Catalytic Activity Of Adenylate Cyclase Research Articles

Transduction of aminergic and peptidergic signals in enteric neurones of the guinea-pig.

1. The biogenic amines 5-hydroxytryptamine (5-HT) and histamine, and the peptides bombesin, gastrin-releasing peptide (GRP), vasoactive intestinal peptide (VIP), cholecystokinin (CCK), substance P and calcitonin gene-related peptide (CGRP) each mimicked slow synaptic excitation (slow e.p.s.p.) when applied to myenteric neurones of the guinea-pig small intestine. 2. Stimulation of the catalytic activity of adenylate cyclase by forskolin and intraneuronal elevation of cyclic 3',5'-adenosine monophosphate (cyclic AMP) also mimicked the slow e.p.s.p. and the actions of the aminergic and peptidergic messengers. 3. Adenosine prevented stimulation of adenylate cyclase by forskolin and abolished the slow e.p.s.p.-like actions of forskolin. 4. Exposure of the neurones to adenosine prior to or during application of bombesin, GRP, VIP, CCK or histamine blocked the actions of these substances. 5. Pre-treatment with adenosine did not suppress the slow e.p.s.p.-like actions of substance P, CGRP or 5-HT. 6. The results suggest that signal transduction for bombesin, GRP, VIP, CCK and histamine involves stimulation of adenylate cyclase and second messenger function of cyclic AMP. Transduction mechanisms for 5-HT, substance P and CGRP appear not to involve second messenger function of cyclic AMP.

Purinergic inhibition in the small intestinal myenteric plexus of the guinea-pig.

1. The actions of adenosine on electrical behaviour of myenteric neurones were investigated with intracellular recording methods in guinea-pig small intestine. 2. The actions of adenosine were: membrane hyperpolarization, decreased input resistance, enhancement of post-spike hyperpolarizing potentials and suppression of excitability. These effects were observed exclusively in AH/type 2 myenteric neurones. 3. The presence of adenosine in the micromolar range of concentrations prevented or suppressed excitatory responses to forskolin. It also aborted the effects of forskolin when added in combination with this activator of adenylate cyclase. 4. Adenosine (100 microM) did not affect the excitatory actions of intracellularly injected cyclic AMP, membrane-permeant analogues of cyclic AMP, inhibitors of cyclic nucleotide phosphodiesterase or elevation of Mg2+ and reduction of Ca2+ in the bathing medium. 5. The results suggest that the mechanism of the inhibitory action of adenosine is suppression of the catalytic activity of adenylate cyclase and consequent reduction of intraneuronal levels of cyclic AMP.

Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate cyclase.

Treatment of rats with thyrotropin (TSH) reduces the adrenoceptor sensitivity of adenylate cyclase from cerebral cortex

Prolonged treatment of rats with TSH attenuates the noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. This effect can be measured in slices from brain cortex and with a membraneous, cellfree adenylate cyclase preparation from the same brain region. The development of this downregulation is time (7 d treatment required) and dose dependent (EC(50) = 3.5 mU/kg for 9 d). After discontinuation of treatment, about 12 d were required for reversal of this effect. Density of ?-adrenoceptors in cerebral cortex as measured by (?) [ (3)H]dihydroalprenolol (DHA) binding was reduced by TSH treatment (22% reduction). The catalytic activity of adenylate cyclase as tested with guanosinemonophosphate phosphoimidophosphate (GMPPNP) was unaffected by TSH treatment. It is suggested that TSH-mediated feedback mechanisms on TRH levels and receptors in brain are responsible for the changes of the central adrenoceptor system. The data are discussed with respect to the blunted TSH-response to TRH observed in certain psychiatric illnesses.

Stimulation of specific GTP binding and hydrolysis activities in lymphocyte membrane by interleukin-2.

Interleukin-2 (IL-2) is a polypeptide growth factor which stimulates the proliferation and differentiation of T lymphocytes. The receptor for IL-2 is expressed on activated T lymphocytes, cloned IL-2 dependent cells and several other cell types. Analysis of the primary structure and of immune-precipitated receptor suggests that this molecule has no intrinsic signal transduction function, unlike other growth factors. IL-2 interaction with a high affinity receptor has been shown, however, to activate the calcium/phospholipid-dependent protein kinase C (PK-C) presumably via phosphoinositide hydrolysis. Members of a family of closely related guanine nucleotide binding proteins (G proteins) regulate a diverse group of metabolic events. Two of them, Gs and Gi, stimulate and inhibit adenylate cyclase activity respectively, and other G proteins are involved in diverse signal transduction system. Another member, Go, has no known function and activation of phospholipase C has been attributed to the action of an unidentified G protein, Gp. Since it has been observed that IL-2 inhibits the catalytic activity of adenylate cyclase and that agents such as PGE2 which stimulate adenylate cyclase activity inhibit the lymphoproliferative response to IL-2, association of GTP binding proteins with IL-2 signal transduction was investigated. In this report we describe for the first time the participation of a GTP binding protein in the action of a polypeptide growth factor, interleukin-2.

Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin.

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.

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

1. 1. 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. 2. 2. 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. 3. 3. 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.

Dopaminergic inhibition of anterior pituitary adenylate cyclase activity and prolactin release: the effects of perturbing calcium on catalytic adenylate cyclase activity.

The dopaminergic inhibition of anterior pituitary adenylate cyclase activity, cAMP accumulation, and prolactin release was studied in the presence of the Ca2+ channel activator, maitotoxin. In isobutylmethylxanthine (IBMX)-treated cells, maitotoxin stimulated prolactin secretion within 30 s and cAMP accumulation within 1 min. Although dopamine reduced cAMP accumulation and prolactin release, the effectiveness of the catecholamine was reduced in the presence of maitotoxin. When hemipituitary glands were exposed for 10 min to 100 ng maitotoxin/ml, their membranes showed increased adenylate cyclase activity. The hypothesis that maitotoxin stimulates adenylate cyclase activity by increasing Ca2+ availability was supported by the observation that, at concentrations up to 100 microM, Ca2+-stimulated anterior pituitary adenylate cyclase activity. Although dopamine decreased basal and maitotoxin-stimulated pituitary cAMP accumulation, via changes in adenylate cyclase activity, the decrement in cyclic nucleotide production, but not prolactin release, can be ascribed to the effect of the catecholamine on the basal activities of these parameters. These data provide additional evidence that an increased Ca2+ flux is stimulating to cAMP generation and prolactin release, whereas dopamine is inhibitory to these processes.

The A1 adenosine receptor. Identification of the binding subunit by photoaffinity cross-linking.

Adenosine modifies the catalytic activity of adenylate cyclase through both inhibitory (A1 or Ri) as well as stimulatory (A2 or Ra) cell surface receptors. We developed 125I-labeled N6-2-(4-aminophenyl)ethyladenosine as a selective ligand to probe the structure of A1 receptors. The binding of this radioligand to rat cerebral cortex or adipocyte membranes is saturable, reversible, and of high affinity (KD approximately 2 nM). A1 receptor agonists antagonize binding stereoselectivity and with a potency order appropriate for A1 receptors. The heterobifunctional cross-linking reagent N-succinimidyl-6-(4-azido-2-nitrophenylamino)hexanoate covalently couples the radioligand to a protein of Mr = 38,000 in both tissues as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Inhibition of covalent labeling by adenosine analogs exhibited the stereoselectivity and potency order typical of A1 receptor ligands. Guanine nucleotides reduced both specific binding and covalent incorporation of the radioligand, evidence that the radioligand is an A1 receptor agonist. These results suggest that the A1 receptor binding subunit of both brain and adipocytes resides on a protein of Mr = 38,000. The new radioligand should prove useful in studying the structure and regulation of A1 receptors.

Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase.

Human platelet membrane proteins were phosphorylated by exogenous, partially purified Ca2+-activated phospholipid-dependent protein kinase (protein kinase C). The phosphorylation of one of the major substrates for protein kinase C (Mr = 41 000) was specifically suppressed by the beta subunit of the inhibitory guanine-nucleotide-binding regulatory component (Gi, Ni) of adenylate cyclase. The free alpha subunit of Gi (Mr = 41 000) also served as an excellent substrate for the kinase (greater than 0.5 mol phosphate incorporated per mol of subunit), but the Gi oligomer (alpha X beta X gamma) did not. Treatment of cyc- S49 lymphoma cells, which are deficient in Gs/Ns (the stimulatory component) but contain functional Gi/Ni, with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C, did not alter stimulation of adenylate cyclase catalytic activity by forskolin, whereas the Gi/Ni-mediated inhibition of the cyclase by the hormone, somatostatin, was impaired in these membranes. The results suggest that the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory component of adenylate cyclase may be a physiological substrate for protein kinase C and that the function of the component in transducing inhibitory hormonal signals to adenylate cyclase is altered by its phosphorylation.

Comparison of Cyclic AMP Accumulation and Morphological Changes Induced by β-Adrenergic Stimulation of Cultured Vascular Smooth Muscle Cells and Fibroblasts

Cyclic 3',5'-adenosine monophosphate (cAMP) accumulation and morphological changes induced by isoproterenol (ISO) on cultured vascular smooth muscle cells (SMC) and vascular fibroblasts derived from spontaneously hypertensive rats, their stroke-prone strain and normotensive Wistar Kyoto rats were investigated. At the time points studied, ISO-induced cAMP accumulation in SMC reached a peak level at 5 min. Accumulation was dose-dependent and was maximal at a concentration of 10(-5) M ISO. Maximal cAMP levels were approximately 600-fold higher than basal levels. Maximal cAMP accumulation or half maximal stimulatory ISO concentrations were similar in SMC from the three strains. ISO had no effects on cyclic 3',5'-guanosine monophosphate (cGMP) levels in SMC. Phenylephrine had no effects on cAMP or on cGMP levels. In contrast to SMC, beta-adrenergic stimulation of vascular fibroblasts resulted in only a 4-fold increase of cAMP levels. 1.5 h after administration of ISO to SMC cultures, the morphological changes were apparent in SMC but not in fibroblasts. Morphological changes induced by ISO were reversible and morphological appearances returned to normal 16 h after exposure to ISO. 10(-3) M dibutyryl cAMP had similar effects on the morphologies of both SMC and fibroblasts. These effects were antagonized by 5 X 10(-6) M colchicine, an inhibitor of microtubule assembly. These results indicate that cultured vascular SMC possess the ability to increase markedly their cellular cAMP level in response to beta-adrenergic stimulation, while fibroblasts are less responsive to the stimulation. Furthermore, cAMP accumulation results in morphological changes of SMC and fibroblasts probably through the alteration of intracellular microtubule systems. As the morphological response to intracellular cAMP (or its lipophilic derivatives) is similar in both SMC and fibroblasts, the difference in each cell line's responsiveness to ISO may be due to a difference in: (1) the density or sensitivity of beta-adrenergic receptors on the plasma membrane of each cell type, or (2) the catalytic activity of adenylate cyclase itself.

Effects of human erythrocyte guanine nucleotide-binding regulatory protein on parathyroid hormone-responsive adenylate cyclase from canine renal cortex.

We studied the effects of the guanine nucleotide-binding regulatory protein (Gs) from human erythrocytes on PTH-responsive adenylate cyclase from partially purified membranes of canine renal cortex (CRC). Extracts of erythrocyte membranes, containing soluble Gs, was obtained by treatment with a detergent (Lubrol PX). Gs did not stimulate adenylate cyclase activity by itself, but amplified the response of adenylate cyclase in CRC membranes to both synthetic bovine PTH-(1-34) [bPTH-(1-34)] and to the hydrolysis-resistant GTP analog 5'-guanylimido-diphosphate [Gpp(NH)p]. Gs increased PTH stimulation of adenylate cyclase activity in both the presence and absence of Gpp(NH)p. In the absence of Gpp(NH)p, the potentiating effect of Gs occurred only when the concentration of bPTH-(1-34) was greater than 10 ng/ml. bPTH-(1-34), Gpp(NH)p, and Gs each enhanced the catalytic activity of adenylate cyclase when added separately or in combination by increasing the apparent maximum velocity (Vmax) of the enzyme without altering the apparent Km for MgATP. The effect of Gs on CRC membrane adenylate cyclase activity in the presence of NaF (10 mM) and forskolin (100 microM) was also examined. NaF- and forskolin-stimulated enzyme activities were significantly increased by Gs in both the presence and absence of Gpp(NH)p (100 microM). Analysis of double reciprocal plots of substrate concentration and enzyme activity revealed that NaF and forskolin increased the Vmax of the catalytic activity and did not alter the apparent Km of the enzyme for MgATP. These data support the role of Gs as a regulator of the response of adenylate cyclase to hormones, guanyl nucleotides, NaF, and forskolin. Our studies address the relative functional stoichiometry between Gs and catalytic unit present in CRC membranes and suggest that the CRC adenylate cyclase system must contain insufficient Gs to couple with all available catalytic units. These results are consistent with the possibility that deficiency of Gs impairs hormonal stimulation by diminishing the apparent Vmax of the catalytic unit and does not alter the apparent affinity of the enzyme for MgATP.

Fat cell beta-adrenergic receptor in the hypothyroid rat. Impaired interaction with the stimulatory regulatory component of adenylate cyclase.

Fat cells from the hypothyroid rat fail to synthesize cyclic AMP in response to beta-adrenergic agonists, although possessing normal amounts of beta-adrenergic receptors (R) and catalytic adenylate cyclase activity. Membranes of hypothyroid rat fat cells contain Mr = 42,000 (major form), 46,0000, and 48,000 (minor forms) peptides of the stimulatory guanine nucleotide-binding regulatory component (Ns) radiolabeled in the presence of cholera toxin and [32P]NAD+. Maps of fragments generated by partial proteolysis of these radiolabeled peptides are virtually identical in hypothyroid and euthyroid preparations. Two-dimensional gel electrophoresis showed that the size and charge of the Mr = 42,000, 46,000, and 48,000 radiolabeled peptides are similar in euthyroid and hypothyroid rat fat cell membranes. Extracts of hypothyroid rat fat cell membranes express normal amounts of Ns activity as measured by their ability to reconstitute the adenylate cyclase of membranes of S49 mouse lymphoma cyc- mutant cells which lack functional Ns activity. Hybridization of hypothyroid rat fat cells with donor membranes of normal rat fat cells, rat hepatocytes, or S49 cyc- cells restores the beta-adrenergic response of these fat cells. Pretreating the donor membranes with a beta-adrenergic antagonist covalent label blocks the ability of these membranes to restore the response of the cells. Rat hepatocytes pretreated with a beta-adrenergic antagonist covalent label do not accumulate cyclic AMP in response to isoproterenol. Hybridization of these receptor-deficient hepatocytes with fat cell ghosts of euthyroid rats restores beta-adrenergic stimulation of cyclic AMP accumulation, whereas hybridization with fat cell ghosts of hypothyroid rat does not restore this response. Ns of pigeon erythrocyte membranes radiolabeled with cholera toxin and [32P]NAD+, extracted in cholate, and reconstituted with fat cell membranes interacts with fat cell R. The ability of R to interact with Ns of pigeon erythrocyte membranes is impaired when the reconstitution is performed with membranes from the hypothyroid rat fat cell. Hypothyroidism appears to affect the ability of R to interact productively with Ns, without affecting either R number or Ns structure and function.

Elimination of GTP biphasic regulation of synaptosomal adenylate cyclase by manganese and solubilization.

Effects of divalent cations and solubilization with Lubrol-PX were studied on guanine nucleotide regulation of synaptosomal adenylate cyclase activity of the rat caudate nucleus. In the presence of Mg2+, both GTP and Gpp(NH)p exerted biphasic actions on the membrane-bound adenylate cyclase activity. The K0.5 value for the GTP stimulation of the cyclase was 47 nM, and the value for the GTP inhibition was 4.5 microM. One hundred microM dopamine selectively enhanced the stimulatory phase of the GTP action, whereas 10 microM morphine selectively enhanced the inhibitory phase of the GTP action. When Mg2+ was replaced by Mn2+, the inhibition of the membrane-bound adenylate cyclase by these nucleotides and morphine was completely abolished; but the catalytic activity of adenylate cyclase was not impaired. These results suggest that the inhibitory action of GTP is responsible for the morphine inhibition of synaptosomal adenylate cyclase. Lubrol-solubilized adenylate cyclase prefered Mn2+ to Mg2+ for its activity. The stimulation of adenylate cyclase by either GTP or Gpp (NH)p was eliminated in the Sepharose 6B-fractionated solubilized preparation in the presence of either Mg2+ or Mn2+. Ten mM NaF also failed to activate the fractionated adenylate cyclase. In the fractionated solubilized preparation, GTP and Gpp(NH)p failed to inhibit adenylate cyclase. These results indicate that GTP and Gpp(NH)p are unable to inhibit the resolved catalytic unit of the synaptosomal adenylate cyclase.

ELIMINATION OF GTP BIPHASIC REGULATION OF SYNAPTOSOMAL ADENYLATE CYCLASE BY MANGANESE AND SOLUBILIZATION

Effects of divalent cations and solubilization with Lubrol-PX were studied on guanine nucleotide regulation of synaptosomal adenylate cyclase activity of the rat caudate nucleus. In the presence of Mg2+, both GTP and Gpp(NH)p exerted biphasic actions on the membrane-bound adenylate cyclase activity. The K0.5 value for the GTP stimulation of the cyclase was 47 nM, and the value for the GTP inhibition was 4.5 μM. One hundred μM dopamine selectively enhanced the stimulatory phase of the GTP action, whereas 10 μM morphine selectively enhanced the inhibitory phase of the GTP action. When Mg2+ was replaced by Mn2+, the inhibition of the membrane-bound adenylate cyclase by these nucleotides and morphine was completely abolished; but the catalytic activity of adenylate cyclase was not impaired. These results suggest that the inhibitory action of GTP is responsible for the morphine inhibition of synaptosomal adenylate cyclase. Lubrol-solubilized adenylate cyclase prefered Mn2+ to Mg2+ for its activity. The stimulation of adenylate cyclase by either GTP or Gpp (NH)p was eliminated in the Sepharose 6B-fractionated solubilized preparation in the presence of either Mg2+ or Mn2+. Ten mM NaF also failed to activate the fractionated adenylate cyclase, in the fractionated solubilized preparation, GTP and Gpp(NH)p failed to inhibit adenylate cyclase. These results indicate that GTP and Gpp(NH)p are unable to inhibit the resolved catalytic unit of the synaptosomal adenylate cyclase.

Formycin 5'-triphosphate, a fluorescent analog of ATP, as a substrate for adenylate cyclase.

Formycin 5'-triphosphate (FoTP), a fluorescent analog of ATP, is shown to be a substrate for the membrane-bound adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rat osteosarcoma cells. The formation of the adenylate cyclase reaction product, 3',5'-cyclic formycin monophosphate (cFoMP), was followed by the conventional radioimmunoassay (RIA) procedure used to detect cAMP and by an assay procedure in which the reaction product was separated from the substrate by reverse-phase high-pressure liquid chromatography (HPLC) and the reaction product was detected by fluorometry. Because the HPLC--fluorometric procedure can determine the amount of cFoMP present in the reaction mixture within 6 min, the enzymatic conversion of FoTP to cFoMP can be followed directly during the course of a typical 15-min incubation. The amount of cFoMP detected by this procedure was found to be within 2% of the values obtained by the RIA. The rate of product formation with FoTP was similar to that observed with ATP and the activity of the enzyme was enhanced about 5-fold with guanyl-5'-yl imidodiphosphate when either ATP or FoTP was used as the substrate. Kinetic studies revealed values for the Vmax of 120 pmol/min per mg of protein and apparent Km values of 220 microM with both substrates. In addition to suggesting that the recognition of the substrate by the adenylate cyclase may not require a specific chemical structure of the 5-membered ring of the base or a unique configuration about either the glycosyl or the C(5')-C(4') bond, the results of this study are consistent with the idea that the cytotoxicity observed with the adenosine analog formycin may be the result of its metabolism to cFoMP. Furthermore, these studies indicate that the fluorescent analog FoTP can be used, in combination with HPLC, to provide an alternative, nonradioactive direct method for the assay of adenylate cyclase catalytic activity.

Regulation of responsiveness of cultured adrenal cells to adrenocorticotropin and prostaglandin E1: cell density, cell division, and inhibitors of protein synthesis.

In cultured bovine adrenocortical cells, responsiveness to ACTH, as assessed by the maximal rate of ACTH-stimulated cAMP production, has been found to depend on cell density and cell proliferation, while the maximal rate of prostaglandin E1 (PGE1)-stimulated cAMP production was constant. The combination of low cell density and normal cell proliferation caused a specific decline in responsiveness to ACTH. Responsiveness did not decline at any density when proliferation was inhibited by mitomycin C treatment. Specific declines in responsiveness to ACTH were also seen when cultures were treated with cycloheximide or sodium butyrate. When protein synthesis was completely inhibited by cycloheximide treatment, responsiveness to ACTH declined with a half-life of 20.5 h and responsiveness to PGE1 declined with a half-life of 75h. Because PGE1-stimulated cAMP production indicates intact adenylate cyclase catalytic activity, changes in ACTH-stimulated cAMP production appear to be due to specific changes in functional ACTH receptors.

Characterization of adenylate cyclase activity in asthmatic neutrophil sonicates.

It has been postulated that bronchial asthma is associated with a defect of the β-adrenergic receptor. Previous studies using intact neutrophil (polymorphonuclear) preparations have shown decreased cyclic adenosine monophosphate responses to isoproterenol and histamine in active bronchial asthma. Using the β-adrenergic antagonist 3H-dihydroalprenolol, we have recently reported normal numbers and affinity of β-adrenergic receptors in homogenates of polymorphonuclear leukocytes from patients with bronchial asthma. In the present study using polymorphonuclear sonicates from 25 patients with bronchial asthma and 23 normal control subjects, we assessed several steps distal to receptor binding, including adenylate cyclase coupling to the β-adrenergic receptor and adenylate cyclase catalytic activity after hormonal (isoproterenol, histamine, and prostaglandin E1) and NaF stimulation. The mean ± SEM concentration of isoproterenol that displaced 50 % 3H-dihydroalprenolol (Kd) was 2.31 ± 0.33 eM for the asthmatic s...

Differences in the cation sensitivity of adenylate cyclase from heart and skeletal muscle: Modification by guanyl nucleotides and isoproterenol

Low concentrations of Mn 2+ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg 2+; at saturating concentrations the cyclase activities obtained with Mg 2+ or Mn 2+ were similar. In contrast, Mg 2+ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn 2+; at saturating concentrations of either metal ion the Mg 2+-supported cyclase activities were 5- to 10-fold greater than Mn 2+-supported activities. Further, compared to Mg 2+, Mn 2+ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg 2+ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn 2+ in heart as well as skeletal muscle appeared to be greater compared to those for Mg 2+. The skeletal muscle cyclase displayed greater apparent affinity for MnATP 2− (app. K m 0.10 m m) compared to MgATP 2− (app. K m 0.32 m m) whereas the heart enzyme displayed greater apparent affinity for MgATP 2− (app. K m 0.07 m m) compared to MnATP 2− (app. K m 0.19 m m). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn 2+ (0.15 to 2 m m) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg 2+. With the heart enzyme the relatively greater potency of Mn 2+ persisted following preactivation. Significant enhancement in the Mn 2+-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me 2+ (Mg 2+ or Mn 2+) without influencing the apparent K m for MeATP 2− (MgATP 2− or MnATP 2−). Evidences were obtained to show that the poor effectiveness of Mn 2+ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn 2+ of adenosine-mediated inhibition of the cyclase, (b) Mn 2+-induced lability of the cyclase, (c) indirect effects of Mn 2+ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn 2+ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.

Conversion of basal 6.0 S adenylate cyclase into 7.4 S by guanyl nucleotide treatment of membrane bound enzyme

Membrane-bound adenylate cyclase from pig kidney medulla was pretreated with either guanyl-5′-yl-imidodiphosphate (Gpp(NH)p) or NaF and solubilized in the presence of Triton X100 ( 0.7 % v v ). Pretreatment by Gpp(NH)p, but not by NaF, shifted the standard sedimentation constant of the solubilized enzyme from 6.0 S to 7.4 S. This effect depended on the Gpp(NH)p concentration used and correlated to the nucleotide-induced increase in adenylate cyclase catalytic activity. Both the 6.0 S and 7.4 S activity peaks had similar Stokes radii.