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Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

ABSTRACT: Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.

SUBMITTER: Hausleithner V

PROVIDER: S-EPMC1152908 | biostudies-other | 1985 Dec

REPOSITORIES: biostudies-other

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Project description:1. The inhibition of hexokinase by glucose 6-phosphate has been investigated in crude homogenates of guinea-pig cerebral cortex by using a sensitive radio-chemical technique for the assay of hexokinase activity. 2. It was observed that 44% of cerebral-cortex hexokinase activity did not sediment with the microsomal or mitochondrial fractions (particulate fraction), and this is termed soluble hexokinase. The sensitivities of soluble and particulate hexokinase, and hexokinase in crude homogenates, to the inhibitory actions of glucose 6-phosphate were measured; 50% inhibition was produced by 0.023, 0.046 and 0.068mm-glucose 6-phosphate for soluble, particulate and crude homogenates respectively. 3. The optimum Mg(2+) concentration for the enzyme was about 10mm, and this appeared to be independent of the ATP concentration. In the presence of added glucose 6-phosphate, raising the Mg(2+) concentration to 5mm increased the activity of hexokinase, but above this concentration Mg(2+) potentiated the glucose 6-phosphate inhibition. When present at a concentration above 1mm, Ca(2+) ions inhibited the enzyme in the presence or absence of glucose 6-phosphate. 4. When the ATP/Mg(2+) ratio was 1.0 or below, variations in the ATP concentration had no effect on the glucose 6-phosphate inhibition; above this value ATP inhibited hexokinase in the presence of glucose 6-phosphate. ATP had an inhibitory effect on soluble hexokinase similar to that on a whole-homogenate hexokinase, so that the ATP inhibition could not be explained by a conversion of particulate into soluble hexokinase (which is more sensitive to inhibition by glucose 6-phosphate). It is concluded that ATP potentiates glucose 6-phosphate inhibition of cerebral-cortex hexokinase, whereas the ATP-Mg(2+) complex has no effect. Inorganic phosphate and l-alpha-glycerophosphate relieved glucose 6-phosphate inhibition of hexokinase; these effects could not be explained by changes in the concentration of glucose 6-phosphate during the assay. 5. The inhibition of hexokinase by ADP appeared to be independent of the glucose 6-phosphate effect and was not relieved by inorganic phosphate. 6. The physiological significance of the ATP, inorganic phosphate and alpha-glycerophosphate effects is discussed in relation to the control of glycolysis in cerebral-cortex tissue.

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Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

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