Abstract
Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.
Highlights
EVIDENCE FOR A NOVEL MECHANISM MEDIATED BY THE extracellular signal-regulated kinase (ERK) PATHWAY*¶ Supported by Charitable Foundation of Guy’s and St. Thomas’ Hospitals Grant R010217
Activity of the Na؉/H؉ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H؉ with an allosteric modifier site in the transport domain
This apparent NHE stimulatory effect of sustained acidosis is more clearly illustrated in Fig. 2B, which shows the change in dpHi/dt (⌬dpHi/dt) during recovery from the second acid pulse relative to that during recovery from the first; ⌬dpHi/dt was close to zero in the control group, again reflecting the absence of a temporal increase in NHE activity, but increased significantly in myocytes exposed to 3 or 6 min of acidosis during the second acid pulse
Summary
¶ Supported by Charitable Foundation of Guy’s and St. Thomas’ Hospitals Grant R010217 We present evidence that a moderate extension of the duration of intracellular acidosis (to Ն3 min) produces a marked increase in sarcolemmal NHE activity in both neonatal and adult rat ventricular myocytes. This effect appears to occur through a novel mechanism that is mediated by acidosis-induced activation of the ERK pathway, the inhibition of which inhibits the stimulation of sarcolemmal NHE activity. Our data suggest that this mechanism may have relevance to the regulation of plasma membrane NHE activity in non-myocyte cells since similar observations were made in COS-7 cells (African Green monkey kidney cells), which exhibit native NHE1 expression [15]
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