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The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.
Phosphorus NMR can measure myocardial tissue pH from the chemical shift of inorganic phosphate (Pi) in isolated buffer-perfused hearts, but in vivo the Pi peak originating from the myocardium is obscured by the resonance of 2,3-diphosphoglycerate (DPG) in the blood, making pH difficult to determine. Taking advantage of the fact that most of the interfering DPG is within the cardiac chambers and is rapidly flowing out of the sensitive volume of our coil, we developed a pulse sequence which would separate myocardial Pi signal from interfering DPG. We tested this method on a flow phantom and in living rat heart, using exogenous glycerol phosphate as a blood-pool marker. The results indicated that signal from moving and nonmoving substances could be separated, and derived values for myocardial pH and PCr/Pi ratio were consistent with previous estimates. This method should be useful for studying myocardial acid-base physiology with NMR.
Systemic acidemia may impair cardiac contractility and predispose to arrhythmias. Moreover, bicarbonate treatment may further depress cardiac performance and increase mortality. Whether changes in myocardial intracellular pH or energy metabolism underlie this diminished performance has not been clarified in the in vivo setting. Thus we investigated the effect of lactic acidosis and two proposed treatments on myocardial energetics and intracellular pH in anesthetized living rats. A previously validated 31P-labeled nuclear magnetic resonance (31P-NMR) spectroscopic technique using saturating pulses was used to follow myocardial intracellular pH, phosphocreatine (PCr), ATP, and inorganic phosphate (Pi). After obtaining baseline values, we infused lactic acid to achieve a level greater than 5 mM. We then added an infusion of either bicarbonate (n = 7) or saline (n = 5). During lactic acid infusion, arterial pH declined (from 7.27 to 7.07, P less than 0.0001), but myocardial intracellular pH did not change (7.13 vs. 7.07, P not significant). The ratio of PCr to Pi, however, decreased with acidemia (from 3.13 to 2.24, P = 0.004), suggesting impaired energy metabolism. Compared with saline, bicarbonate infusion restored systemic pH (from 7.08 to 7.29), but myocardial pH was unaltered. In addition, PCr/Pi declined further following bicarbonate treatment (1.41 vs. 2.42, P = 0.08) but not following saline. Thus, despite reversal of systemic acidemia, bicarbonate treatment was associated with more severe impairment of energy metabolism than saline. This suggests a mechanism for previously reported adverse cardiac effects of bicarbonate treatment.