Renal sodium/calcium exchange; a vasodilator that is defective in salt-sensitive hypertension.

The Na+ : Ca2+ exchanger is an important plasma membrane ion transport pathway that plays a major role in controlling [Ca2+]i. In smooth muscle cells, it may function as a Ca2+ extrusion pathway and may help lower [Ca2+]i in response to vasoconstrictor-induced increases in [Ca2+]i. It may also extrude [Ca2+]i and lead to vasodilation in response to vasodilators. Our recent studies have been performed to determine the existence and regulation of the Na+ : Ca2+ exchanger in renal contractile cells which include afferent and efferent arterioles and mesangial cells. Exchanger activity is present in all three of these contractile elements but is higher in afferent arterioles vs. efferent arterioles. We have also examined the role of altered regulation of the exchanger in the SHR and in salt-sensitive hypertension. With the establishment of high blood pressure, Na+ : Ca2+ exchanger activity is reduced in afferent but not in efferent arterioles in both models of hypertension. Other works in cultured mesangial cells and freshly dissected afferent arterioles, have shown that protein kinase C (PKC) up-regulates the Na+ : Ca2+ exchanger from Dahl/Rapp salt-resistant rats while it fails to do so in arterioles and mesangial cells from salt-sensitive rats. This defect in PKC regulation of Na+ : Ca2+ exchange is the result of a loss of PKC-mediated translocation of the exchanger to the plasma membrane in S mesangial cells. Thus, a defect in the PKC-Na+ : Ca2+ exchanger-translocation pathway may cause dysregulation of [Ca2+]i and help explain the dramatic decrease in GFR that occurs in this model of hypertension.

Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats.

The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+ exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs were cultured, grown on coverslips, and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope-based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 h. Baseline [Ca2+]i values measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]i values were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM, which resulted in movement of Na+ out of and Ca2+ into these cells (reverse-mode Na+/Ca2+ exchange). PKC was activated in these cells with 15-min exposure to 100 nM phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i (Delta[Ca2+]i) with reduction in Nae was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+]i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Delta[Ca2+]i in R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction of R but not S MCs, an increase which was abrogated by prior 24-h PMA pretreatment and corresponded to reduction in the 70-kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+/Ca2+ exchange activity in MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+ exchange regulation pathway in MCs of S rats.

Altered sodium-calcium exchange in afferent arterioles of the spontaneously hypertensive rat.

Studies were performed to determine if there is a derangement in Na-Ca exchange activity in afferent (AA) and efferent (EA) arterioles from 3- and 9-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Cytosolic calcium concentration ([Ca2+]i) was assessed using microscope-based photometry in fura-2 loaded arterioles bathed in a Ringer's solution. Baseline [Ca2+]i was similar in the AA of 3- and 9-week-old WKY and SHR. In AA from 3-week-old rats, [Ca2+]i increased by 89 +/- 15 nM in WKY and by 73 +/- 13 nM in SHR during decreases in bath sodium concentration ([Na+]e) from 150 to 2 mM (Na+ replaced with n-methyl-D-glucamine). In 9-week-old hypertensive SHR (SBP = 150 mm Hg), increases in [Ca2+]i were attenuated (24 +/- 3 nM) relative to 3-week-old WKY and SHR, and 9-week-old WKY (90 +/- 9 nM; P < 0.05). Likewise, the rate of removal of Ca2+ in the continued presence of 2 mM Nae (Ca2+ sequestration and/or extrusion) was markedly reduced in AA of 9-week-old SHR (-0.15 +/- 0.03 nM/second) versus 3-week-old SHR (-0.72 +/- 0.12 nM/second) and 3- and 9-week-old WKY (-0.49 +/- 0.10 and -0.67 +/- 0.14 nM/second). In other experiments, AAs were preincubated in 1 mM ouabain to increase intracellular [Na+]. This maneuver augmented the increase in [Ca2+]i obtained with removal of Na+e; however, the responses obtained in 9-week-old SHR arterioles were still attenuated compared to those obtained in arterioles for 3- and 9-week-old WKY and 3-week-old SHR. These results suggest that exchanger number and/or sensitivity to the transmembrane Na gradient was reduced in the SHR AA. In EA, baseline [Ca2+]i was similar in 3- and 9-week-old WKY and SHR. In contrast to AA, the magnitude of Na-dependent and Na-independent changes in [Ca2+]i was not different in the EA of 3- and 9-week-old WKY and SHR. These results indicate that regulation of Na-Ca exchange activity may differ between AA and EA segments. Furthermore, diminished Na-Ca exchange and Na-independent Ca2+ sequestering/extrusion mechanisms could contribute to altered AA [Ca2+]i in the SHR.

A non-radioactive fluorescent method for measuring protein kinase C activity.

The purpose of this study is to report the development of a non-radioactive fluorescent peptide assay for measuring protein kinase C activity (PKC). The assay is based on a glycogen synthase derived fluorescent peptide that is phosphorylated by PKC. Phosphorylation causes the peptide to migrate toward the anode while the non-phosphorylated peptide migrates toward the cathode during agarose gel electrophoresis. Quantitation of PKC activity can be accomplished by excision of the appropriate bands and measuring their relative fluorescence. Using this assay, PKC activity was measured in whole cell homogenates from cultured renal mesangial cells. The enzyme(s)-substrate system followed Michaelis-Menten kinetics under limited conditions and, therefore, Lineweaver-Burk plots were used to obtain Michaelis constant and maximum velocity values. An apparent KM value of 40 microM was obtained for the fluorescent peptide substrate with a control Vmax value of 300 pmol/min. Addition of phorbol 12-myristate 13-acetate increased Vmax to 380 pmol/min.

Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex.