Na-H Exchange Is a Major Pathway for Na Influx in Rat Vascular Smooth-Muscle

Rat aortic segments and aortic smooth muscle cells in primary culture were used to examine the importance of the Na-H exchange system in transporting Na into smooth muscle. Ethylisopropylamiloride was ~100 times more potent than amiloride at inhibiting Na influx into smooth muscle. In a 135 mM Na-containing medium ~80% of the Na influx rate could be inhibited by 100 μM ethylisopropylamiloride. The rate of Na entry into cells was markedly influenced by extracellular and intracellular pH. Elevating extracellular pH from 6.0 to 8.0 increased the Na influx rate. The dependence of the Na influx rate on intracellular pH was demonstrated by acidification of cells with nigericin or preincubation with ammonium chloride. These two procedures increased Na influx rate by about 3.5-fold. In both instances the increases in Na influx rate could be completely attenuated by ethylisopropylamiloride. Increases in Na influx rate via the Na-H exchange also increased the activity of the Na-K pump, thereby maintaining intracellular Na content approximately constant. These results indicate that Na-H exchange is a major influx pathway for Na in rat vascular smooth muscle. Activation of this system activates the Na-K pump, which maintains intracellular Na constant

Amiloride Analogs Cause Endothelium-Dependent Relaxation in the Canine Coronary-Artery Invitro - Possible Role of Na+/ca2+ Exchange

A number of amiloride analogues were used to test the proposal that Na/Ca exchange may play a role in the secretion of endothelium‐derived relaxing factor (EDRF). The analogues used were those substituted on either the 5‐amino group or the terminal guanidino nitrogen atom. The former block both Na /Ca and Na /H exchange whilst the latter block the Na channel and the Na /Ca exchange. Both series of compounds caused relaxation in isolated rings of dog coronary artery (EC values, 1–10 μM) presumably due to release of EDRF since removal of endothelium greatly attenuated the response. Amiloride (1–100 μM) had little effect on either endothelium‐intact or denuded arteries. The guanidino substituted analogues also appeared to block selectively the relaxation response to acetylcholine in the coronary artery, independently of their EDRF‐releasing activity. It is proposed that endothelial cells have an active Na /Ca exchange operating in the forward mode to extrude Ca. This mechanism may be important in the control of EDRF release. 1988 British Pharmacological Societ

Na-H Antiport in Cultured Rat Aortic Smooth-Muscle - its Role in Cytoplasmic Ph Regulation

We have investigated the role of the Na-H antiport in the regulation of intracellular pH (pH(i)) in vascular smooth muscle. Experiments were conducted on contractile-state rat aortic smooth muscle cells grown in primary culture and loaded with the pH-sensitive, fluorescent indicator 2',7',-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Cells equilibrated in a normal physiological salt solution (PSS) containing 135 mM Na, pH 7.4 at 37°C, had a pH(i) of 7.16 ± 0.04 (means ± SE; n=8). 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a concentration-dependent fall in pH(i). Removal of extracellular Na caused an intracellular acidification that was rapidly reversed on replacement of Na. The rate of recovery from NHCl-induced intracellular acidosis was dependent on extracellular Na concentration (K(m) 14.6 ± 2.8 mM) and was accelerated by increasing the transmembrane Na gradient and slowed by decreasing it. Recovery from acidosis was completely abolished by either EIPA or the absence of extracellular Na. These results demonstrate that the Na-H antiport is an important mechanism for the maintenance and regulation of pH(i) in vascular smooth muscle cells. The BCECF fluorescence technique provides an ideal method for further studies on the mechanisms for pH(i) regulation in these cells

Intracellular Ph in Human Arterial Smooth-Muscle - Regulation by Na+/h+ Exchange and a Novel 5-(N-Ethyl-N-Isopropyl)Amiloride-Sensitive Na+-Dependent and Hco3--Dependent Mechanism

We investigated in a physiological salt solution (PSS) containing HCO the intracellular pH (pH(i)) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Na influx rates. The recovery of pH(i) from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO/HCO-buffered PSS than in HEPES-buffered PSS. Recovery of pH(i) was dependent on extracellular Na (K(m), 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO-dependent mechanisms. Recovery instead apparent mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na/H exchange and a previously undescribed EIPA-sensitive Na- and HCO-dependent mechanism. Differentiation between this HCO-dependent mechanism and Na/H exchange was achieved after depletion of cellular ATP. Under these conditions, the NHCl-induced Na influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO/HCO-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na, the EIPA inhibition curve for the intracellular acidosis-induced Na influx was monophasic (IC, 39 nM), whereas in an identical CO/HCO-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC, 37.3 nM and 312 μM). Taken together, the results indicate that Na/H exchange and a previously undescribed EIPA-sensitive Na- and HCO-dependent mechanism play an important role in regulating the pH(i) of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50% at lesser, more physiological, levels of induced acidosis