Endothelin-1 augments Na⁺/H⁺ exchange activity in murine pulmonary arterial smooth muscle cells via Rho kinase.

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH(i)), typically due to activation of Na(+)/H(+) exchange (NHE). In the systemic vasculature, ET-1 increases pH(i), Na(+)/H(+) exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH(i) and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH(i) and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3-5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH(i) and NHE activity induced by increasing concentrations of ET-1 (10(-10) to 10(-8) M). We found that application of exogenous ET-1 increased pH(i) and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH(i) or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH(i)

Effect of PKC activation on intracellular pH (pH_i) and Na⁺/H⁺ exchanger (NHE) activity.

<p>Representative traces show pH_i measured during ammonium pulse in the <b>A)</b> absence and <b>B)</b> presence of PMA (1 μmol/L). <b>C and D)</b> Bar graphs show mean±SEM for <b>C)</b> the change (Δ) in baseline pH_i (n = 7) and <b>D)</b> NHE activity (n = 8) in response to PMA.</p

Effect of ROCK inhibition on intracellular pH (pH_i) and Na⁺/H⁺ exchanger (NHE) activity.

<p><b>A)</b> Bar graph showing mean±SEM values for basal pH_i measured in cells in the absence (n = 4) or presence (n = 4) of Y-27632 (10 μmol/L; 30 min). <b>B)</b> Bar graph shows mean±SEM values for the change (Δ) in baseline pH_i in response to ET-1 (10^-8 mol/L) in the absence (n = 5) and presence (n = 3) of Y-27632. <b>C)</b> Bar graph showing mean±SEM for NHE activity in cells challenged with ET-1 in the absence (n = 11) and presence (n = 5) of Y-27632. * indicates significant difference from control; †indicates significant difference from ET-1 alone.</p

Effect of endothelin-1 (ET-1) on intracellular pH (pH_i) and Na⁺/H⁺ exchanger (NHE) activity.

<p><b>A)</b> Representative traces showing pH_i measured during the ammonium pulse protocol in control cells and cells treated with ET-1 (10^-8 M). <b>B and C)</b> Bar graphs showing mean±SEM values for <b>B)</b> the change (Δ) in baseline pH_i (n = 3-6) and <b>C)</b> NHE activity (n = 5-15) induced by different concentrations of ET-1. * indicates significant difference from control (0 nmol/L ET-1)</p

Schematic illustrating the effect of endothelin-1 (ET-1) on intracellular pH (pH_i) in pulmonary arterial smooth muscle cells.

<p>ET-1 binds to surface receptors, leading to activation of Rho kinase (ROCK). ROCK activation in turn results in enhanced Na⁺/H⁺ exchanger (NHE) activity, increased H⁺ efflux and an alkaline shift in pH_i. </p

Effect of dimethyl amiloride (DMA; 1 μmol/L) on intracellular pH (pH_i) and Na⁺/H⁺ exchanger (NHE) activity.

<p><b>A and B)</b> Representative traces showing pH_i measured during the ammonium pulse protocol in cells treated with ET-1 (10^-8 M) in the <b>A)</b> absence and <b>B)</b> presence of DMA. <b>C and D)</b> Bar graphs showing mean±SEM values for <b>C)</b> the change (Δ) in baseline pH_i (n = 3-6) and <b>D)</b> NHE activity (n = 5-15) in control (Con) cells or in cells treated with DMA alone (n =  4 for pH_i and n = 6 for NHE activity) , ET-1 (10^-8 M; n =  5 for pH_i and n = 11 for NHE activity) and ET-1 + DMA (n = 6 for pH_i and n = 5 for NHE activity). * indicates significant difference from control; †indicates significant difference from ET-1 alone.</p

Rabaptin-5 regulates receptor expression and functional activation in mast cells

Rab5 is a small GTPase that regulates early endocytic events and is activated by RabGEF1/Rabex-5. Rabaptin-5, a Rab5 interacting protein, was identified as a protein critical for potentiating RabGEF1/Rabex-5's activation of Rab5. Using Rabaptin-5 shRNA knockdown, we show that Rabaptin-5 is dispensable for Rab5-dependent processes in intact mast cells, including high affinity IgE receptor (FcϵRI) internalization and endosome fusion. However, Rabaptin-5 deficiency markedly diminished expression of FcϵRI and β1 integrin on the mast cell surface by diminishing receptor surface stability. This in turn reduced the ability of mast cells to bind IgE and significantly diminished both mast cell sensitivity to antigen (Ag)-induced mediator release and Ag-induced mast cell adhesion and migration. These findings show that, although dispensable for canonical Rab5 processes in mast cells, Rabaptin-5 importantly contributes to mast cell IgE-dependent immunologic function by enhancing mast cell receptor surface stability

Roles of RabGEF1/Rabex-5 domains in regulating FcϵRI surface expression and FcϵRI-dependent responses in mast cells

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (−/−) mast cells and examined FcϵRI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in −/− mast cells, including decreased basal FcϵRI expression, slowed FcϵRI internalization, elevated IgE + Ag–induced degranulation and IL-6 production, and the decreased ability of −/− cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface FcϵRI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen–activated −/− mast cells. By contrast, correction of these −/− phenotypes required a functional Vps9 domain. Thus, FcϵRI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity