The Inositol- 1,4,5=Trisphosphate System Is Involved in Rapid Effects of Aldosterone in Human Mononuclear Leukocytes

There is increasing evidence for rapid steroid action on electrolyte transport in human mononuclear leukocytes (HML). In HML, aldosterone stimulates the Na+/H+ antiporter within a few minutes. Because a variety of hormones and growth factors activate the Na+/H+ antiporter via protein kinase C and inositol phospholipids, a possible involvement of inositol-1,4,5-trisphosphate (IP3) in the rapid effects of aldosterone in HML was investigated. The stimulation of IP3 generation was started by the addition of aldosterone, concanavalin A, or other steroids. A significant increase in IP3 levels by aldosterone (1 nmol/L, P < 0.05) was found after 1 min, similar to that found after concanavalin A (25 micrograms/mL). Aldosterone caused a concentration-dependent elevation of IP3 levels, with an apparent EC50 of approximately 0.1 nmol/L. Fludrocortisone stimulated IP3 generation at similar concentrations, whereas a weaker IP3 stimulation by glucocorticoids (hydrocortisone, dexamethasone) occurred at micromolar concentrations only. Canrenone, a potent inhibitor of classical aldosterone action, was not effective up to a concentration of 100 nmol/L. These findings show kinetic and pharmacological similarities with both the functional data on Na+/H+ antiport stimulation by aldosterone and the studies of 125I-aldosterone binding to plasma membranes of HML. Thus, these data are the first to indicate an involvement of the phosphoinositide pathway in the rapid membrane effects of aldosterone

GTP and Ca2+ Modulate the Inositol 1,4,5-Trisphosphate-Dependent Ca2+ Release in Streptolysin O-Permeabilized Bovine Adrenal Chromaffin Cells

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half-maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+

Decavanadate displaces inositol 1,4,5-trisphosphate (IP3) from its receptor and inhibits IP3 induced Ca2+ release in permeabilized pancreatic acinar cells

Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 μM IP3 is half maximally inhibited with approximately 5 μM decavanadate. Complete inhibition is achieved with around 20 μM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 μM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor. EGTA, ethylene-glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol; IP3, inositol 1,4,5-trisphosphate; MOPS, morpholinopropane sulfonic acid; HEPES, N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethan

Agonist/Inosital Trisphosphate-Induced Release of Calcium from Murine Keratinocytes: A Possible Link with Keratinocyte Differentiation

Extracellular calcium concentrations markedly affect the pattern of proliferation and differentiation in cultured keratinocytes. When medium contains 0.1 mM calcium or above, the cells lose their proliferative ability, rapidly stratify, and terminally differentiate. Because 1,25(OH)2D3 (a modulator of Ca++ homeostasis) enhances the differentiation of keratinocytes, we investigated whether a link exists between 1 ,25(OH)2D3-induced release of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) from Ptdlns 4,5-P2 and intracellular calcium [Ca++]i release from keratinocytes. Specifically, primary culture of keratinocytes were loaded with fluorescence dye Fura-2AM (10 μM) and changes in fluorescence intensity were monitored at the excitation wavelengths of 340 and 380 nm and emission wavelength of 505 nm. Additions of two agonists, 1,25(OH)2D3 (1.2 × 10-9 M) and 13-Cis retinoic acid (0.2 × 10-9 M), to dye-loaded keratinocytes induced rapid release of [Ca++]i, respectively, followed by gradual return to the prestimulated state. Addition of Ins(1,4,5)P3 (10) to saponin-treated (leaky) keratinocytes also resulted in a rapid release of [Ca]. In contrast, the addition of inositrol-1,3,4,5-tetrakisphosphate. Ins(1,3,4,5)P at similar concentration exerted negligible effect. Taken together, these results support the view that 1,25(OH)2D3-induced [Ca++]i release in keratinocytes may be via the Ins(1,4,5)P3 induced early release of intracellular [Ca++]. This may explain, at least in part, 1,25(Oh)2D3-enhanced keratinocyte differentiatin

Mechanisms of agonist synergism in human isolated platelets

The synergism between various platelet agonists which results in an aggregation response has been described frequently in vitro, however there are no satisfactory explanations for the mechanisms controlling this response. In vivo, platelets are exposed to many aggregating stimuli at low concentrations at an injury site and synergism between such stimuli would enable an intensive aggregation and release response to occur. This study has investigated the intracellular mechanisms associated with agonist synergism in human isolated platelets using the following agonist combinations; adrenaline and thrombin, adrenaline and LDL. The action of LDL alone in activating platelets was also studied. Subthreshold concentrations of adrenaline (5-20 μM), thrombin (0.006-0.02 U/ml) and LDL (0.1-0.2 mg protein/ml), which individually did not cause an aggregation response were combined in pairs to produce a synergistic aggregation response, which was not inhibited by the presence of aspirin. There was no hydrolysis of phosphatidylinositol 4,5, bisphosphate or production of inositol trisphosphate associated with the synergism. Using quin 2, no increases in intracellular calcium concentration ([Ca2+]i ) were detected during the synergistic aggregation responses, however small [([Ca2+]i increases were detected using aequorin. No release or metabolism of arachidonic acid from membrane phospholipids was observed. In agreement with the calcium results, there was no phosphorylation of the 20 kDa protein. However, despite no significant diacylglycerol production, the 47 kDa protein was clearly phosphorylated following synergism between both the agonist combinations. Whether the phosphorylation of the 47 kDa protein is important in the initiation of a synergistic response remains to be determined and the presence of other pathways must be considered. Activation of platelets with 2.75 mg protein/ml LDL was accompanied by a slow increase in inositol trisphosphate production and intracellular calcium levels. Low LDL concentrations (<0.25 mg protein/ml) did not stimulate either aggregation or any intracellular mechanism