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

The interaction between the Ca 2+ -binding protein, calmodulin, and guanyl nucleotides was investigated in a rat striatal paniculate 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′-(3,7-imido)triphosphate (GppNHp), calmodulin could further activate adenylate cyclase only at concentrations less than 0.2 p.M 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 cal-modulin-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. The finding that GTP was unable to activate the calmodulin-sensitive component suggests that this component has either a different mode of binding to a GTP-binding protein or inherently higher GTPase activity than has the calmodulin-insensitive component. The results suggest there are two components of adenylate cyclase activity that can be differentiated by their sensitivities to calmodulin and guanyl nucleotides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65160/1/j.1471-4159.1983.tb00838.x.pd

ROLE OF CALMODULIN IN STATES OF ALTERED CATECHOLAMINE SENSITIVITY

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72490/1/j.1749-6632.1980.tb29620.x.pd

Inhibition of a Low K m GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca 2+ -binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5′-(Β, Γ-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 Ca 2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca 2+ 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 K m GTPase activity. The inhibition of GTPase activity by calmodulin was Ca 2+ -dependent and was maximal at 0.12 Μ M free Ca 2+ . Maximal inhibition by calmodulin was 40% in the presence of 10 −7 M GTP. The IC 50 for calmodulin was 100 n M. 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 Ca 2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related. Inhibition of a low K m GTPase activity by calmodulin could represent an action of calmodulin in increasing the association of a GTP-binding protein with the catalytic subunit activity resulting in a reduction of the “turn-off” GTPase activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66214/1/j.1471-4159.1985.tb05444.x.pd

Effects of Calmodulin on Rat Striatal Adenylate Cyclase Activity (Dopamine, Guanyl Nucleotide, Parkinson's Disease).

This thesis describes our investigation of the effects of calmodulin, a 16,800 dalton calcium-binding protein, on adenylate cyclase activity in rat striatal membranes. Rat striatal adenylate cyclase activity is stimulated by the action of dopamine on specific D-1 receptors. Receptor activation is mediated through a GTP-transducing mechanism to increase the activity of the catalytic moiety of adenylate cyclase. We have studied the interaction between calmodulin and dopamine in the activation of adenylate cyclase. In rat striatal membranes depleted of endogenous calmodulin, adenylate cyclase activity was stimulated by calmodulin in a calcium and dose dependent manner. The apparent K(,a) for calmodulin in the activation of adenylate cyclase was 70 nM, and stimulation was maximal at a free calcium concentration of 10('-7) M. Dopamine-stimulated adenylate cyclase activity was enhanced by the addition of calcium and calmodulin; the apparent K(,a) for dopamine decreased 3-fold from 1.1 (mu)M to 0.35 (mu)M. We have distinguished between calmodulin-stimulated and calmodulin-insensitive components of adenylate cyclase by their differential response to the guanyl nucleotide triphosphates, guanosine triphosphate (GTP) and a non-hydrolyzable analog, guanosine-5'-((beta),(gamma)-imido) triphosphate (GppNHp). Adenylate cyclase activity is regulated in part by the action of a high affinity GTPase activity which inactivates the GTP-stimulated enzyme. We found that calmodulin inhibited a high affinity GTPase activity at concentrations of calcium and calmodulin identical to those that stimulated adenylate cyclase activity. Adenylate cyclase activity in the striatum from rats chronically treated with dopamine receptor antagonists (antipsychotic drugs) exhibited an increased sensitivity to dopamine, calmodulin, and GppNHp as compared to saline treated controls. We conclude that calmodulin and dopamine share a common locus of action, and suggest that calmodulin plays a regulatory role in the dopamine-sensitive adenylate cyclase system in rat striatum.Ph.D.PharmacologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/161041/1/8612640.pd