Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data.

Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 2532] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC.

Analysis of effects of adrenaline and collagen on the activity of prostaglandin E1-stimulated human platelet adenylyl cyclase.

A mathematical model relating the activity of adenylyl cyclase (AC) with concentrations of inhibitors, equilibrium dissociation constants, specific activity and efficacies of AC depending on the states of binding sites of the receptors was used for analysis of the data on inhibition of PGE1-stimulated AC of human platelet membranes (Farndale et al. Biochem J 1992; 282; 25-32). The equilibrium dissociation constant, x2 characterizing the affinity of adrenaline for its receptor, was estimated to be 0.14 microM; this constant is a better characteristic of adrenaline's potency than IC50, the concentration corresponding to half-asymptotic inhibition of AC. Collagen does not affect the affinity of adrenaline for its receptor. G protein involved in inhibition of AC activity by collagen is different from Gi2 mediating inhibition by alpha 2-adrenergic agonists. The model applied for the analysis may be thought to be the best means presently to relate dose-response dependencies to what is known or can be hypothesized about the mechanisms underlying inhibition of AC activity.

Analysis of trichloroacetic acid in the urine of workers occupationally exposed to trichloroethylene by capillary gas chromatography.

A gas chromatographic procedure is described for the determination of trichloroacetic acid in urine, the major metabolite of trichloroethylene exposure. Trichloroacetic acid was derivatised to its methyl ester with BF3/methanol reagent and then extracted into toluene and analysed by capillary gas chromatography using electron-capture detection. The response was linear in the range 0.4-100 mg/l of trichloroacetic acid in urine and showed a relative recovery of 99.6%. The procedure is suitable for monitoring occupational exposure to trichloroethylene.

Analysis of effects of corticotropin, forskolin and fluoride on activity of adenylate cyclase of bovine adrenal cortex.

A mathematical model relating the activity of adenylate cyclase (AC) with concentrations of stimulators, equilibrium dissociation constants, specific activity and efficacies of AC depending on the states of its binding sites has been developed and used for analysis of the data on activation of AC of bovine adrenal cortex plasma membranes presented in (De Foresta et al. (1987) FEBS Lett. 216, 107-112). Equilibrium dissociation constants. chi h and chi l, corresponding to high- and low-affinity forskolin-binding sites were estimated to be 0.37 and 17 microM: these constants characterize forskolin's potency more adequately than does ED50, the concentration eliciting half-asymptotic activity of AC. Corticotropin does not affect the affinity of AC for forskolin whereas fluoride increases this affinity, thus augmenting forskolin's potency. Hormone receptor of adenylate cyclase of bovine adrenal cortex has been suggested to have two or more binding sites for corticotropin. Some unidentified factor(s) may be responsible for the differences found in adenylate cyclase activity in different experiments carried out under similar conditions. The model applied for the analysis may be thought to be the best means for the moment to relate dose-response dependencies with what is known or can be hypothesized about the mechanisms underlying activation of adenylate cyclase.