Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization.

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium.

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.

Calcium and osmotic regulation of the Na+/H+ exchanger in neonatal ventricular myocytes.

Intracellular pH regulation in primary cultures of neonatal cardiac myocytes has been characterized. Myocytes were exposed to hyperosmolar solutions to examine the effects on pH regulation by the Na+/H+ exchanger. Exposure to 100 mM NaCl, sorbitol, N-methyl-D-glucamine, or choline chloride all caused significant increases in steady state pHi in myocytes. Omission of extracellular calcium or administration of calmodulin antagonists reduced the osmotic activation of the exchanger. The myosin light-chain inhibitor ML-7 completely blocked osmotic activation of the exchanger suggesting that myosin light-chain kinase is involved in osmotic activation of the exchanger in the myocardium. The calmodulin-dependent protein kinase II inhibitor KN-93 inhibited the rate of recovery from an acute acid load as did trifluoperazine (TFP) and the calmodulin blocker W7, [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. Addition of the calcium ionophore ionomycin caused a large increase in resting pHi in isolated myocytes. However, this effect was largely resistant to HMA (5-(N,N-hexamethylene)-amiloride) indicating that an alternative mechanism of pHi regulation is responsible. The results demonstrate that the Na+/H+ exchanger of the neonatal myocardium is responsive to calcium and osmotically responsive pathways and that myosin light-chain kinase is a key protein involved in mediating the osmotic response.

Cell cycle behavior and MyoD expression in response to T3 differ in normal and mdx dystrophic primary muscle cell cultures.

Since mdx limb muscle regeneration in vivo is accompanied by rapid myoblast proliferation and differentiation compared to normal, we tested the possibility that proliferation and differentiation were differentially regulated in normal and mdx dystrophic muscle cells. Cell cycle behavior, MyoD expression, and the effects of thyroid hormone (T3) treatment were examined in primary cultures. Using a 4-hour pulse time for bromodeoxyuridine (BrdU) incorporation during S-phase, the phases of the cell cycle (early S, late S, G(2)/M, and G(0)/G(1)) were separated by 2-colour fluorescence (BrdU/PI) analysis using flow cytometry. The G(0)/G(1)-early S and the late S-G(2)/M transitions were examined under the influence of T3 in cycling normal and mdx muscle cell cultures over a 20-hour time period. Myogenesis and differentiation were assessed morphologically and by immunostaining for MyoD protein. Mdx cultures had fewer cells in G(0)/G(1) at 20 hours and more cells in early and late S-phase compared to normal cultures. T3 significantly increased the proportion of normal cells in early S-phase by 20 hours, and reduced the proportions in G(2)/M phase. Over the same time interval in parallel cultures, the proportion of MyoD+ normal cells decreased significantly. In the absence of T3, mdx cell cultures showed greater proportions of cells in S-phase than normal cultures, and similar increases in S-phase and loss of MyoD expression over time. However, mdx cultures had no change in the proportion that were MyoD+ during T3 treatment. The results confirm that T3 in primary cultures increased proliferation and prevented the de-differentiation of mdx cells to a greater degree than was typical of normal cells. The different susceptibilities to T3-related shifts between proliferation and differentiation observed in vitro support the idea that committed mdx myoblasts may be more activated and proliferative than normal myoblasts during regeneration in vivo.

Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.

We examined regulation of the Na(+)/H(+) exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 178 amino acids of the Na(+)/H(+) exchanger were expressed in Escherichia coli fused with glutathione S-transferase. The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays. Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter. Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein. In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH(i). These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na(+)/H(+) exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter.

Levels of MyoD protein expression following injury of mdx and normal limb muscle are modified by thyroid hormone.

Thyroid hormone (T3) affects muscle development and muscle regeneration. It also interacts with the muscle regulatory gene MyoD in culture and affects myoblast proliferation. We studied the localization of MyoD protein using a well-characterized polyclonal antibody for immunohistochemistry. Relative numbers of myogenic precursor cells per field were identified by their MyoD expression during muscle regeneration in normal and mdx dystrophic mice, with particular reference to the expression in mononuclear cells and myotubes at various T3 levels. In regeneration by normal muscles, relatively few MyoD+ nuclei per field were present in mononuclear cells of euthyroid and hypothyroid mice. MyoD staining of mononuclear cell nuclei was approximately doubled in fields of regenerating muscles of normal hyperthyroid compared to euthyroid mice, and was observed in precursors that appeared to be aligned before fusion into myotubes. In mdx regenerating muscle, twofold more mononuclear cells positive for MyoD were present in all three treatment groups compared to normal muscles regenerating under the same conditions. Localization was similar to the pattern in normal euthyroid mice. However, in muscles regenerating in hyperthyroid mdx mice, both mononuclear cell nuclei and centrally located nuclei in a subpopulation (about 15%) of new myotubes formed after the crush injury were intensely stained for MyoD protein. The changes observed are consistent with reports on T3-induced alteration of muscle repair, and propose a link between MyoD regulation and the accelerated differentiation during regeneration under high T3 conditions. (J Histochem Cytochem 46:59-67, 1998)