Adenylate Cyclases in Vertebrate Retina: Enzymatic Characteristics in Normal and Dystrophic Mouse Retina

Adenylate cyclase activity and the effects of various activators and inhibitors of this enzyme were measured in retinas from normal mice (C57BL/6J) and congenic animals with photoreceptor dystrophy. In normal retina, approximately 250 microM-ATP was required for half-maximal stimulation of the enzyme. Activity was supported by Mg2+ and Mn2+, but Ca2+ was ineffective. The enzyme was inhibited by EGTA and stimulated by 5'-guanylylimidodiphosphate (GPP(NH)P), dopamine, and NaF. The stimulatory effects of GPP(NH)P and dopamine were greater in the presence of EGTA. Examination of microdissected normal retinas revealed that the inner (neural) retina had adenylate cyclase activity four times that of the photoreceptor cell layers, and that EGTA inhibited activity in the inner retina, but had no effect in the outer retina. In dystrophic retinas basal enzyme activity was 60% higher than that in normal retina. The enzyme in this tissue was stimulated by EGTA, GPP(NH)P, and dopamine, and their effects were additive. These results indicate that adenylate cyclase activity in vertebrate retina is under complex regulation by substrate, divalent cations, guanine nucleotides, dopamine, and perhaps calmodulin. In addition, the data demonstrate that adenylate cyclase is not evenly distributed in the retina and that it is regulated differently in the inner and outer retina. Finally, the present results indicate that regulation of this enzyme in dystrophic retina may be qualitatively and quantitatively different from that in normal retina.     

Calmodulin-Sensitive and Calmodulin-Insensitive Components of Adenylate Cyclase Activity in Rat Striatum Have Differential Responsiveness to Guanyl Nucleotides

The interaction between the Ca2+-binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal particulate fraction. We found that the ability of calmodulin to stimulate adenylate cyclase in the presence of guanyl nucleotides depends upon the type and concentration of the guanyl nucleotide. Adenylate cyclase activity measured in the presence of calmodulin and GTP reflected additivity at every concentration of these reactants. On the contrary, when the activating guanyl nucleotide was the nonhydrolyzable analog of GTP, guanosine-5'-(beta,gamma-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 microM GppNHp. Kinetic analysis of adenylate cyclase by GppNHp was compatible with a model of two components of adenylate cyclase activity, with over a 100-fold difference in sensitivity for GppNHp. The component with the higher affinity for GppNHp was competitively stimulated by calmodulin. The additivity between calmodulin and GTP in the striatal particulate fraction suggests that they stimulate different components of cyclase activity. The calmodulin-stimulatable component constituted 60% of the total activity. Our two-component model does not delineate, at this point, whether there are two separate catalytic subunits or one catalytic subunit with two GTP-binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat

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

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Pertussis toxin reverses Gpp(NH)p inhibition of basal and forskolin activated adipocyte adenylate cyclase

Inhibition of basal adenylate cyclase by GTP or guanyl-5'-yl imidodiphosphate was abolished in membranes isolated from rat adipocytes previously incubated with pertussis toxin. Forskolin (0.1 microM) stimulated adenylate cyclase about 4-fold and inhibition of cyclase by GTP or guanyl-5'-yl imidodiphosphate was also abolished by pertussis toxin treatment of rat adipocytes. Forskolin (1 microM) increased adenylate cyclase activity at least ten-fold and the inhibitory effect of GppNHp was reduced but not abolished by pertussis toxin. In rabbit adipocytes, pertussis toxin reversed the inhibition of adenylate cyclase activity by GppNHp to the same extent as that by GTP in the presence of 1 microM forskolin. The present results indicate that pertussis toxin can reverse the inhibition of adipocyte adenylate cyclase by nonhydrolyzable GTP analogs as well as that by GTP.     

Inhibition by S-adenosyl-L-homocysteine of dopamine dependent adenylate cyclase in rat retina

S-adenosyl-L-homocysteine (S-AH), a potent inhibitor of biological transmethylation, decreased the response of rat retina adenylate cyclase to dopamine and to 2-amino-6, 7-dihydroxytetrahydronaphtalene (ADTN). This effect appeared for 10^?7M of S-adenosyl-L-homocysteine and was linear for concentration ranging to 10^?4M. S-adenosyl-L-homocysteine did not decrease the cyclic AMP accumulation with sodium fluoride, a non specific adenylate cyclase activator. On the other hand, the incorporation of methyl group was reduced in rat retina homogenates by S-adenosyl-L-homocysteine. These findings suggest that the activity of the dopamine dependent adenylate cyclase is linked to a methylation process.     

Adenylate Cyclases in the Vertebrate Retina: Distribution and Characteristics in Rabbit and Ground Squirrel

Adenylate cyclase activity and the effects of EGTA, 5'-guanylylimidodiphosphate (GPP(NH)P), and dopamine were measured in microdissected layers of rod-dominant (rabbit) and cone-dominant (ground squirrel) retinas, The distribution of basal enzyme activity was similar in both species, with the highest levels found in the inner plexiform and photoreceptor cell inner segment layers, EGTA inhibited adenylate cyclase in the inner retina of both species and stimulated activity in rabbit outer and inner segment layers, but had no effect in these layers from ground squirrel. Enzyme activity was stimulated in all regions by GPP(NH)P, except in the outer segments of the photoreceptors. Dopamine stimulated the enzyme in the outer and inner plexiform and inner nuclear layers in rabbit, but only in the inner plexiform layer in ground squirrel. These data demonstrate that the enzymatic characteristics of adenylate cyclase vary extensively from region to region in vertebrate retina and suggest that cyclic AMP may have multiple roles in this tissue. A model for the distribution of the different forms of adenylate cyclase in retina is proposed.     

Calmodulin activation of rat lung adenylate cyclase is independent of the cytoplasmic factors modulating the enzyme.

Adenylate cyclase activity in the rat lung membranes washed with 150 microM-EGTA was stimulated by calmodulin in the presence of 100 microM-Ca2+. The calmodulin activation of the enzyme was concentration-dependent; however, at high concentrations the activation was diminished. Activation of adenylate cyclase by calmodulin was immediate, reversible and due to an increase in the Vmax. without apparent effect on the affinity of the enzyme for ATP. The rat lung supernatant produced additive activation of the adenylate cyclase that was already maximally stimulated by calmodulin, indicating that either calmodulin and cytoplasmic factors act at different sites on adenylate cyclase or different adenylate cyclases may be involved. The data further support our previous conclusion that calmodulin is not involved in the activation of adenylate cyclase by cytoplasmic factors in rat lungs.     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Adenylate Cyclase of Torpedo Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Abstract: Cholinergic synaptosomes isolated from the electric organ of Torpedo contain membrane-bound adenylate cyclase activity (∼6 pmol/mg proteidmin), which is dependent on the presence of guanine nucleotides. The activity is strongly dependent on temperature and only slightly affected by NaCl. The Torpedo adenylate cyclase is completely inhibited by low levels of free Ca²⁺ (K₀∼ 0.5 μ M ). This effect is not altered by either trifluoperazine or addition of exogenous calmodulin. Ca³⁺ has no effect on the activation step of the adenylate cyclase by guanyl-5'-yl imidodiphosphate (GppNHp), and Mn²⁺ abolishes the Ca²⁺-dependent inhibition of cyclic AMP synthesis. These findings suggest that Ca²⁺ exerts its effect by direct interaction with a site located on the catalytic subunit. Torpedo synaptosomes contain presynaptic inhibitory muscarinic receptors. The binding of muscarinic agonists to the receptors is modulated (to lower affinity) by GTP. However, muscarinic ligands, examined under a variety of assay conditions, have no effect on adenylate cyclase activity. These results suggest that although both the muscarinic receptor and the adenylate cyclase are coupled to G proteins, they either interact with different G proteins or are situated in different regions of the presynaptic membrane.     

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) stimulates postjunctional D-1 and D-2 dopamine receptors

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) mimicks the ability of dopamine either to enhance adenylate cyclase activity in homogenates of goldfish retina or to inhibit adenylate cyclase activity in homogenates of the intermediate lobe (IL) of the rat pituitary gland previously treated with cholera toxin. Both the dopamine stimulated adenylate cyclase activity in the fish retina and the dopamine inhibited adenylate cyclase activity in the rat IL are associated with cells postjunctional to the dopaminergic neurons innervating these tissues. Therefore, the present data do not support the contention that TL-99 is a selective presynaptic dopamine receptor agonist.     

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4-dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photo-receptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark-adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light-adaptive pigment dispersion in isolated dark-adapted bullfrog RPE-eyecups incubated in constant darkness in normal Ringer's solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark-adapted bullfrog RPE-eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine-induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE-eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport.(ABSTRACT TRUNCATED AT 400 WORDS)     

Indoleamine-Sensitive Adenylate Cyclase in Rabbit Retina: Characterization and Distribution

Previous histological, electrophysiological, and biochemical reports have addressed the hypothesis that serotonin functions as a neurotransmitter in mammalian retinas. We have tested the effect on the levels of cyclic AMP of the application of exogenous serotonin, 5-methoxytryptamine, melatonin, and 5-methoxydimethyl-tryptamine to isolated, incubated rabbit retinas. All indoleamines tested significantly elevated intracellular levels of cyclic AMP in both light- and dark-adapted, incubated, intact retinas, provided a phosphodiesterase inhibitor was present. In homogenates of rabbit retina, all indoleamines tested also markedly increased adenylate cyclase activity over basal levels. Maximal activity was observed with 50 microM indoleamine; addition of GTP augmented this increase. The increase in enzyme activity persisted in the presence of known antagonists of dopamine and serotonin 5-HT2-receptors, but was blocked by the mixed 5-HT1, 5-HT2-antagonist lysergic acid diethylamide. The retinal locations of this response have also been identified using layer microdissection techniques on freeze-dried samples obtained from rabbit eyecups suprafused with indoleamine plus phosphodiesterase inhibitor. Cyclic AMP levels were measured in discrete retinal layers of both light- and dark-adapted suprafused eyecups, and increased levels were observed primarily in the inner and outer plexiform layers, which contain the synapses of the retinal neurons.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Octopamine- and dopamine-sensitive adenylate cyclase in the brain ofLocusta migratoria during its development

Octopamine- and dopamine-sensitive adenylate cyclases were studied in the brain of Locusta migratoria during its metamorphosis. In the adult brain the effects of octopamine and dopamine on adenylate cyclase were additive, suggesting the presence of separate populations of adenylate cyclase-linked receptors for octopamine and dopamine. There are no separate receptors for noradrenaline. Octopamine stimulates adenylate cyclase in both adult and larval brain; however, in adult brain octopamine is more potent than in larval brain. Dopamine stimulates adenylate cyclase activity only in adult brain. The sensitivity of adenylate cyclase to octopamine changes during the development of the animal. Phentolamine and cyproheptadine are potent antagonists of octopamine-stimulated adenylate cyclase, while propranolol has a weak effect. No cytosol factor which would modulate either basal or octopamine-stimulated adenylate cyclase was found. The effect of GTP and octopamine on adenylate cyclase was synergistic in adult brain but not in larval brain, while the effect of GppNHp and octopamine was synergistic in both adult and larval brains.     

Calmodulin stimulation of adenylate cyclase in rat brain membranes does not require GTP

Calcium stimulates adenylate cyclase activity in rat cerebral cortical membranes with either ATP or AppNHp as substrate. In contrast, isoproterenol stimulates the cerebral cortical enzyme with ATP as substrate but not with AppNHp as substrate unless exogenous GTP is added. In rat striatal membranes, calcium or dopamine stimulate adenylate cyclase activity with ATP as substrate, but not with AppNHp as substrate. GTP restores the dopamine but not the calcium response. The inhibitory guanine nucleotide GDP-βS antagonizes dopamine and GppNHp stimulation of the brain adenylate cyclases, but not stimulation by calcium of either rat cerebral cortical or striatal enzymes. Results indicate that GTP is not requisite to calcium-calmodulin activation of adenylate cyclases in brain membranes. In addition, calcium-calmodulin cannot activate striatal adenylate cyclases with a nonphosphorylating nucleotide, AppNHp, as substrate.     

Subcellular distributions of calcium/calmodulin-stimulated and guanine nucleotide-regulated adenylate cyclase activities in the cerebral cortex

The subcellular distribution of Ca²⁺/calmodulin-stimulated adenylate cyclase activity was studied in comparison with that of guanine nucleotide-stimulated cyclase activity. The distributions of these activities were similar among the crude fractions but differed among the purified subsynaptosomal fractions. The specific activity of Ca²⁺/calmodulin-stimulated cyclase was highest in a light synaptic membrane fraction, which has few, if any, postsynaptic densities, whereas that of guanine nucleotide-stimulated cyclase was highest in a heavier synaptic membrane fraction rich in postsynaptic densities. These results suggest that the Ca²⁺/calmodulin-stimulated cyclase has, at least in part, a different cellular or subcellular location than the guanine nucleotide-stimulated cyclase.Abbreviations used CaM calmodulin - GppNHp guanosine 5-(,-imino) triphosphate