OBJECTIVE— Endogenous expression of apolipoprotein E (apoE) has a significant impact on adipocyte lipid metabolism and is markedly suppressed in obesity. Adipose tissue oxidant stress is emerging as an important mediator of adipocyte dysfunction. These studies were undertaken to evaluate the role of oxidant stress for regulation of adipocyte apoE. RESEARCH DESIGN AND METHODS— ApoE gene and protein expression in 3T3-L1 adipocytes or mature adipocytes and adipose tissue from C57/BL6 mice was evaluated after induction of oxidant stress. The response of adipose tissue and adipocytes from obese compared with lean mice to antioxidants was also assessed. RESULTS— Oxidant stress in 3T3-L1 cells or adipocytes and adipose tissue from lean mice significantly reduced apoE mRNA and protein level. Inclusion of an antioxidant eliminated this reduction. Oxidant stress was accompanied by activation of the nuclear factor-κB (NF-κB) transcription complex, and its effect on apoE was eliminated by an NF-κB activation inhibitor. Treatment of freshly isolated adipose tissue or mature adipocytes from obese mice with antioxidant increased apoE expression but had no effect on cells or tissue from lean mice. Incubation of freshly isolated adipocytes from lean mice with stromovascular cells from obese mice significantly suppressed adipocyte apoE compared with incubation with stromovascular cells from lean mice, but this suppression was reversed by inclusion of antioxidant or a neutralizing antibody to tumor necrosis factor-α. CONCLUSIONS— Oxidant stress significantly modulates adipose tissue and adipocyte apoE expression. Furthermore, oxidant stress contributes to suppression of adipocyte apoE in obesity. This suppression depends on interaction between adipose tissue stromovascular cells and adipocytes.

Cholinergic agents are known to affect the epithelial transport of H2O and electrolytes in the kidney. In proximal tubule cells, cholinergic agonists increase basolateral Na-HCO(3) cotransport activity via M(1) muscarinic receptor activation. The signaling intermediates that couple these G protein-coupled receptors to cotransporter activation, however, are not well defined. We therefore sought to identify distal effectors of muscarinic receptor activation that contribute to increased NBC activity in cultured proximal tubule cells. As demonstrated previously for acute CO2-regulated cotransport activity, we found that inhibitors of Src family kinases (SFKs) or the classic mitogen-activated protein kinase (MAPK) pathway prevented the stimulation of NBC activity by carbachol. The ability of carbachol to activate Src, as well as the proximal (Raf) and distal [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] elements of the classic MAPK module, was compatible with these findings. Cholinergic stimulation of ERK1/2 activity was also completely prevented by overexpression of a dominant negative mutant of Ras (N17-Ras). Taken together, these findings suggest a requirement for the sequential activation of SFKs, Ras, and the classic MAPK pathway [Raf-->MAPK/ERK kinase (MEK)-->ERK]. These findings provide important insights into the molecular mechanisms underlying cholinergic regulation of NBC activity in renal epithelial cells. They also suggest a specific mechanism whereby cholinergic stimulation of the kidney can contribute to pH homeostasis.

Regulation of renal Na-HCO cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO(2)) requires both Src family kinase (SFK) and classic MAPK pathway activation. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) couples discrete G protein-coupled receptor and growth factor receptor signaling to SFK activation. We examined the role of Pyk2-SFK interaction in coupling these stimuli to increased NBC1 activity in opossum kidney cells. Carbachol increased tyrosine autophosphorylation of endogenous Pyk2 and ectopically expressed wild-type Pyk2 and were abrogated by kinase-dead mutant (Pyk2-KD) overexpression. Pyk2 phosphorylation was calcium/calmodulin dependent, and Pyk2 associated with Src by means of SH2 domain interaction. Pyk2 phosphorylation and Pyk2-Src interaction by carbachol were mimicked by both ANG II and CO(2). To correlate Pyk2 autophosphorylation and Pyk2-Src interaction with NBC activity, cotransporter activity was measured in untransfected cells and in cells overexpressing Pyk2-KD in the presence or absence of carbachol, ANG II, or CO(2). In Pyk2-KD-overexpressing cells, the effect of carbachol, ANG II, and CO(2) was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO(2) to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2-Src interaction.

Angiotensin II (AII) plays an important role in renal proximal tubular acidification via the costimulation of basolateral Na/HCO3 cotransporter (NBC) and apical Na/H exchanger (NHE) activities. These effects are mediated by specific G protein-coupled AII receptors, but their corresponding downstream effectors are incompletely defined. Src family tyrosine kinases (SFKs) contribute to the regulation of both transport activities by a variety of stimuli and are coupled to classic mitogen-activated protein kinase (MAPK) pathway activation in this cell type. We therefore examined these signaling intermediates for involvement in AII-stimulated NBC activity in cultured proximal tubule cells. Subpressor concentrations of AII (0.1 nM) increased NBC activity within minutes, and this effect was abrogated by selective antagonism of AT1 angiotensin receptors, SFKs, or the classic MAPK pathway. AII directly activated Src, as well as the proximal (Raf) and distal (ERK) elements of the classic MAPK module, and the activation of Src was prevented by AT1 receptor antagonism. An associated increase in basolateral membrane NBC1 content is compatible with the involvement of this proximal tubule isoform in these changes. We conclude that AII stimulation of the AT1 receptor increases NBC activity via sequential activation of SFKs and the classic MAPK pathway. Similar requirements for SFK/MAPK coupling in both cholinergic and acidotic costimulation of NBC and NHE activities suggest a central role for these effectors in the coordinated regulation of epithelial transport by diverse stimuli.

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