Search

Cyclooxygenase inhibitors, such as indomethacin and diclofenac, have well-described effects to enhance renal water reabsorption and urinary concentrating ability. Concentrating ability is regulated in part at the level of the thick ascending limb of Henle's loop, where active NaCl absorption drives the countercurrent multiplication mechanism. We used semiquantitative immunoblotting to test the effects of indomethacin and diclofenac, given over a 48-h period, on the expression levels of the ion transporters responsible for active NaCl transport in the thick ascending limb. Both agents strongly increased the expression level of the apical Na-K-2Cl cotransporter in both outer medulla and cortex. Neither agent significantly altered outer medullary expression levels of other thick ascending limb proteins, namely, the type 3 Na/H exchanger (NHE-3), Tamm-Horsfall protein, or alpha1- or beta1-subunits of the Na-K-ATPase. Administration of the EP3-selective PGE(2) analog, misoprostol, to indomethacin-treated rats reversed the stimulatory effect of indomethacin on Na-K-2Cl cotransporter expression. We conclude that cyclooxygenase inhibitors enhance urinary concentrating ability in part through effects to increase Na-K-2Cl cotransporter expression in the thick ascending limb of Henle's loop. This action is most likely due to elimination of an EP3-receptor-mediated tonic inhibitory effect of PGE(2) on cAMP production.

The intracellular messenger mitogen-activated protein kinase (MAPK) is activated in vascular smooth muscle cells (SMC) by various growth factors as well as by agonists that have no proliferative effect. We explored the hypotheses that SMC proliferation is associated with a specific pattern of MAPK activation by evaluating the kinetics of MAPK activation and tyrosine phosphorylation and the intracellular location of MAPK in SMC following addition of agonists of varying mitogenic potential. A peak in MAPK activation and tyrosine phosphorylation occurred 3-10 min after the addition of agonists to SMC derived from human saphenous vein (early phase), followed by a plateau of activity, which was variable in duration (late phase). A correlation was not found between mitogenicity and the degree to which MAPK became activated or tyrosine phosphorylated in the early phase. However, the duration of MAPK activation and tyrosine phosphorylation correlated strongly with the ability of agonists to stimulate SMC proliferation. Nuclear translocation of MAPK was associated with SMC proliferation, although the degree to which each agonist induced nuclear translocation did not parallel its mitogenic potential. The relative dependency of all three events on protein kinase C differed for each agonist and was greater in the late versus the early phase. Thus, in human SMC, nuclear translocation of MAPK and prolonged activation and tyrosine phosphorylation of MAPK are associated with growth factor-induced mitogenesis.

Although nitric oxide (NO), one of the endothelium-derived relaxing factors, prevents formation of platelet aggregates, the mechanism by which this occurs is not fully understood. Accordingly, the effect of NO on signal transduction of gel-filtered human platelets was measured and compared with that of a cell-permeant guanosine 3',5'-cyclic monophosphate (cGMP) analogue, 8-bromo-cGMP (8-BrcGMP). NO inhibited the rise in intracellular Ca2+ concentration ([Ca2+]i), phosphorylation of the 47-kDa substrate (p47) of protein kinase C (PKC), serotonin secretion, and phosphatidic acid production induced by thrombin or the endoperoxide analogue U-46619. Similar effects were seen with 8-BrcGMP, and NO induced a concentration-related rise in cGMP. Neither NO nor 8-BrcGMP inhibited platelet aggregation, [Ca2+]i mobilization, or serotonin secretion induced by the Ca2+ ionophores A23187 or ionomycin or directly activated PKC purified from platelets. However, both NO and 8-BrcGMP enhanced p47 phosphorylation induced by the Ca2+ ionophores without augmenting phosphatidic acid production. Thus, if [Ca2+]i is elevated, a rise in cGMP enhances PKC activation. Both NO and 8-BrcGMP, however, prevent Ca2+ mobilization and platelet aggregation induced by receptor-mediated agonists by interfering with signal transduction at a point proximal to phospholipase C activation.

In the presence of extracellular Ca2+, epinephrine induces a rise in cytoplasmic Ca2+ ([Ca2+]i) that is associated with fibrinogen binding to the platelet surface, platelet aggregation, and enhancement of the thrombin-stimulated [Ca2+]i rise and protein phosphorylation. Whether the [Ca2+]i rise induced by epinephrine results from Ca2+ entry associated with fibrinogen binding to its receptor on the platelet surface, the glycoprotein (gp) IIb-IIIa complex, is unknown. To determine the importance of the occupancy of the gp IIb-IIIa receptor on platelet function after epinephrine administration, we studied the effects of two monoclonal antibodies (M-148 and 7E3) and two synthetic peptide analogues to fibrinogen (synthetic tetrapeptides Arg-Gly-Asp-Ser (RGDS) and dodecapeptide His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val [gamma-(400-411)]), all of which bind to gp IIb-IIIa and inhibit fibrinogen binding and platelet aggregation on the epinephrine-induced rise in [Ca2+]i and enhancement of thrombin's phosphorylation of the 47-kDa substrate of protein kinase C (p47). None of the gp IIb-IIIa ligands significantly enhanced or inhibited the epinephrine-induced [Ca2+]i rise or its augmentation of p47 phosphorylation after thrombin administration; however, the synergistic [Ca2+]i rise that follows addition of both epinephrine and thrombin was reduced by both antibodies and both peptides. Thus ligand binding of gp IIb-IIIa does not influence the epinephrine-induced [Ca2+]i rise or its promotion of protein kinase C activation by thrombin; these events can be dissociated from the synergistic [Ca2+]i rise.

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

The concentration of cytoplasmic ionized Mg2+ ([Mg2+]i) varies considerably among different cell types. It has not been measured in platelets. Incorrect estimates of this value could markedly affect many intracellular investigations, including calibration of measurements of platelet cytoplasmic ionized Ca2+ concentration ([Ca2+]i) with the photoprotein aequorin and other Ca2+-sensitive probes. [Mg2+]i was measured in washed, gel-filtered human platelets suspended in modified Tyrode buffer by two methods: 31P-nuclear magnetic resonance (NMR) spectroscopy of intact platelets and null-point titration in platelets selectively permeabilized with digitonin. The 31P-NMR spectra demonstrated that the [Mg2+]i, as calculated from the chemical shift values of ATP resonances, was 0.23 +/- 0.02 (SD) mM in unstimulated platelets. The mean [Mg2+]i as determined by null-point titration was 0.3 +/- 0.1 mM (range: 0.1-0.6 mM). When this [Mg2+]i value was used to construct a Ca2+-calibration curve for aequorin, the indicated [Ca2+]i values in resting and stimulated platelets were lower than those obtained from curves based on previously assumed values for [Mg2+]i (1.0-1.25 mM). This finding largely resolves the discrepancy between resting [Ca2+]i as determined by aequorin or by quin2, fura-2, and indo-1.

The protein kinase C activators phorbol ester 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (DAG) cause platelet aggregation, secretion, and a rise in aequorin-indicated cytoplasmic Ca2+ ([Ca2+]i), but the importance of this action to platelet activation by these agonists has not been established. We found that the previous addition of PMA or DAG either enhanced or inhibited the platelet response if thrombin was subsequently added, depending on the latter's concentration. The effects of PMA or DAG on the response to thrombin were obtained only if the agonists were added in concentrations sufficient to elevate [Ca2+]i themselves. A [Ca2+]i rise also occurred after the second agonist (thrombin), but its magnitude did not necessarily correlate with subsequent aggregation, secretion, or the activation of protein kinase C as reported by the phosphorylation of a 47-kDa protein (p47). The protein kinase C inhibitor sphingosine inhibited aggregation and p47 phosphorylation caused by PMA or DAG alone or with thrombin, but the [Ca2+]i rise in response to the first agonist was not affected. PMA-induced aggregation and p47 phosphorylation were inhibited by quin2, which also inhibited protein kinase C activity in a cell-free system. We conclude that a rise in aequorin-indicated [Ca2+]i is necessary for PMA or DAG to activate platelets or to alter the subsequent platelet response to thrombin; this [Ca2+]i rise may be a prerequisite for activation of protein kinase C.