ß-Adrenergic Receptor Activation Increases Acetylcholine Receptor Number in Cultured Skeletal Muscle Myotubes

Treatment of embryonic chick muscle myotubes with the beta-adrenergic agonist isoproterenol increased the number of surface membrane nicotinic cholinergic receptors. Receptor degradation was unaffected by isoproterenol, suggesting that receptor synthesis was increased. The effect of isoproterenol appears to be mediated by the beta-adrenergic receptor adenylate cyclase system for the following reasons: (a) The response to isoproterenol was dose-dependent and stereospecific. (b) The response to catecholamines followed the order isoproterenol greater than epinephrine greater than norepinephrine. (c) Alprenolol, a beta-adrenergic antagonist, but not phentolamine, an alpha-antagonist, abolished the effect. (d) The maximal effects of isoproterenol and cholera toxin, an activator of adenylate cyclase, were not additive. These results suggest that under certain physiological states catecholamines may play an important role in the regulation of cholinergic receptors.     

On the role of guanyl nucleotides in adenylate cyclase system of embryonic skeletal muscle

The effects of guanyl nucleotides on the catalytic and catecholamine-stimulated activities of adenylate cyclase in developing chick skeletal muscles were studied. GTP and guanylyl imidodiphosphate stimulate the cyclase catalytic activity already at the early embryonic stages without having potentiating influence on the catecholamine-stimulated activity in embryonic muscle. In a distinct and regular form this effect can be observed only after hatching. Therefore during embryogenesis the coupling function of the GTP-binding component of adenylate cyclase system characteristic of its mature state is not manifested. The effects of the nucleotide suggest that they occur as two independent processes.     

In vitro studies of skeletal muscle membranes

Summary Sarcolemmal membranes were prepared from slow-twitch (red) and fasttwitch (white) skeletal muscle of the rat. A sensitive adenylate cyclase assay was used and basal, fluoride- and catecholamine-stimulated activities measured. The greaterin vivo sensitivity of red muscle to the effects of catecholamines correlates, in the present study, with approximately a twofold stimulation of its sarcolemmal adenylate cyclase with isoproterenol (10 m). The white muscle enzyme, on the other hand, is only minimally stimulated (20%) at the same concentration of -adrenergic agonist. Fast-twitch muscle is known to be physiologically insensitive to catecholaminein vivo.A course of sciatic nerve denervation was followed to further distinguish these two metabolic types of skeletal muscle and their respective adenylate cyclases. The slow-twitch muscle enzyme activities were completely and permanently lost on denervation. The white muscle enzyme, however, recovered almost completely after an initial reduction in specific activity the first week. Interestingly, the NaF-stimulated activity lagged behind both the basal and hormone-stimulated activities of the white muscle enzyme, in returning to control levels. The activities of cyclic nucleotide phosphodiesterase were evaluated in homogenates of the two muscle types in innervated rats and following denervation, in order to further define the neural influence on skeletal muscle cyclic nucleotide metabolism.The results suggest that the motor nerve may regulate some of the metabolic properties of slow-twitch muscle (which may involve cyclic AMP) by controlling the responsiveness of its sarcolemmal-bound adenylate cyclase system.Presented in part at the 60th Annual Meeting, Federation of American Societies for Experimental Biology, April, 1976, Anaheim, California.     

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase.

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5'-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the Ka was 0.05 microM. 5'-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, KD 0.07 microM. Beta adrenergic amines accelerated the rate of 5'-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol approximately soterenol approximately salbutamol greater than epinephrine greater than norephrine. Catecholamine activation was antagonized by propranolol and the beta2 antagonist butoxamine; the beta1 antagonist practolol was inactive. [3H]Dihydroalprenolol bound to the membranes and binding was antagonized by beta adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the beta2 type.     

Ontogenetic study of the regulatory and coupling function of guanyl nucleotide-binding proteins in the hormone-sensitive adenylate cyclase system

Reconstitution of catecholamine-sensitive adenylate cyclase from chick embryonic muscle membranes and guanyl nucleotide-binding proteins of mature rabbit muscle makes it possible to reveal the coupling (potentiating) effect of these nucleotides 1 week earlier than in the native condition. The effective insertion of guanyl-nucleotide-binding proteins into the embryonic membrane coincides with the onset of a pronounced increase in membrane lipid fluidity during the course of embryogenesis. The different ontogenetic time-courses for the origination of the two guanyl nucleotide effects, on catalytic adenylate cyclase activity (in early embryogenesis) and on the coupling process (in postembryonic life), suggest the existence in this system of two separate guanyl-nucleotide-binding proteins performing regulatory and coupling functions, respectively.     

In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase.

[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.     

Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscles.

Cyclic AMP content, adenylate cyclase (EC 4.6.1.1) activity and phosphodiesterase I (EC 3.1.4.1) activity of the hind leg skeletal muscle and cardiac muscle in 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters were examined. In 60-day-old myopathic animals, cardiac cyclic AMP levels were higher and phosphodiesterase I activity was lower, without any changes in the basal adenylate cyclase activity, whereas in 150-day-old myopathic hamsters, cardiac cyclic AMP and basal adenylate cyclase activity were lower, without any changes in the homogenate phosphodiesterase I activity. On the other hand, basal adenylate cyclase and phosphodiesterase I activities in the skeletal muscle homogenate from 60- and 150-day-old myopathic animals were not different from the normal values but the skeletal muscle cyclic AMP levels were significantly less in 60-day-old myopathic hamsters only. The plasma cyclic AMP levels in 60-day-old myopathic hamsters, unlike 150-day-old myopathic animals, were higher than the normal. Although these results reveal differences in myopathic cardiac and skeletal muscles, it is concluded that changes in adenylate cyclase-cyclic AMP system in myopathy are dependent upon the degree of disease.     

Different Natures of Supersensitivity of Adenylate Cyclase Stimulated by Calcitonin Gene-Related Peptide and Isoproterenol in Rat Diaphragm After Denervation and Reserpine Treatment

In skeletal muscles, calcitonin gene-related peptide (CGRP) released from motor nerve terminals and humoral catecholamines stimulate adenylate cyclase (AC) and enhance muscle contraction. The effects of denervation and treatment with reserpine on twitch contraction and the AC system in rat diaphragm were investigated. The basal levels of twitch contraction and AC activity of the diaphragm of rats were both increased 2 weeks after phrenic nerve denervation but were not altered by treatment with reserpine. Reserpine treatment provoked supersensitivity of AC to isoproterenol, without affecting the response to CGRP. On the other hand, denervation decreased the activation of AC and enhancement of twitch contraction by CGRP, without affecting the responses to isoproterenol. These data suggest that denervation causes up-regulation of AC as a result of loss of CGRP release from nerve terminal and that depletion of catecholamines by reserpine treatment supersensitizes the responses at the beta-adrenoceptor level. Thus, nervous and humoral factors regulate the AC system in striated muscle by different mechanisms.     

A new mechanism of inhibiting adenylate cyclase involving a beta-adrenergic receptor]

The previously unknown mechanism of adenylate cyclase activity inhibition by catecholamines has been found. It is realized through a beta-adrenoreceptor in the smooth muscle of fresh-water mollusc Anodonta cygnea. As to its ligand-binding characteristics (one class of binding sites with Kd = 0.35 + 0.06 nM, a competitive series of ligands substitution: isoproterenol greater than adrenalin greater than propranolol greater than noradrenaline greater than serotonin = dopamine greater than phentolamine) as well as to negative regulation of the GTP affinity this receptor is similar to beta-adrenoreceptors of higher vertebrates. The dose-dependent inhibiting effect (to 50-60%) of isoproterenol and noradrenaline on the basal, GTP- and serotonin-stimulated activity of adenylate cyclase and cAMP level which is removed only by beta-adrenergic blockers is shown in vitro and in vivo. It is concluded that inhibition of adenylate cyclase activity by catecholamines in the muscular tissue of the mollusc is realized via beta-adrenoreceptor.     

Changes in cyclic AMP, adrenylate cyclase and protein kinase levels during the development of embryonic chick skeletal muscle.

The levels of cyclic AMP (cAMP) and activities of adenylate cyclase and protein kinase have been examined in chick skeletal muscle tissue between the 7th and 15th day of its embryonic development. The tissue cAMP levels were found to increase in two main phases; from 8–10 days and from 12–15 days of development. Parallel increases between the 8th and 10th day of development were also found in the basal enzyme activities of both adenylate cyclase and protein kinase. The maximum values of all three parameters coincided with the onset of cell fusion in the tissue. The results are compared with the findings of a similar study carried out on differentiating myoblasts cultured in vitro, and are assessed in terms of the possibility that cAMP levels control the expression of myoblast differentiation.     

Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the K_a was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K_D 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β₂ antagonist butoxamine; the β₁ antagonist practolol was inactive. [³H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β₂ type.     

Calcitonin gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger.

Recent immunocytochemical studies have shown that calcitonin gene-related peptide (CGRP) coexists with the neurotransmitter acetylcholine in spinal motoneurons of the chick. Moreover, CGRP causes an increase in the number of acetylcholine receptors on the surface of cultured chick myotubes. CGRP might thus serve as one of the signals by which motoneurons regulate endplate development. In a search for the second messengers involved, we now demonstrate that CGRP stimulates accumulation of cyclic AMP (cAMP) in cultured chick myotubes. This effect is, at least in part, mediated by an increase in cAMP synthesis, as the peptide also activates adenylate cyclase in chick muscle membranes. Nanomolar concentrations of CGRP elicit significant increases in both cellular cAMP levels and acetylcholine receptor numbers. The present findings suggest that cAMP is one of the second messengers which mediate the increase in acetylcholine receptor number elicited by CGRP. Furthermore, CGRP might be implicated in other trophic actions mediated by cAMP in skeletal muscle cells.     

Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. Interaction between two second messenger systems

Calcitonin gene-related peptide (CGRP) has previously been shown to coexist with acetylcholine in spinal cord motoneurons and to stimulate adenylate cyclase in skeletal muscle cells. We now demonstrate that in cultured chick myotubes whose phosphoinositides have been labeled with [3H]inositol, CGRP enhanced the accumulation of [3H]inositol mono-, bis-, and trisphosphates. Rat CGRP-I (rCGRP) (0.1 microM) elicited a transient increase in [3H]inositol 1,4,5-trisphosphate, as well as a more sustained elevation of [3H]inositol 1,3,4-trisphosphate levels. In the presence of Li+, rCGRP evoked an approximately 3-fold increase of [3H]inositol monophosphate levels, which persisted for up to 1 h. This effect of rCGRP was concentration-dependent, the half-maximal response being obtained at 1 nM. Since rCGRP also accelerated the rate of synthesis of [3H]inositol-containing lipids, it appears that the peptide acts by stimulating phosphoinositide turnover in chick myotubes. Agents that either mimic or elevate intracellular cyclic AMP also enhanced the synthesis of [3H]inositol-containing lipids, and the accumulation of inositol phosphates, suggesting that the effects of rCGRP are mediated, at least in part, via the activation of adenylate cyclase. This hypothesis was strengthened by the non-additivity of the inositol phosphate responses elicited by rCGRP and other cAMP-mobilizing agents, and by the sensitivity of these responses to various pharmacological treatments. The present results provide an example of positive interaction between cAMP and the phosphoinositide signaling system. They further suggest that a coexisting neuropeptide may exert pleiotropic actions upon its target cell by stimulating multiple signal transduction pathways.     

Functional characteristics of the adenylate cyclase system of developing skeletal muscles in the rat

The functional development of hormone-sensitive adenylate cyclase system of rat skeletal muscles was studied. It was shown that within 15-17 embryonic days the plasma membrane of the muscle cell contains catecholamine-sensitive adenylate cyclase (isoproterenol greater than epinephrine greater than norepinephrine) which on these ontogenetic stages is represented by functionally active catalytic, regulatory and receptory components. The coupling component, which, according to the authors' view, is presumably an independent (fourth) functional subunit of adenylate cyclase system, is formed only in the postnatal period. A suggestion is put forward that the above process is due to the fact that guanyl nucleotide-binding protein(s) responsible in the mature target cell for the coupling of receptory and catalytic components may appear in the membrane only after birth.     

The molecular size of adenylate cyclase in the absence and presence of nucleotide and hormone effectors

The molecular size of adenylate cyclase solubilized from frog erythrocyte membranes by digitonin extraction has been determined by chromatography on Sepharose 6B. Regardless of whether the membranes are exposed to catecholamines, GPP(NH)P, NaF or no effector prior to solubilization, the apparent molecular size of the adenylate cyclase enzyme is the same. Furthermore, a similar elution profile for the enzyme is observed when the catalytic activity in the eluates is measured in the presence of Mn++, rather than Mg++. Since it is generally assumed that the persistent activation of adenylate cyclase by GPP(NH)P requires interaction of the catalytic moiety with the guanine nucleotide regulatory site, it appears that the adenylate cyclase activity detected in the column eluates represents an intact catalytic-regulatory site complex. The adenylate cyclase activity derived from catecholamine pretreated frog erythrocyte membranes does not co-elute with catecholamine-occupied beta-adrenergic receptors, indicating that the agonist-promoted increase in apparent receptor size observed here and in earlier studies does not represent a physical coupling of the receptor and the adenylate cyclase enzyme.     

Effect of immobilized adrenaline on the state of the adenylate cyclase system in the cardiac and skeletal muscles of rats in the recuperative period after physical exertion

It is shown that in the rehabilitation period after a single long physical load the adenylate cyclase activity and cAMP content in the skeletal muscle are low and normalize by the 48th and 36th h, respectively. These indices in the myocardium in the nearest rehabilitation period increase considerably, gradually normalizing by 24th and 48th h, respectively. Immobilized epinephrine accelerates normalization of the adenylate cyclase activity and cAMP content in the skeletal muscle and in myocardium in the rehabilitation period after physical load, the phosphodiesterase activity being unchanged.     

Postnatal development of glycogen- and cyclic AMP-metabolizing enzymes in mammalian skeletal muscle

Glycogen and cyclic AMP-metabolizing enzymes of rabbit skeletal muscle were examined during postnatal development. Glycogen synthase I, glycogen phosporylase and lactate dehydrogenase activity increased 7-fold by the 6th--8th postnatal week while glycogen synthase D was present in the neonate at one-half adult levels. Cyclic AMP phosphodiesterase decreased; adenylate cyclase increased 10-fold for both the epinephrine and NaF-stimulated states of the enzyme.     

1-Methylisoguanosine: An orally active marine natural product with skeletal muscle and cardiovascular effects

1-Methylisoguanosine is a marine natural product with skeletal muscle relaxant, hypothermic and cardiovascular effects following oral administration in mice and rats. The cardiovascular effects (hypotension associated with bradycardia) are similar to those of adenosine. Structure-activity studies with a series of 1-methylisoguanosine analogues suggest a reasonable correlation between ability of the compounds to stimulate adenosine-sensitive adenylate cyclase and effects on skeletal muscular and cardiovascular responses. Thus, it appears that 1-methylisoguanosine may function as a long-acting adenosine analogue.     

Role of the hypothalamus and pituitary gland in the development of pancreatic islet sensitivity to glucose in fetal rats

Differences in the pattern of the development of three enzymes of the plasma membrane have been established. The activity of Na, K-ATPase progressively increases, that of adenylate cyclase decreases, whereas the activity of 5-nucleotidase undergoes only slight changes during embryogenesis. Differences between these enzymes were also found with respect to the development of their sensitivity to the regulatory effects of catecholamines. Adrenaline reactivity of adenylate cyclase may be detected already in embryogenesis; it is lower than that in definite muscle tissue increasing during further ontogenesis. Catecholamine reactivity was not found in Na, K-ATPase and 5-nucleotidase up to the 17th day of incubation of chick embryos. The effect of adrenalin was observed at later stages of ontogenesis, it may be initiated by exogeneous cAMP and protein kinase. At postembryonic stages, similarity in the behavior of these enzymes was found with respect to the presence and pattern of their reaction to adrenalin (stimulation), as well as with respect to temporal dynamics of the effect. The data obtained indicate the existence of close connections between these enzymes, which are realized in the sequence adrenoreceptor-adenylate cyclase-cAMP-protein kinase-effector proteins.     

Alteration of starfish oocyte adenylate cyclase properties in gametogenesis

1. The properties of the oocyte membrane adenylate cyclase system of the starfish Aphelasterias japonica at different stages of gametogenesis were studied.2. The adenylate cyclase activity of fully grown oocytes was insensitive to catecholamines whereas activity in growing oocytes was increased.3. Forskolin and NaF stimulated catalytic activity of growth and fully grown oocytes to various degrees. The stimulated effect of these modifiers was more considerable in grown oocytes.4. We believe that the interaction between components of the adenylate cyclase system is altered in gametogenesis.