Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels.

Abstract

SUMMARY1. The pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in segments of rat resistance vessels (internal diameter about 200 ,tm) with the vessels mounted in a myograph for simultaneous measurements of isometric contraction.2. BCECF loaded slowly into the vessels over 1 h and did not affect the maximal contractility of the vessels. There was a loss of dye with time which, however, was very slow when the segments were only excited for 2 s/min, suggesting that the loss was mainly due to dye bleaching with only a very slow leak.3. The ratio of the emissions (at 540 nm) with excitation at 495 and 450 nm was calibrated in terms of pH using the K+-H+ ionophore nigericin. This calibration gave a pHi value of 7-15 +0'02 (n = 20), suggesting that hydrogen ions are not in electrochemical equilibrium in these vascular smooth muscles which have a membrane potential of about -60 mV.4. Addition of 10 mM-NH4Cl caused a transient alkalinization and wash-out of 10 mM-NH4Cl a transient acidification. Increasing CO2 with maintained bicarbonate caused a rapid acidification followed by an incomplete recovery. Removal of CO2 and bicarbonate (HEPES-buffered solution) with constant extracellular pH caused a transient alkalinization but steady-state pHi was not significantly altered.5. In bicarbonate-free buffer the Na+-H+ exchange blocker 5-(N-ethyl-Nisopropyl) amiloride (EIPA) and sodium-free conditions caused a slow acidification. In bicarbonate buffer (PSS) EIPA had no detectable effect after 10 min but the anion exchange blocker diisothio-cyanatostilbenedisulphonic acid (DIDS) caused a small acidification over that time course.6. The rate of recovery after an acid load was about 50 % lower in HEPES buffer compared to PSS and it was inhibited by EIPA. In PSS amiloride and EIPA each had a small inhibitory effect on the pH recovery after an acid load. DIDS also inhibited the recovery from an acid load in PSS and this effect was additive to that of EIPA. DIDS and EIPA also had additive inhibitory effects on the 22Na+ influx stimulated by the acid loading, while in HEPES buffer DIDS had no effect on either pH recovery or 22Na+ influx. These results suggest that a Na+-H+ exchange and an C. AALKJ?ER AND E. J. CRAGOE influx of bicarbonate coupled to sodium influx are of importance for pHi control in these vessels.7. Noradrenaline-induced contractions in HEPES buffer were associated with a fall in pHi while in PSS pHi was not significantly altered. Contraction induced by high-K+ solutions was associated with an acidification which was larger in HEPESbuffered solution than in PSS. The acidification in high-K+ solutions was only partly explained by the reduction of Na+. 8. These results suggest that the bicarbonate influx is of importance for maintaining pHi during a contraction and since pHi falls during a noradrenaline contraction in HEPES buffer they also suggest that a possible noradrenaline-induced resetting of the Na+-H+ exchange cannot prevent a fall in pH...

Proceedings of the Physiological Society, 15‐16 December 1989, King's College London Meeting: Communications

“…This therefore suggests that Na-H exchange is largely responsible for the extrusion of acid equivalents. Similar findings have been reported in cultured aortic cells (Korbmacher et al 1988) but in resistance vessels (Aalkjaer & Cragoe, 1988) and in ureter (Aickin, 1988) recovery is accelerated by the presence of CO2, and amiloride is only strongly inhibitory in the nominal absence of CO2.…”

“…Em depolarized by about 2 mV. Similar behaviour has been observed in resistance vessels (Aalkjaer & Cragoe, 1988) while a steady-state acidosis of about 0-2 units has been found in cultured aortic cells (Korbmacher et al 1988) and in vas deferens and ureter (Aickin, 1984(Aickin, , 1988.…”

“…Peculiarly, but as in other smooth muscles studied (see Aickin, 1988;Aalkjaer & Cragoe, 1988), intracellular buffering power was unaffected by the presence or nominal absence of CO2. This therefore suggests that Na-H exchange is largely responsible for the extrusion of acid equivalents.…”

“…Since various tissues have been studied and different methods used, it seemed important to establish whether the lack of consistency stemmed from the methodology or from genuine differences between tissues. I have therefore used double-barrelled pH-sensitive microelectrodes in segments of guinea-pig femoral artery, making comparison both with my previous work using microelectrodes in guinea-pig vas deferens (Aickin, 1984) and ureter (Aickin, 1988) and with results from fluorescent indicators in cultured aortic cells (Korbmacher et al 1988) and in segments of smaller, resistance vessels (Aalkjaer & Cragoe, 1988).…”

Na+/H+ Exchange and Hypoxic Pulmonary Hypertension

“…1), the relative significance of these various transport mechanisms to control of pH i under basal conditions appears to vary by experimental organism, tissue, and cell type. All four of these ion transporters have been described in various types of vascular smooth muscle [1][2][3][4] and, with the exception of Na + -HCO 3 − cotransport, modulate pH i in pulmonary arterial smooth muscle cells (PASMCs). [5][6][7] Of these, NHE plays a significant role in regulating PASMC pH i , under both resting and stimulated conditions.…”