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Acetylcholine (ACh) causes vascular smooth muscle relaxation by releasing endothelium-derived relaxing factor (EDRF) from endothelial cells (EC). Although a pivotal role for cytosolic free Ca2+ ([Ca2+]i) has been implicated in the generation and/or release of EDRF by various agonists, there is no conclusive evidence showing that ACh increases [Ca2+]i in EC. In the present study, using the Ca2+-sensitive fluorescent indicator fura-2, we show for the first time that ACh (10(-5) M) increases [Ca2+]i six- to sevenfold above prestimulus levels in primary cultures of rabbit aortic EC (RbAEC). ACh effects are dose dependent [effective concentration producing 50% of the maximum response (EC50) approximately 9 X 10(-7) M] and are blocked by atropine, a selective muscarinic receptor antagonist. The [Ca2+]i increase is due both to the mobilization of intracellular Ca2+ and to the influx of extracellular Ca2+. A 5-min incubation of RbAEC with 4 beta-phorbol 12-myristate 13-acetate (10(-7) M) inhibits ACh-induced [Ca2+]i transients, suggesting that the signaling pathway involved in ACh receptor signal transduction may be modulated via protein kinase C. These cultured EC provide a unique in vitro model system for studying mechanisms involved in ACh-induced EDRF release.

To evaluate the influence of intracellular alkalinization on basal and agonist-induced vascular tone, we studied the effect of NH4Cl on rat aorta. NH4Cl induced a gradually developing contraction in a dose-dependent manner. Although the contractile response to 20 mM NH4Cl was associated with a latent period (LP) of 23.4 +/- 2.8 min, intracellular pH (pHi) measurements in cultured rat aortic smooth muscle cells showed that NH4Cl-induced intracellular alkalinization was immediate and transient, returning to basal pHi levels in about 30-35 min. Agents that elevate Ca2+, such as A23187 and high KCl, significantly reduced the LP associated with 20 mM NH4Cl-induced contraction. NH4Cl-induced contractions were sensitive to extracellular Ca2+ removal and to the addition of forskolin (1 microM); however, NH4Cl by itself did not cause Ca2+-influx as shown by 45Ca-uptake studies. Addition of 20 mM NH4Cl to precontracted tissues resulted in a transient relaxation, which was complete in approximately 10 min, followed by a contraction above the original level of tone. NH4Cl pretreatment caused time-dependent alterations in both the rapid and slow phases of phenylephrine and angiotensin II contractions. Rapid-phase of phenylephrine and angiotensin II contractions. Rapid-phase responses were diminished at shorter NH4Cl incubation times (10 min), whereas slow-phase response was augmented after a longer incubation (20 min). Overall, the vasorelaxant and vasoconstrictor effects induced by NH4Cl suggest a complex relationship between intracellular alkalinization and arterial contractility.

Regulation of intracellular pH (pHi) via a Na(+)-H+ exchange-dependent mechanism was studied in cultured human umbilical vein endothelial cells (HEC) using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, as well as measuring 22Na influx. Basal pHi of HEC incubated in a bicarbonate-free Na+ medium was 6.99 +/- 0.03. In HEC that had been acid-loaded using nigericin or a NH4Cl prepulse, pHi recovery occurred via a Na(+)-dependent mechanism that was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The potency of amiloride derivatives to inhibit 22Na influx was EIPA greater than 5-(N,N-dimethyl)amiloride greater than amiloride [Ki (extracellular Na = 30 mM) = 17 nM, 150 nM, and 8.8 microM, respectively]. EIPA-sensitive 22Na influx in acid-loaded HEC was a saturable function of the external Na+ concentration (0-130 mM), exhibiting an approximate Km and Vmax of 19.70 +/- 0.14 mM and 34.01 +/- 2.2 nmol.10(6) cells-1.min-1, respectively. H+ efflux was also dependent on external Na+ and blocked by EIPA. At resting pHi, HEC Na(+)-H+ exchange was slightly stimulated by increases in medium osmolality. However, when HEC were acid-loaded in the presence of hypertonic (sucrose) medium, Na(+)-H+ exchange activity (22Na influx or pHi recovery) increased markedly. Overall, these data indicate that pHi in cultured HEC can be regulated by a Na(+)-H+ exchanger and that its activity can be markedly influenced by osmolality at acidic pHi.