Forms of adenylate cyclase, activation and/or potentiation by forskolin

Activation of different forms of adenylate cyclases (AC) by forskolin and displacement of [14,15-3H]dihydroforskolin binding from membranes by forskolin in the absence or presence of specific stimulatory hormone and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p) have been studied. These conditions have been used to generate forskolin dose-response curves of AC activation. A plot of enzyme activation versus apparent forskolin-binding showed a linear and a nonlinear relationship, respectively, in the absence or presence of the other two stimulators. The latter relationship can be fitted by two linear regression lines with a defined intercept, the slopes of which represent two distinct binding-activation (B-A) effects. The B-A effects of forskolin for rat adipocyte and liver membranes in the absence of stimulatory hormone and Gpp(NH)p were 10 and 8 (pmol X min-1) X (pmol)-1, respectively. The B-A effects for the same membranes in the presence of the other two stimulators were 69 (high) and 13 (low) (pmol X min-1) X (pmol)-1 for adipocyte membrane, and 83 (high) and 9 (low) (pmol X min-1) X (pmol)-1 for liver membrane. The ratio of potentiation of forskolin-activated enzyme activity to the unmodified forskolin-stimulated activity (P-A ratio) was determined without the binding data. At 3 microM forskolin, with and without 230 epinephrine and 10 microM Gpp(NH)p, the P-A ratio was 3.7, decreasing to 1.1 with the addition 100 microM forskolin. The line representing a high B-A effect and a resulting high P-A ratio appears to describe the interactions between forskolin and the AC stimulated by epinephrine and Gpp(NH)p. The line of low B-A effect may represent the interaction between forskolin and the basal AC. Two peaks of AC activity were eluted from forskolin-Sepharose column. They have apparent differences in sensitivity to Gpp(NH)p and affinity for forskolin. Based on the results available thus far, with consideration for known limitations of the methodology, a working model has been proposed for forskolin activation of AC.     

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.     

A simple test for enhanced guanyl nucleotide exchange in brain adenylate cyclase systems activated by neurotransmitters

The mechanism of receptor-induced activation of adenylate cyclase has been proposed to involve an enhanced exchange of GDP for GTP. The kinetics of this process have not been investigated so far in the brain due to a spontaneous activation of the enzyme by guanyl nucleotides, which precludes the ability to follow receptor-dependent events. We show that it is possible to investigate the mechanism of receptor action in such systems by using a combination of guanosine 5'-(beta-gamma-imino)triphosphate (Gpp(NH)p) and guanosine 5'-(2-O-thio)diphosphate (GDP beta S). In pineal membranes, beta-adrenergic agonists increase the rate of adenylate cyclase activation by 10 or 100 microM Gpp(NH)p about 40-fold (0.023-0.9 min-1 kact) and decrease the inhibitory potency of GDP beta S nearly 1000-fold. As a result, 100 microM GDP beta S which blocks 90% of the activation by 10 microM Gpp(NH)p has no inhibitory effect in the presence of 10 microM Gpp(NH)p and 10 microM noradrenaline or isoproterenol. In caudate nucleus, dopamine does not appear to increase the rate of activation of adenylate cyclase by 10 microM Gpp(NH)p. Nevertheless, 100 microM GDP beta S blocks 90% of the activation by 10 microM Gpp(NH)p but has no inhibitory effects in the presence of dopamine. Thus, one can demonstrate that even weakly activating receptors have the capacity to facilitate a functional exchange of GDP beta S for Gpp(NH)p and measure the efficacy of the interaction between the receptor and the functionally linked guanyl nucleotide subunit.     

Forskolin-induced change of the size of adenylate cyclase

Forskolin, a potent activator of cyclic AMP generating systems, has been proposed to act directly on the catalytic unit of adenylate cyclase. Nevertheless, some arguments indicate a possible role of the guanosine triphosphate-binding regulatory protein in forskolin action on adenylate cyclase. In this study, we have observed an increase in the apparent sedimentation coefficient of solubilized adenylate cyclase, elicited by forskolin, both in rat liver (from 6.4 +/- 0.1 to 7.2 +/- 0.1 S) and rat striatum (from 6.7 +/- 0.1 to 7.6 +/- 0.1 S). On both systems, a similar increase in the sedimentation coefficient was observed after preactivation of the enzyme with guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p). In contrast to the Gpp(NH)p effect, the forskolin action was found to be reversible. Simultaneous pretreatments of adenylate cyclase with forskolin and Gpp(NH)p did not induce additive increases of the apparent sedimentation coefficient of adenylate cyclase. The modification of the size of solubilized adenylate cyclase was corroborated by gel filtration studies. In rat liver membranes, the Stokes radius of the solubilized enzyme increased from 59 +/- 1 A for basal state to 65 +/- 1 A for forskolin preactivated state. A possible explanation of our findings is that forskolin may stabilize the complex between the GTP-binding regulatory protein and the catalytic unit of adenylate cyclase in a reversible manner.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

Adenylate cyclase of platelets from spontaneously hypertensive rats: reduction of the inhibition by GTP

We have compared the effects of Gpp[NH]p on adenylate cyclase activity of platelet membranes in SHR and WKY rats. In the presence of 50 microM forskolin, low concentrations of Gpp[NH]p (0.01 to 0.3 microM) inhibited the enzyme activity in both strains, but the maximal level of inhibition was significantly lower in SHR (- 20%). In the absence of forskolin, 0.1 microM Gpp[NH]p was inhibitory only in WKY and the adenylate cyclase activity was greater in hypertensive rats at this nucleotide concentration. Increasing Gpp[NH]p from 0.1 to 3 microM induced the same increase of enzyme activity in both strains. In SHR, GTP itself induced a lower inhibition of the enzyme stimulated by 50 microM forskolin or 0.1 microM prostaglandin E1. These results suggest that the modulatory effect of the guanine nucleotide inhibitory protein on adenylate cyclase may be reduced in platelets from SHR.     

Effect of GDP on transducin interaction with cyclic nucleotide phosphodiesterase and rhodopsin from bovine retinal rods

In the presence of guanyl nucleotides and rhodopsin-containing retinal rod outer segment membranes, transducin stimulates the light-sensitive cyclic nucleotide phosphodiesterase 5.5-7 times. The activation constant (Ka) for GTP and Gpp(NH)p is 0.25 microM, that for GDP and GDP beta S is 14 and 110 microM, respectively. GDP purified from other nucleotide contaminations at concentrations up to 1 mM does not stimulate phosphodiesterase but binds to transducin and inhibits the Gpp(NH)p-dependent activation of phosphodiesterase. The mode of transducin interaction with bleached rhodopsin also depends on the nature of the bound guanyl nucleotide: in the presence of GDP rhodopsin-containing membranes bind 70-100% of transducin, whereas in the presence of Gpp(NH)p the membranes bind only 13% of the protein. The experimental results suggest that GDP and GTP convert transducin into two different functional states, i.e., the transducin X GTP complex binds to phosphodiesterase causing its stimulation, while the transducin X GDP complex is predominantly bound to rhodopsin.     

Differential Sensitivity of Basal and Opioid-Stimulated Low Km GTPase to Guanine Nucleotide Analogs

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.     

Forskolin Potentiates the Stimulation of Rat Striatal Adenylate Cyclase Mediated by D-1 Dopamine Receptors, Guanine Nucleotides, and Sodium Fluoride

We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 microM GTP, 100 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two- to fourfold) in the EC50 value for forskolin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM-100 microM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194-1,825%, 122-1,141%, and 208-938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4-dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 microM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 microM) and SKF38393 (1 microM). At higher concentrations of forskolin (10-100 microM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium inhibition of cardiac adenylyl cyclase. Evidence for two distinct sites of inhibition

Increasing the free calcium concentration from 10(-8) M to 10(-4) M inhibited cardiac sarcolemmal adenylyl cyclase activated by the addition of 5 X 10(-4) M forskolin or 1 X 10(-4) M GTP or Gpp(NH)p. The calcium inhibition curve in the presence of all three activators was shallow and best fit by a two site model of high affinity (less than 1.0 microM) and low affinity (greater than 0.1 mM). Gpp(NH)p appeared to decrease the sensitivity of adenylyl cyclase to inhibition by calcium at the high affinity site. Similar inhibition constants were obtained with each of the activators. Calmodulin content of native freeze-thaw vesicles was 76.2 +/- 14.2 ng/mg. Treatment of the vesicles with 1 mM EGTA to remove calmodulin significantly reduced calmodulin content to 19.7 +/- 1.35 ng/mg. This treatment had no significant effect on the calcium inhibition profile. Increasing free calcium to 3 X 10(-6) M was shown to have no effect on the EC50 estimated for either Gpp(NH)p or forskolin but did slightly increase the EC50 estimated for Mg2+ in the presence of maximal concentrations of either activator. Nevertheless, maximally stimulating concentrations of Mg2+ were unable to overcome calcium inhibition. Pretreatment of sarcolemmal membranes with pertussis toxin was shown to have no significant effect on calcium inhibition of adenylyl cyclase. The results suggest that the overall inhibitory action of calcium was most likely calmodulin independent and involved a direct interaction with the catalytic subunit at two distinct sites of high and low affinity. At the low affinity site calcium most likely competes with Mg2+ for an allosteric divalent cation binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of a GTP-binding protein in coupling of a muscarinic cholinergic receptor and Na,K-ATPase in myocardial sarcolemma]

The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme.     

Forskolin activation of adenylate cyclase in mouse parotid membranes

In mouse parotid membranes forskolin activated adenylate cyclase four-fold; maximal activation of the enzyme occurred with 10 microM forskolin. Activation was not dependent on the guanyl nucleotide GTP nor on the inhibitory guanine nucleotide 5'-0-(2-Thiodiphosphate), GDP beta S. In contrast, stimulation of adenylate cyclase by isoproterenol required GTP and was antagonized by GDP beta S in a dose-dependent manner. These results indicate that the guanyl-binding protein of mouse parotid adenylate cyclase is not a requisite for forskolin activation and lends support for direct interaction of forskolin at the catalytic subunit.     

Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes.

The guanine nucleotides guanosine 5'[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H )inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Purification and partial characterization of two forms of urinary trypsin inhibitor

The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.     

Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides

Binding of thyrotropin-releasing hormone (TRH) to specific receptors on membranes isolated from GH4C1 pituitary cells was inhibited by monovalent cations and guanyl nucleotides. NaCl and LiCl inhibited TRH binding by 70%, with half-maximal inhibition at 30 mM; RbCl and KCl inhibited only 10% at concentrations up to 150 mM. NaCl decreased both the apparent number and the affinity of TRH receptors and increased the rate of dissociation of TRH from both membrane and Triton X-100-solubilized receptors. Guanyl nucleotides inhibited TRH binding up to 80%, with guanyl-5'-yl imidodiphosphate (Gpp(NH)p) approximately GTP much greater than GDP approximately ATP greater than GMP. GTP and Gpp(NH)p exerted half-maximal effects at 0.3 microM and decreased receptor affinity to one-third of control but did not change receptor number. Gpp(NH)p accelerated the dissociation of TRH from membranes but not from solubilized receptors. The effects of NaCl were independent of temperature, while GTP and Gpp(NH)p were much more inhibitory at 22 degrees C (70%) than at 0 degrees C (10%). Inhibition by NaCl could be reversed by washing the membranes, and inhibition by GTP was reversed if membranes were chilled to 0 degrees C. The inhibitory effects of low concentrations of NaCl and Gpp(NH)p were additive. Neither monovalent cations nor GTP prevented the TRH-receptor complex from undergoing transformation from a state with rapid dissociation kinetics to a slower dissociating form. The results suggest that sodium ion regulates TRH binding by interacting with a site on the receptor, while guanyl nucleotides regulate TRH binding indirectly.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)