND, Jr., E.W. (1971). Cyclic AMP, pp. 204-210. New York: Academic Press. ROSSUM, J.M (1963). Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Archs int. Pharmacodyn., 143, 299-330.
Serum protein determination
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