Human platelets as the object of investigation of the molecular mechanisms underlying nongenomic effects of glucocorticoid hormones

Abstract

The effect of hydrocortisone (100 nM-10 gM) on the major biochemical parameters of platelet activity (intracellular free calcium concentration, thromboxane B 2 content, and baseline and stimulated levels of cAMP and cGMP) is examined. The results obtained indicate that the inhibitory effect of glucocorticoids on platelet aggregation is mediated by activation of the adenylate cyclase system and suppression of the calcium response. Presumably, neither guanylate cyclase nor phospholipase A2-dependent systems are the targets of nongenomic actions of glucocorticoid hormones. Platelets can serve as a convenient tool for the investigation of nongenomic effects of glucocorticoid hormones. Key Words: hydrocortisone; calcium; cGMP; cAMP; thromboxane; inhibitors and activators of plateletsNongenomic effects contribute considerably to the physiological and pharmacological activity of steroid hormones. Molecular targets of steroid hormones are usually located in the cytoplasma membrane. The following trigger mechanisms of nongenomic effect of steroid hormones have been demonstrated: 1) stimulation of Na+/K+-ATPase in the kidney tubular epithelium [12,13]; 2) activation of rapid calcium entry by progesterone, testosterone, and lc~,25-dihydroxyvitamin D 3 [3-5,9,14]; 3) modification of the metabolism of second messengers in normal and transformed cells [6,7,11]; 4) modulation of the activity of neuronal Cl-channels coupled to GABA Areceptors [8]. The nongenomic effects of steroid hormones are characterized by latency, duration of their action (within 30 min), and tolerance to the blockers of RNA and protein syntheses. Steroids induce a membranotropic effect without penetrating the plasma Department of Pharmacology and Radiobiology, Russian State Medical University, Moscow membrane, which has been used to distinguish between genomic (mediated by intracellular receptors) and nongenomic effects. For example, the membrane stage in the action of steroid hormones can be identified with the use of these hormones immobilized on a high-molecular-weight carrier [1]. Anuclear ceils (erythrocytes and platelets) provide another approach to the investigation of the mechanisms underlying the membrane-mediated effects of steroids.The aim of the present study was to develop a theoretical and experimental basis for the evaluation of the nongenomic effect of the glucocorticoid hydrocortisone (cortisol) on platelet activity. MATERIALS AND METHODSThe procedures of platelet isolation, loading with the fluorescent probe Fura 2-AM, and calculation of the intracellular free calcium content were described elsewhere [2]. The content of cAMP, cGMP, and thromboxane B 2 was measured by the radioligand

Early stages in the mechanism of action of glucocorticoids on human platelets. Effect of hydrocortisone on intracellular cAMP and Ca2+ content

“…The rise of [Ca2 § results in activation of Ca2+-dependent membrane-bound enzymes, phospholipases Ph and C, stimulation of cell contractile apparatus followed by secretion release from the secretory granules, and expression of adhesive protein receptors on the platelet surface [11]. The platelet Ca-response involves the calcium-phosphoinositide system and three types of Ca channels in the plasma membrane: Ca 2 § channel of P2x-purine receptor (activated by ADP and ATP), Ca 2 § channels regulated by second messenger (calcium ions, inositol-l,4,5-tris-phosphate); and potential-independent G-protein-operated Ca channels (activated by (z-subunit of G-protein) [ by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and membrane-permeable dibutyryl derivative of cAMP (dbcAMP) [6].Hydrocortisone (HC) inhibits platelet activity [2]. In concentrations of 1-25 gM it inhibits collageninduced platelet aggregation and potentiates adenosineinduced platelets disaggregation.…”