Showing papers on "Cyclase published in 1975"

Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance.

Abstract: Narcotics affect adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in two opposing ways, both mediated by the opiate receptor. The first process is the readily reversible inhibition of the enzyme by narcotics; the second is a compensatory increase in enzyme activity which is delayed in onset and relatively stable. Late positive regulation of the enzyme counteracts the inhibitory influence of morphine and is responsible for narcotic dependence and tolerance. The coupled inhibitory and positive regulatory mechanisms for adenylate cyclase provide a means of activating and deactivating neural circuits hours after the initial event and thus may play a role in a memory process.

Morphine receptors as regulators of adenylate cyclase activity

Abstract: Morphine inhibits adenylate cyclase (EC 4.6.1.1) activity of neuroblastoma times glioma hybrid cells. The inhibition is stereospecific and is reversed by the antagonist, naloxone. The relative affinities of narcotics for the opiate receptor agree well with their effectiveness as inhibitors of adenylate cyclase. Morphine-sensitive and -insensitive cell lines were found, and the degree of sensitivity was shown to be dependent upon the abundance of narcotic receptors. Thus, morphine receptors are functionally coupled to adenylate cyclase. A molecular mechanism for narcotic addiction and tolerance is proposed.

Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase.

Abstract: An activating factor of adenylate cyclase (EC 4.6.1.1) HAS BEEN OBTAINED FROM DETERGENT-DISPERSED PREPARATIONS OF PORCINE CEREBRAL CORTEX BY COLUMN CHROMATOGRAPHY ON ECTEOLA-cellulose. The factor was identified by acrylamide gel electrophoresis and by enzyme activation studies as the Ca2+-binding protein that regulates the activity of a brain cyclic nucleotide phosphodiesterase. This Ca2+-binding protein confers a Ca2+-dependent activation upon the adenylate cyclase, which is reversed by the subsequent addition of egta in excess of the free Ca2+. It is proposed that this Ca2+-dependent regulator controls enzymatic activities responsible for the synthesis of adenosine 3':5'-monophosphate and for the hydrolysis of guanosine 3':5'-monophosphate.

Catecholamine-induced subsensitivity of adenylate cyclase associated with loss of beta-adrenergic receptor binding sites

Abstract: Injection of frogs with beta-adrenergic catecholamines for 1-24 hr produces marked subsensitivity of the erythrocyte membrane adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1.] to in vitro stimulation by isoproterenol. The subsensitization is specific for catecholamine stimulation, since basal and fluoride-stimulated enzyme activity are unaffected. Maximum isoproterenol-stimulated adenylate cyclase activity declines by 75% in the isoproterenol-treated animals (P less than 0.001). The concentration of isoproterenol causing one-half maximal activation of adenylate cyclase, however, is unaltered. (-)[3H]Alprenolol, a potent competitive beta-adrenergic antagonist, was used to study directly the beta-adrenergic receptor binding sites in the erythrocyte membranes from control and subsensitized animals. A highly significant (P less than 0.005) 60% fall in the number of the beta-adrenergic receptor binding sites ("specific"(-)[3H]alprenolol binding sites) in the treated animals was found. The binding affinity of the sites was not markedly altered. These data suggest that beta-adrenergic catecholamines are able to regulate catecholamine sensitivity of tissues in vivo, by regulating the properties of the beta-adrenergic receptor binding sites.

Differentiation of neuroblastoma cells in culture.

Abstract: Summary 1 An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E1 by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G1-stage of the cell cycle 2 Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP 3 Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other 4 Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells 5 A working hypothesis for the malignancy of nerve cells has been proposed This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells 6 A new experimental therapeutic model for the treatment of neuroblastoma is proposed This involves the administration of sodium butyrate followed by the injection of l-dihydroxyphenylalanine (l-dopa) and prostaglandin E1 in the presence of cyclic AMP phosphodiesterase inhibitor 7 Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells

Selection of a variant lymphoma cell deficient in adenylate cyclase

Abstract: Isoproterenol, a stimulator of adenylate cyclase, was used to select a stable variant clone of mouse lymphosarcoma cells deficient in the enzyme. The inability of four different stimulators to activate cyclic adenosine monophosphate synthesis in the variant, in contrast to its wild-type parent, implies that in normal cells one type of adenylate cyclase molecular can respond to different activators.

Rapid changes in rat pineal beta-adrenergic receptor: alterations in l-(3H)alprenolol binding and adenylate cyclase.

Abstract: The properties of the beta-adrenergic receptor which regulates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing)8 EC 4.6.1.1] in the pineal gland are similar to the properties of the sites which specifically bind l-[3H]alprenolol, a potent beta-adrenergic antagonist. Stimulation of the beta-adrenergic receptor results in a 30-fold increase in the activity of N-acetyltransferase (= arylamine acetyltransferase; acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), an enzyme involved in the synthesis of thepineal hormone melatonin. In the normal diurnal light-dark cycle there is greater physiological stimulation of the beta-adrenergic receptor in the pineal during the night than during the day. Pineals from rats kept in constant light for 24 hr possess more hormone-sensitive adenylate cyclase and specifically bind more l-[3H]alprenolol than do pineals from rats kept in the dark overnight. When rats, exposed to light for 24 hr, are treated with the beat-adrenergic agonist isoproterenol, there is a rapid loss of both hormone-sensitive adenylate cyclase activity and specific l-[3H]alprenolol binding sites. There is no change in the affinity of adenylate cyclase for isoproterenol or for its substrate, ATP. Similarly, although there are fewer binding sites, there is no change in the affinity of the remaining sites for either agonist or antagonist. Inhibition of protein synthesis with cycloheximide does not affect the loss of either adenylate cyclase activity or specific binding sites. The data suggest that stimulation of the beta-adrenergic receptor causes a rapid decrease in the number of available receptors and in hormone-sensitive adenylate cyclase activity; conversely, lack of stimulation causes an increase in these parameters. It is suggested that these changes contribute to the phenomena of super- and subsensitivity in the pineal gland by regulating the capacity of the pineal to synthesize cyclic AMP in response to beta-adrenergic stimulation.

Direct identification and characterisation of beta-adrenergic receptors in rat brain.

Abstract: MODULATION of cellular function by the autonomic nervous system is accomplished, at least in part, by the stimulation of β-adrenergic receptors by catecholamines at neuroeffector junctions1. Activation of these receptors results in stimulation of adenylate cyclase with increased intracellular levels of cyclic AMP. The β-adrenergic receptors of a variety of non-neural tissues have been characterised indirectly by measurement of altered adenylate cyclase activity or of intracellular cyclic AMP in response to β-adrenergic agonists and antagonists. Direct characterisation of the β-adrenergic receptors in avian2,3 and amphibian4 erythrocytes and in canine heart5 has been accomplished by binding assays using radioactive β-adrenergic antagonists.

Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus.

Abstract: — An adenylate cyclase present in the caudate nucleus of rat brain, which is selectively stimulated by low concentrations of dopamine, and which is believed to mediate dopaminergic synaptic transmission, has been characterized with respect to several properties. The parameters studied included temperature, pH, ATP concentration, Mg/ATP ratio, and metal ion specificity. The effects of other compounds, including EGTA, NaF and several guanosine nucleotides, were also tested on the dopa-mine-sensitive adenylate cyclase. In addition, the subcellular distribution of the enzyme was studied. The highest specific activity was found in subcellular fractions enriched in nerve endings. A half-maximal increase in the activity of the enzyme in a subcellular fraction occurred in the presence of 4 × 10−6 M dopamine. Fluphenazine, a dopamine antagonist, competitively inhibited the activity of the enzyme in this fraction, with a calculated inhibition constant (Ki) of 8 × 10−9M.

Development of a Mn-2+-sensitive, "soluble" adenylate cyclase in rat testis.

Abstract: A distinctive Mn-2+-sensitive adenylate cyclase [ATP pyrophosphate-lyase(cyclizing), EC 4.6.1.1] system insensitive to fluoride has been found in rat seminiferous tubules and epididymal sperm. The development of this distinctive adenylate cyclase in testis was studied during spermatogenesis. It was first detectable in seminiferous tubules in immature rats at about the time of the first reductive divisions and the appearance of spermatid cells. The specific activity of the enzyme increased substantially during the period of spermatogenesis when spermatids develop into mature spermatozoa, and reached maximal values in the testis of adult rats. After centrifugation of testis tissue homogenates at 105,000 X g for 60 min, the Mn-2+-sensitive adenylate cyclase activity was found in the cytosol. The enzyme remains in solution after centrifugation at 300,000 X g for 5 hr or at 180,000 X g for 24 hr and passes through a 0.22 mum Millipore filter. Electron microscopic examination showed no visible membrane fragments or vesicles in the filtered supernatant. The Mn-2+-sensitive adenylate cyclase system is also present in epidiymal sperm. However, in the sperm obtained from either the caput or the cauda of epididymis, the adenylate cyclase is membrane-associated and found in particulate fractions of sperm homogenates. It therefore appears that the Mn-2+-sensitive adenylate cyclase is initially present in the cytoplasm either unattached or loosely bound to intracellular membranes and becomes firmly attached to sperm membranes later in development. This occurs either during the process of maturation of spermatids into sperm or during the transport of the testicular sperm into the epididymis.

Fatty acids as modulators of membrane functions: catecholamine-activated adenylate cyclase of the turkey erythrocyte.

Abstract: Activation of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1[ from turkey erythrocytes by isoproterenol decreased precipitously below 26 degrees. Certain unsaturated fatty acids enhanced the activation by isoproterenol up to 25-fold at reduced temperatures. The fatty acid also enhanced the formation of a persistent active state of the enzyme which was produced by preincubation with guanosine 5'-(beta,gamma-imino)triphosphate [Gpp(NH)p]. Once the enzyme had been activated by Gpp(NH)p plus isoproterenol the reaction rate was no longer as temperature sensitive and the fatty acid had little effect. The synthetic Gpp(NH)p apparently substituted for the natural GTP, which is known to play a regulatory role in the adenylate cyclase system. The findings suggest that the function of GTP which is mediated by the hormone is the temperature-sensitive event which is enhanced by the fatty acid. The use of free fatty acid to probe membrane-associated reactions in intact cells and in isolated membrane preparations is proposed.

Effects of adenosine on levels of adenosine cyclic 3',5'-monophosphate in human blood platelets in relation to adenosine incorporation and platelet aggregation.

Abstract: Cyclic 39, 59-[14C]AMP was measured in platelets that had first been incubated with [14C]adenine. Maximum increases of 2-4-fold were observed 0.5 min after addition of 10-40 µM adenosine. Smaller increases were obtained with higher concentrations of adenosine. In 0.5-min incubations 2-chloroadenosine was less effective than adenosine at concentrations below 20 µM and more effective at concentrations above 100 µM. Incorporation of 1-10 µM adenosine into platelets was inhibited at least 96% by p-nitrobenzylthioguanosine without any effect on the increase in cyclic [14C]AMP caused by these concentrations of adenosine, suggesting that adenosine acts at an extracellular site. With higher adenosine concentrations, p-nitrobenzylthioguanosine was less effective in inhibiting incorporation of adenosine but blocked the decline in cyclic [14C]AMP levels observed on increasing the adenosine concentration above 40 µM. This inhibitory effect of high adenosine concentrations on the accumulation of cyclic [14C]AMP was more easily detected when adenosine was added with prostaglandin E1 and represents a second, possibly intracellular action of adenosine unrelated to its effect in increasing cyclic AMP levels. Papaverine markedly potentiated the increase in platelet cyclic [14C]AMP observed with all concentrations of adenosine, indicating that adenosine activates platelet adenylate cyclase. The kinetics of this activation were studied in intact platelets incubated for short intervals in the presence of papaverine. Adenosine (K A = 1 µM) activated platelet adenylate cyclase up to a maximum of 8-10-fold. This action of adenosine was competitively inhibited by caffeine (Ki = 72 µM) or theophylline (Ki = 25 µM). No inhibitory effect of high adenosine concentrations on cyclic [14C]AMP formation was observed in intact platelets in the presence of papaverine. The plateletaggregating agents ADP and epinephrine, but not vasopressin, markedly inhibited the increase in platelet cyclic [14C]AMP with adenosine. ADP was found to be a noncompetitive inhibitor (Ki = 0.9 µM) of the effect of adenosine on adenylate cyclase in intact platelets. Some close correlations were observed between the effects of adenosine on platelet cyclic [14C]AMP levels and on platelet aggregation. Caffeine partially blocked the inhibition of aggregation by adenosine. As a whole the results show that platelets possess a specific extracellular membrane receptor for adenosine, which is distinct from that for ADP and which mediates the inhibition of platelet function by adenosine by activating platelet adenylate cyclase.

Melanocyte-stimulating hormone promotes activation of pre-existing tyrosinase molecules in cloudman s91 melanoma cells.

Abstract: TYROSINASE activity is greatly enhanced in cultured Cloudman S91 melanoma cells following addition of melanocyte-stimulating hormone (MSH) to the culture medium1. The increased activity occurs in the G2 phase of the cell cycle2 because membrane receptors for MSH are available only in this phase3. The response to MSH is mediated through cyclic AMP (refs 1–4). It is well documented that many peptide hormones function by activating membrane-bound adenyl cyclase molecules, causing net increases in intracellular cyclic AMP concentrations. These increases in turn have profound effects on cell division, morphology, and the expression of differentiated functions in a wide variety of cells and tissues. Little is known, however, about the levels at which genetic expression is regulated or the molecular intermediates involved in the regulation. For example, it is generally assumed that cyclic AMP-dependent protein kinases are involved in many of the responses because most tissues contain such enzymes5. The regulation of glycogenolysis is the best known example supporting this assumption6–7. But it cannot be taken as fact that in eukaryotes all cyclic AMP-mediated processes are post-translational in nature.

Vasopressin dependent adenylate cyclase in single segments of rabbit kidney tubule.

Abstract: AVP dependent adenylate cyclase activity was measured in single pieces of 8 different tubular segments isolated from collagenase treated rabbit kidneys.

Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions.

Abstract: Rat liver membrane adenylate cyclase (EC 4.6.1.1) that has been stimulated more than 10-fold by cholera toxin (choleragen) has a 3-fold greater sensitivity to stimulation by glucagon. Choleragen similarly increases the sensitivity of cyclase to other peptide (ACTH, vasoactive intestinal polypeptide) and nonpeptide (catecholamines) hormones in this and other tissues. The rate of 125I-labeled glucagon-membrane dissociation is decreased about 2-fold in toxin-treated liver membranes. Toxin-activated cyclase activity of fat cell membranes is retained upon solubilization with Lubrol PX. Provided 125I-labeled choleragen is first incubated with cells under conditions resulting in enzyme activation, the solubilized cyclase activity migrates with a component of 125I-labeled choleragen on gel filtration chromatography. Agarose derivatives containing the "active" subunit (molecular weight 36,000) of the toxin can specifically adsorb solubilized adenylate cyclase. Toxin-stimulated cyclase can be immunoprecipitated with antitoxin or anti-"active" subunit antibodies. There is a large excess of membrane receptors (ganglioside GM1) which, with the use of choleragenoid, can be shown to be functionally equivalent with respect to cyclase activation. Choleragenoid, an inactive competitive antagonist of toxin binding, can occupy and block a large proportion of toxin receptors without affecting toxin activity. A scheme of toxin action is proposed that involves lateral membrane diffusion of the initially inactive toxin-receptor complex with subsequent direct interaction with and modulation of adenylate cyclase. The basic features of this scheme may be pertinent to the mechanisms by which hormone receptors normally modulate adenylate cyclase.