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Pregnancy and estradiol decrease GTPase activity in the guinea pig uterine artery. Am J Physiol Heart Circ Physiol 281: H2168-H2175, 2001.--The mechanisms by which pregnancy redistributes cardiac output in an organ-specific manner are poorly understood. We propose that it is consequential to estrogen-mediated alterations in G protein-mediated signal transduction. Aortas and uterine (UAs) and mesenteric arteries (MAs) were obtained from late-pregnant, nonpregnant, or ovariectomized guinea pigs chronically treated with 17[beta]-estradiol. High-affinity GTPase activity was assayed enzymatically. The cGMP generated in response to the endothelium-dependent agonist ACh was measured in UAs incubated with or without cholera toxin (CTX, which inhibits [G.sub.s][alpha]). Pregnancy significantly decreased UA but not aorta or MA GTPase activity. 17[beta]-Estradiol decreased UA GTPase activity compared with untreated ovariectomized animals. ACh increased cGMP in pregnant but not nonpregnant UAs. Pretreatment of nonpregnant UAs with CTX increased ACh-induced cGMP levels similar to pregnancy. Thus pregnancy and estradiol decrease the GTPase activity of a CTX-sensitive G protein in UAs, increasing receptor-dependent cGMP release. This alteration in receptor-mediated G protein coupling in UAs may contribute to the characteristic cardiovascular adaptation to pregnancy. G proteins; nitric oxide; acetylcholine; estrogen

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr69/Thr70 (Thr69 in mice and Thr70 in humans) and Ser321/Ser322 (Ser321 in mice and Ser322 in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr69 in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-DL-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3=,4=-(methylenedioxy) benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr69 dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSCs administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies char by dysregulated Akt. [ABSTRACT FROM AUTHOR]

Discusses a study that examined the expression of tumor necrosis factor (TNF)-α in mouse neonatal cardiomyocytes. Materials and methods used; Effect of nuclear factor (NF)-κ B inhibitors and activators on TNF-α secretion by lipopolysaccharide-stimulated cardiomyocytes; Importance of identifying the intracellular signaling cascades that underlie the production of TNF-α.

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1∗±0.8 vs. 1.4∗±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2. [ABSTRACT FROM AUTHOR]

The National Academies of Science stresses the importance of research mentoring. We assessed the internal consistency and application of a novel 33 item mentor evaluation survey and explored differences across subgroups. The survey was administered annually to mentees. The response rate was 17.8% for a sample of 710 respondents. The survey exhibited strong internal validity with Cronbach Alpha > 0.89 for each subscale. Overall scores across the three domains were high. Basic Science trainees scored their mentor significantly lower than those in Translational or Clinical Science across domains (0.11-0.25 points). Underrepresented Racial Ethnic Groups (UREG) trainee scores were significantly lower in academic guidance and personal communication. Women had lower scores in 4 out of 5 domains. The survey is a modified instrument to assess mentee experience, although further validation against mentee outcomes is needed.

Mutations in the LIM homeobox transcription factor 1-beta (LMX1B) are a cause of nail patellar syndrome, a condition characterized by skeletal changes, glaucoma and focal segmental glomerulosclerosis. Recently, a missense mutation (R246Q) in LMX1B was reported as a cause of glomerular pathologies without extra-renal manifestations, otherwise known as nail patella-like renal disease (NPLRD). We have identified two additional NPLRD families with the R246Q mutation, though the mechanisms by which LMX1B R246Q causes a renal-specific phenotype is unknown. In this study, using human podocyte cell lines overexpressing either myc-LMX1B WT or myc-LMX1B R246Q , we observed dominant negative and haploinsufficiency effects of the mutation on the expression of podocyte genes such as NPHS1, GLEPP1, and WT1. Specifically, we observed a novel LMX1B R246Q -mediated downregulation of WT1(-KTS) isoforms in podocytes. In conclusion, we have shown that the renal-specific phenotype associated with the LMX1B R246Q mutation may be due to a dominant negative effect on WT1(-KTS) isoforms that may cause a disruption of the WT1 (-KTS):(+KTS) isoform ratio and a decrease in the expression of podocyte genes. Full delineation of the LMX1B gene regulon is needed to define its role in maintenance of glomerular filtration barrier integrity.