Beta-adrenergic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0--8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the beta-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na+K+(Mg2+)-ATPase activity and a 6--8 fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2--3 fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological beta 2 type response with isoproterenol (1.8 . 10(-8) M) > epinephrine (1.1 . 10(-7) M) > norinephrine (3.2 . 10(-6) M). In contrast, binding studies employing (-)-[3H]dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (-)-[3H]dihydroalprenolol by catecholamine agonists. Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of Mr 80000 and 86000, whereas in the adult only a single Mr 113000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.     

Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.     

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate)

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.     

Dopamine stimulated increase of GTP levels in the rat retina

Dopamine stimulates a 7-10-fold increase of GTP concentration in whole rat retina maintained in vitro. Half-maximal stimulation of GTP levels were obtained with 10(-6) M dopamine, and significant increases in GTP levels were seen with 10(-7) M dopamine. Intracellular GTP levels were significantly increased within 4 min after exposure to dopamine and maximal effects were reached within 30 min. Dopamine agonists, apomorphine and bromocriptine, also stimulate a 7-10-fold increase in GTP concentration, whereas other catecholamines (norepinephrine, epinephrine, and isoproterenol) were less potent. Several other neurotransmitters present in rat retina (gamma-aminobutyric acid, glycine, glutamine, and taurine) had no effect on GTP levels. Although dopamine also stimulates increases in cyclic AMP levels in the retina, dibutyryl cyclic AMP and 8-bromo-cyclic AMP had no effect on GTP levels, indicating that the dopamine-stimulated increase of GTP is independent of the catalytic production of cyclic AMP by adenylate cyclase. Since dopamine-stimulated adenylate cyclase activity requires GTP, the dopamine-stimulated increase in GTP concentration described in this report may serve to facilitate dopamine stimulation of adenylate cyclase activity.     

Beta-agonist-induced inhibitory-guanine-nucleotide-binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma.

In the sarcolemma fraction of foot muscles of a fresh-water bivalve mollusc, Anodonta cygnea, a direct inhibitory, rather than stimulatory, effect of the beta-adrenergic agonist isoproterenol, at micromolar concentration, on cAMP level and adenylate cyclase activity, was revealed. It was blocked by beta- but not alpha-adrenergic antagonists. A single class of [3H]dihydroalprenolol-binding sites with binding properties of beta-adrenergic receptor was detected in mollusc sarcolemma. Potentiation of the inhibitory effect of isoproterenol on mollusc adenylate cyclase activity by GTP or guanosine 5'-[beta,gamma-imido]triphosphate at micromolar concentrations, and its elimination in the presence of guanosine 5'-[beta-thio]diphosphate, were shown. The pertussis-toxin-induced ADP-ribosylation of sarcolemma 40-kDa protein [immunochemically related in the C-terminal part to pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein (G-protein) alpha subunits of vertebrates], as well as the treatment of mollusc sarcolemma with antisera responsive to the C-terminus of vertebrate inhibitory G-protein (G(i)) alpha subunit led to elimination of the inhibitory effect of isoproterenol on adenylate cyclase activity. The results obtained suggest that beta-agonist-induced inhibition of adenylate cyclase in A. cygnea foot muscle may be realized via the beta-adrenoreceptor/G(i) signalling pathway.     

β-adrenegic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0–8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the β-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na⁺K⁺(Mg²⁺)-ATPase activity and a 6–8-fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2–3-fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological β₂ type response with isoproterenol (1.8 · 10^?8 M) > epinephrine (1.1 · 10^?7 M) > norinephrine (3.2 · 10^?6 M). In contrast, binding studies employing (?)-[³H] dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (?)-[³H]dihydroalprenolol by catecholamine agonists.Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of M_r 80 000 and 86 000, whereas in the adult only a single M_r 133 000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.     

In situ analysis of adenylate cyclase activity in permeabilized rat adipocytes: sensitivity to GTP, isoproterenol, and N6-(phenylisopropyl)adenosine

Adenylate cyclase activity in rat adipocyte suspensions was assayed in situ using a digitonin permeabilization technique. Recovery of activity was dependent on digitonin concentration, reaching a maximum at 20 micrograms/ml digitonin and paralleling the effect on cell permeability. Maximum adenylate cyclase activity recovered in permeabilized cells was 75% of that in comparable homogenates. Isoproterenol, a beta-adrenergic agonist, activated adenylate cyclase by 1.4, 2.2 and 4.5 fold at 10(-6), 10(-5) and 10(-3) M, respectively, despite perturbation of the plasma membrane. Exogenous GTP was not required for expression of beta-adrenergic activation, but 10(-5) M GTP maximally increased both basal and isoproterenol-dependent activity. The response to 10(-6) M isoproterenol was increased 2.1 fold by 10(-5) M GTP. N6-(Phenylisopropyl)adenosine at 10(-6) M inhibited both basal and isoproterenol-dependent adenylate cyclase activity by approximately 30%, demonstrating that the adenosine-dependent inhibitory pathway (Ni) remained functional in the digitonin-permeabilized cells. In situ analysis of adenylate cyclase is not only simple and rapid, but provides a unique approach to studying regulation of this key enzyme.     

Epinephrine-sensitive adenylate cyclase of human fat cell ghosts. Modulation of enzyme activity by GTP

Some of the effects of GTP on human fat cell adenylate cyclase activity were studied. This nucleotide caused a dose-dependent inhibition of basal activity without affecting the hormone-activated rate of cAMP formation. Maximal effects were observed at GTP-concentrations of about 1 x 10(-4) M. The relative extent of hormonal stimulation was about 1.5-fold increased in the presence of 0.1 mM GTP.     

Inhibition of adenylate cyclase activity in human fat cells by alpha-adrenergic agonists

The effects of the alpha 1-adrenergic agonist methoxamine and the alpha 2-adrenergic agonist clonidine on isoproterenol stimulated adenylate cyclase activity were examined in plasma membranes prepared from female human subcutaneous adipose tissue. It was found that in the presence of 10 microM GTP and 100 mM NaCl increasing concentrations of both agonists inhibited basal and isoproterenol-stimulated adenylate cyclase activity. The inhibitory action of 5 x 10(-7) M clonidine could not be overcome by increasing concentrations of isoproterenol. These results suggest both alpha 1- and alpha 2-adrenergic agonists inhibit beta-agonist-stimulated adenylate cyclase activity in human adipose tissue.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Heterologous desensitization of pigeon erythrocyte adenylate cyclase by the catalytic subunit of cAMP-dependent protein kinase

Preincubation of pigeon erythrocyte plasma membranes with the catalytic subunit of cAMP-dependent protein kinase results in the desensitization of erythrocyte adenylate cyclase. The adenylate cyclase activity measured in the presence of 10 microM isoproterenol and 50 microM GTP-gamma-S decreases by 40% after 10 min incubation; that in the presence of 50 microM GTP-gamma-S by 35% (20 min). The decrease of the adenylate cyclase activity is due to the prolongation of the lag phase of the enzyme activation in the presence of a hydrolysis-resistant GTP analog and to the drop in activity in the steady state of the activation. The heterologous desensitization of adenylate cyclase induced by cAMP-dependent protein kinase is also coupled with the decrease of the number of beta-adrenoreceptors capable of acquiring a high affinity for the agonists in the absence of guanyl nucleotides. The effect of the catalytic subunit on adenylate cyclase is fully compatible with the process of the enzyme desensitization in erythrocytes treated with isoproterenol or cAMP.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Two site binding of bepridil and modulation of adenylate cyclase in cardiac sarcolemmal membranes

A preparation of cardiac sarcolemmal membranes is described. These membranes exhibit 9-24-fold purification of (Na+ + K+)-ATPase, potassium-stimulated nitrophenolphosphatase, 5'-nucleotidase, adenylate cyclase, sialic acid content, and beta-receptor number. Sarcolemmal membranes have two classes of binding sites for the calcium entry blocker, bepridil, 70 X 10(12) high-affinity sites/mg, Kd 25-40 nM; and 30 X 10(15) low-affinity sites/mg, Kd 54-70 microM. Binding of bepridil to these sites appears responsible for inhibition of isoprenaline-stimulated and activation of fluoride-stimulated adenylate cyclase. Since basal adenylate cyclase activity is not influenced, bepridil must act not at the catalytic site, but by altering the interactions between beta-receptor and catalytic and regulatory components of adenylate cyclase.     

Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat

Abstract: Adenylate cyclase activity in cell-free homogenates of the rat superior cervical ganglion (SCG) was assayed under a variety of experimental conditions. Adenylate cyclase activity was decreased by approximately one-half when 1 m M EGTA was included in the homogenization buffer and assay mixture, indicating the presence of a Ca²⁺-sensitive adenylate cyclase in the ganglion. In the presence of EGTA, basal adenylate cyclase activity in homogenates of the SCG was 12.9 ± 0.6 pmol cyclic AMP/ganglion/10 min. Enzyme activity was stimulated three- to fourfold by 10 m M NaF or 10 m M MnCl₂, Both GTP and its nonhydrolyzable analog guanylylimidodiphosphate (GppNHp) stimulated adenylate cyclase in a concentration-dependent manner over the range of 0.1–10.0 μ M . Stimulation by GppNHp was five to six times greater than that produced by GTP at all concentrations tested. Decentralization of the ganglion had no effect on basal or stimulated adenylate cyclase activity. Receptor-linked stimulation of adenylate cyclase was not obtained with any of the following: isoproterenol, epi-nephrine, histamine, dopamine, prostaglandin E₂, or va-soactive intestinal peptide. Thus the receptor-linked regulation of adenylate cyclase activity appears to be lost in homogenates of the ganglion.     

Adenylate cyclase of multiple lipomata. Regional differences in adrenaline-responsiveness

Multiple symmetric lipomatosis has been proposed to be associated with impaired catecholamine-responsiveness of hypertrophic adipose tissue at the level of beta-adrenergic receptors or adenylate cyclase respectively. We have studied the regulation of the adenylate cyclase by guanine nucleotides and adrenaline in 5 subjects suffering from multiple encapsulated lipomata. In the presence of GTP (0.1 mmol/l) basal adenylate cyclase activity averaged 0.5 +/- 0.3 nmol cAMP/mg protein/10 minutes in normal adipose tissue and 1.0 +/- 0.4 nmol cAMP/mg protein/10 minutes in hypertrophic adipose tissue respectively. The synthetic GTP-analogue GMP(PNP) (0.1 mmol/l) increased non-stimulated activity by about 100% in both tissues. Adrenaline (1 mumol/l-1 mmol/l) caused a dose-dependent increase of enzymic activity in both tissues which had a maximum of 130% above basal levels in the presence of GTP and of 300% in the presence of GMP(PNP) respectively. In one of the six subjects suffering from gluteal lipomata normal adipose tissue was obtained from the gluteal as well as the abdominal region on two occasions. Maximally effective concentrations of adrenaline (1 mmol/l) induced a 3-fold increase of enzymic activity in abdominal membranes compared with about a 1.7- and 1.75-fold increase in normal and lipomatous tissue from the gluteal region. The results show that encapsulated lipomata contain a normally reactive adenylate cyclase system.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Effect of prostaglandin E1 on the cyclic adenosine-3',5'--monophosphate system and radiosensitivity of B-82 cells cultured in vitro

The cyclic AMP (cAMP) system of mice fibroblasts L, clone B-82 has been studied. B-82 cells possess receptors to prostaglandin E1 (PGE1) and are deficient in beta-adrenoreceptors. It has been shown that 10 mM NaF, 1.10(-4) M GTP and 1.10(-5) M PGE1 stimulate adenylate cyclase of B-82 cells. PGE1 causes increase in the intracellular content of cAMP and protects B-82 cells against irradiation. Specifict beta-agonist isoproterenol fails to modulate adenylate cyclase activity and does not change cAMP content in B-82 cells. Isoproterenol has no effect on the B-82 cells radiosensitivity, although it is known to be a potent radioprotector. It is presumed that the stimulation of cAMP system by radioprotector is necessary for the performance of radioprotective effect.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.