An aspartate conserved among G-protein receptors confers allosteric regulation of alpha 2-adrenergic receptors by sodium

The residue involved in sodium regulation of G-protein-coupled receptors has been identified by site-directed mutagenesis of the alpha 2-adrenergic receptor gene. Mutation of Asp-79 to Asn-79 entirely eliminates allosteric regulation of ligand binding by monovalent cations without perturbing the selectivity of adrenergic binding or allosteric modulation of that binding by amiloride analogs. The high degree of conservation of this aspartate residue in all G-protein-coupled receptors, without even a conservative change to glutamate, underscores the probable importance of this allosteric regulation.     

Na+/H+ exchanger activity in the pig kidney epithelial cell line, LLC-PK1: inhibition by amiloride and its derivatives

Rapidly growing pig-kidney-derived epithelial cells, LLC-PK1, lack detectable amiloride-sensitive Na+/H+ exchange activity when assayed directly. A large 22Na uptake is induced when the cells are acid-loaded prior to assay by incubation with buffer containing ammonium chloride or nigericin. The acid-stimulated sodium uptake is sensitive to amiloride, with half-maximal inhibition at 3.5-4.5 microM in buffer containing 15 mM sodium ion. There is simple competitive interaction between amiloride and sodium ion when the amiloride concentration is below 25 microM and the sodium ion concentration is above 20 mM. Derivatives of amiloride which carry substituents on the 5-amino group are 35- to 175-fold more inhibitory than amiloride itself.     

Species-specific Mn2+/Mg2+ antiport from Mg2(+)-loaded erythrocytes.

Mg2(+)-loaded rat erythrocytes performed Mn2+/Mg2+ antiport, which was nonspecifically stimulated by anions and cations. Mn2+/Mg2+ antiport was shown to operate via the Na+/Mg2+ antiporter because extracellular Na+ and Mn2+ inhibited the intracellular uptake of each other's ions competitively. Furthermore, Mn2+/Mg2+ antiport and Na+/Mg2+ antiport were identically inhibited by various amiloride derivatives. Na+/Mg2+ antiport of chicken and human erythrocytes cannot perform Mn2+/Mg2+ antiport although chicken erythrocytes took up more Mn2+ than rat erythrocytes.     

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Summary Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the unmodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. AtV _m =RP+20 mV andT=12°C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening atV _m =RP+20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.     

A derivative of amiloride blocks both the light-regulated and cyclic GMP-regulated conductances in rod photoreceptors

Vertebrate rod photoreceptors in the dark maintain an inward current across the outer segment membrane. The photoresponse results from a light-induced suppression of this dark current. The light-regulated current is not sensitive to either tetrodotoxin or amiloride, potent blockers of Na+ channels. Here, we report that a derivative of amiloride, 3',4'-dichlorobenzamil (DCPA), completely suppresses the dark current and light response recorded from rod photoreceptors. DCPA also blocks a cyclic GMP-activated current in excised patches of rod plasma membrane and a cGMP-induced Ca++ flux from rod disk membranes. These results are consistent with the notion that the Ca++ flux mechanism in the disk membrane and the light-regulated conductance in the plasma membrane are identical. DCPA also inhibits the Na/Ca exchange mechanism in intact rods, but at a 5-10-fold-higher concentration than is required to block the cGMP-activated flux and current. The blocking action of DCPA in 10 nM Ca++ is different from that in 1 mM Ca++, which suggests either that the conductance state of the light-regulated channel may be modified in high and low concentrations of Ca++, or that there may be two ionic channels in the rod outer segment membrane.     

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.     

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.