Gisela Moehren

Quantification of Short Term Signaling by the Epidermal Growth Factor Receptor

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15–30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

Unilateral nephrectomy selectively stimulates phospholipase D in the remaining kidney

The activation of phospholipase D in the kidney could be detected in vivo in rats treated with ethanol by the accumulation of phosphatidylethanol. Unilateral nephrectomy stimulated the activity of phospholipase D in the remaining kidney as indicated by an increase in the level of phosphatidylethanol. A significant increase in phosphatidylethanol level was observed as early as 5 min after contralateral nephrectomy and peak accumulation (200% of control) was observed after 15 min. The phosphatidylethanol level decreased again to the basal level after 2 h. The accumulation of phosphatidylethanol was specific for kidney and the product was localized primarily in the cortex. Phospholipase D activity in kidney cortical slices from untreated rats was stimulated in vitro by plasma obtained from unilaterally nephrectomized rats, indicating that circulating factors in the plasma are responsible for the activation of phospholipase D. The phospholipase D activation by plasma from uninephrectomized animals was selectively inhibited by the tyrosine kinase inhibitor genistein, but not by the protein kinase C inhibitor H7. It is concluded that phospholipase D activity is stimulated as an early signal transduction event in compensatory kidney growth.

Leupeptin inhibits phospholipases D and C activation in rat hepatocytes

Abstract The relationship between phospholipase D and C activation was studied in intact rat hepatocytes and rat liver plasma membranes. In intact hepatocytes, in the presence of ethanol, vasopressin, phorbol ester, and calcium independently stimulated phosphatidylethanol (PETH) formation, a specific marker of phospholipase D activity. Leupeptin (10–1500 μM) inhibited PETH formation induced by vasopressin, but was ineffective in response to phorbol ester or calcium. Leupeptin also inhibited the formation of inositol phosphates in intact cells in response to vasopressin. In liver plasma membranes, GTP[S] induced the production of phosphatidic acid and, in the presence of ethanol, PETH. Plasma membrane-associated phospholipase D did not require calcium and was insensitive to protein kinase C inhibitors. Leupeptin inhibited PETH formation in response to GTP[S]. The inhibition by leupeptin could be overcome by increasing the concentration of GTP[S]. In plasma membranes, the inhibitory effects of leupeptin on phospholipase D occurred at doses that far exceed those required to maximally inhibit proteolysis. These data highlight a central role for phospholipase C in the activation of phospholipase D, and a minor role for a direct G-protein activation. The findings also demonstrate a novel use of leupeptin as an inhibitor of phospholipases D and C perhaps at the level of a G protein.

The Role of Calcium and Phospholipase A2 in Glucagon-Induced Enhancement of Mitochondrial Calcium Retention

Glucagon affects metabolic activities in different compartments of the liver cell, including the mitochondria. These effects of glucagon are often preserved during isolation of the mitochondria (see 1 for review). The mitochondrial actions of glucagon may not be directly mediated by a rise in the level of cAMP; instead, mitochondrial calcium uptake may be involved in transferring the hormonal signal to the mitochondrial matrix. Glucagon induces a mobilization of calcium from non-mitochondrial stores in hepatocytes, presumably by activation of the phosphoinositide-specific phospholipase C (2,3). This leads to a transient increase in cytosolic free calcium levels, disturbing the balance between matrix and cytosolic calcium concentrations maintained by the calcium transport systems present in the inner membrane. There is evidence that mitochondrial calcium content is increased after glucagon treatment (4).