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.     

Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity.     

Alterations in cerebral β-adrenergic receptor-adenylate cyclase system induced by halothane,ketamine and ethanol

The effect of halothane, ketamine and ethanol on β-adrenergic receptor adenylate cyclase system was studied in the brain of rats. An anesthetic concentration of halothane and ketamine added in vitro decreased the stimulatory effect of norepinephrine on cyclic AMP formation in slices from the cerebral cortex. On the other hand, ethanol increased the basal activity of cerebral adenylate cyclase without affecting on the norepinephrine-stimulated activity. The increase of the basal activity induced by ethanol was not antagonized by propranolol, a β-adrenergic antagonist. In the crude synaptosomal (P₂) fraction, these drugs had no significant effect on the basal adenylate cyclase activity, binding of [³H]dihydroalprenolol to β-receptor, and binding of [³H]guanylylimido diphosphate ([³H]Gpp(NH)p) to guanyl nucleotide binding site. In contrast, the adenylate cyclase activity stimulated by Gpp(NH)p or NaF was significantly inhibited by an anesthetic concentration of these drugs. An anesthetic concentration of these drugs increased the membrane fluidity of P₂ fraction monitored by the fluorescence polarization technique. The addition of linoleic acid (more than 500 μM) also induced not only the increase of fluidity, but also the decrease of Gpp(NH)p- or NaF-stimulated adenylate cyclase activity in the cerebral P₂ fraction. The present results suggest that general anesthetics may interfere with the guanyl nucleotide binding regulatory protein-mediated activation of cerebral adenylate cyclase by disturbing the lipid region of synaptic membrane.     

The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.     

Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

[3H]norepinephrine binding by rat glial cells in culture lack of correlation between binding and adenylate cyclase activation

Subcellular fractions prepared from rat glial cells in culture (clonal line C₆) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [³H]norepinephrine binding and enzyme activation were measured under identical experimental conditions.Binding sites for norepinephrine could be detected; their main characteristics were: apparent K_m : 4 · 10⁻⁶ M, maximal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrenergic agonist) and the blocking agent propranolol were unable to compete with [³H]norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [³H]norepinephrine binding.As in several other systems so far studied, the presence on the glial cell's membrane of a large number of “catechol-binding sites” makes it difficult to characterize the β-adrenergic receptor.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Multiphasic activation of smooth muscle adenylate cyclase by pretreatment with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) suggests multiple enzyme populations

The activation of uterine smooth muscle adenylate cyclase was studied by pretreating the particulate form of the enzyme with the GTP analog guanyl-5′-yl imidodiphosphate (Gpp(NH)p). Pretreatment with Gpp(NH)p left the enzyme in an irreversibly activated state which survived subsequent washing in guanyl nucleotide-free buffer. Activation under these conditions was multiphasic with rapid and slow components. At 23 °C slow activation proceeded at about $\text{1}\text{10}\text{th}$ the rate of rapid activation. The onset of the slow phase took longer at lower temperatures. Routine adenylate cyclase assay conditions (conversion of [³²P]ATP to cyclic [³²P]AMP) carried out without pretreatment probably characterized the rapidly activated component. The simplest kinetic model suggests not only the generally accepted two-step association reaction, but also implies the existence of more than one enzyme form, each of which is characterized by a separate activation rate. The complex kinetics of activation might be explained by a heterogeneous mixture of unassociated and preassociated nucleotide binding and catalytic subunits.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [³H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [³²P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min^?1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [³²P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [³H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. ³H-agonist binding and agonist-promoted [³²P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.     

Association of binding sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Interaction of gastrointestinal hormones

1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.     

Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Dopamine receptors in canine caudate nucleus

Summary Physiological, pharmacological, histochemical and biochemical studies indicate that dopamine receptors are heterogenous in the, central nervous system with each individual functions. This review describes pharmacological and biochemical characteristics of dopamine receptors, particularly in canine caudate nucleus, which have been studied in our laboratory with a brief comparison to the current studies by other workers in similar research fields.Two distinct dopamine receptors have been characterized by means of [³H]dopamine binding to the synaptic membranes from canine caudate nucleus. One of the receptors with a Kd of about 3 M for dopamine may be associated with adenylate cyclase and referred to as D, receptor. The other receptor with a Kd of about 10 nM for dopamine is independent of adenylate cyclase and referred to as D₂. A photochemical irreversible association of [³H]dopamine with the membraneous receptors makes it possible to separate D₁ and D₂ receptors from one another by gel filtration on a Sephadex G-200 column after solubilization with Lubrol PX. On the basis of selective inhibition of [³H]dopamine binding to D₁ and D₂ receptors, dopamine antagonists can be classified into three classes: D₁-selective (YM-09151-2), D₂-selective (sulpiride) and nonselective (haloperidol, chlorpromazine). Effects of these typical antagonists on the metabolism of rat brain dopamine suggest that D₁ receptor is more closely associated with the neuroleptic-induced increase in dopamine turnover. Studies with 28 benzamide derivatives and some classical neuroleptics reveal that apomorphine-induced stereotypy displays a greater association with D₁ than with D₂ receptors.Dopamine-sensitive adenylate cyclase in canine caudate nucleus can be solubilized with Lubrol PX in a sensitive form to either dopamine, Gpp(NH)p or fluoride. Sephadex G-200 gel filtration separates adenylate cyclase from D₁ receptors with a concomitant loss of dopamine sensitivity. Addition of the D₁ receptor fraction to the adenylate cyclase restores the responsiveness to dopamine. The solubilized dopamine-unresponsive adenylate cyclase can be further separated into two distinct fractions by a batch-wise treatment with GTP-sepharose: a catalytic unit which does not respond to fluoride, and a guanine nucleotide regulatory protein. The regulatory protein confers distinct responsiveness to Gpp(NH)p and fluoride upon adenylate cyclase. These results indicate that dopamine-sensitive adenylate cyclase is composed of at least three distinct units; D₁ receptor, guanine nucleotide regulatory protein and adenylate cyclase.     

Evidence for Different States of the Dopamine Dl Receptor: Clozapine and Fluperlapine May Preferentially Label an Adenylate Cyclase-Coupled State of the Dl Receptor

Abstract: It has been shown previously that typical neuroleptics have higher affinities for 3,4-dihydroxyphenyl-ethylamine (dopamine) Dl receptors as labeled by(R)- (+)- 8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 -N-3-benzazepine-7-ol ([³H]SCH 23390) than for inhibiting dopamine-stimulated adenylate cyclase. We now report that the atypical neuroleptics, clozapine and fluperlapine, exhibit characteristics opposite to typical neuroleptics, i.e., they have higher affinity for inhibiting dopamine-stimulated adenylate cyclase than [³H]SCH 23390 binding. A variety of compounds, i.e., clozapine, fluperlapine, and dopamine, were tested for their capacity to affect the rate constants of [³H]SCH 23390 binding; these experiments revealed no effect of any tested compound on on-rate or off-rate of [³H]SCH 23390 binding. Treatment of striatal membranes with phospholipase A₂ (PLA₂) caused a rapid decrease in the B_max value of the [³H]SCH 23390 binding with no effect on the K_d value. The adenylate cyclase, both the unstimulated, the dopamine-, fluoride-, and forskolin-stimulated activity, was far less sensitive than [³H]SCH 23390 binding to PLA₂. Treatment of striatal membranes with filipine and (NH₄SO₄ produced, as did PLA₂ treatment, a rapid decline in [³H]SCH 23390 binding. However, opposite to PLA₂ treatment, these agents stimulated the adenylate cyclase. In conclusion, a comparison of the pharmacological characteristics of [³H]SCH 23390 binding and dopamine-stimulated adenylate cyclase suggests the existence of two different Dl binding sites. The rate experiments exclude the possibility of allosterically coupled sites. Instead our results favor that the Dl receptor exists in different states/conformations, i.e., both adenylate cyclase-coupled and uncoupled, and further, that the atypical neuroleptics clozapine and fluperlapine may have adenylate cyclase-coupled dopamine Dl receptors as target.