Characterization of the expression of PDZ-RhoGEF, LARG and G(alpha)12/G(alpha)13 proteins in the murine nervous system.

Small GTPases of the Rho-family, like Rho, Rac and Cdc42, are involved in neuronal morphogenesis by regulating growth cone morphology or dendritic spine formation. G-proteins of the G12-family, G12 and G13, couple G-protein-coupled receptors (GPCRs) to the activation of RhoA. Recently, two novel Rho-specific guanine nucleotide exchange factors (RhoGEFs), PDZ-RhoGEF and LARG, have been identified to interact with the activated alpha-subunits of G12/G13 and are thus believed to mediate GPCR-induced Rho activation. Although studies in neuronal cell lines have shown that G12/G13 and PDZ-RhoGEF mediate GPCR-induced neurite retraction, the role, as well as the expression of this signalling pathway, in intact brain has not been adequately studied. In the present study, we have characterized systematically the expression of G(alpha)12, G(alpha)13, PDZ-RhoGEF and LARG in various murine tissues as well as their subcellular localization in the central and peripheral nervous systems. By performing immunohistochemistry, using polyclonal antibodies raised against the above proteins, we observed that G(alpha)12, G(alpha)13 and their RhoGEF-effectors are distributed widely in the mammalian nervous system. Moreover, these proteins localize to distinct morphological compartments within neurons. While LARG and G(alpha)12 were mainly found in somata of the neurons, PDZ-RhoGEF and G(alpha)13 were predominantly localized in the neuropil of central neurons. Interestingly, PDZ-RhoGEF is a neural-specific protein, whereas LARG is nearly ubiqoutous. Our data provide evidence that the G12/13-RhoGEF-mediated pathway is present throughout the adult brain and may be involved in regulation of neuronal morphogenesis and function via GPCRs.

Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes.

Myocardial hypertrophy is an adaptational response of the heart to increased work load, but it is also associated with a high risk of cardiac mortality due to its established role in the development of cardiac failure, one of the leading causes of death in developed countries. Multiple growth factors and various downstream signaling pathways involving, for example, ras, gp-130 (ref. 4), JNK/p38 (refs. 5,6) and calcineurin/NFAT/CaM-kinase have been implicated in the hypertrophic response. However, there is evidence that the initial phase in the development of myocardial hypertrophy involves the formation of cardiac para- and/or autocrine factors like endothelin-1, norepinephrine or angiotensin II (refs. 7,8), the receptors of which are coupled to G-proteins of the Gq/11-, G12/13- and Gi/o-families. Cardiomyocyte-specific transgenic overexpression of alpha1-adrenergic or angiotensin (AT1)-receptors as well as of the Gq alpha-subunit, Galphaq, results in myocardial hypertrophy. These data demonstrate that chronic activation of the Gq/G11-family is sufficient to induce myocardial hypertrophy. In order to test whether Gq/G11 mediate the physiological hypertrophy response to pressure overload, we generated a mouse line lacking both Galphaq and Galpha11 in cardiomyocytes. These mice showed no detectable ventricular hypertrophy in response to pressure-overload induced by aortic constriction. The complete lack of a hypertrophic response proves that the Gq/G11-mediated pathway is essential for cardiac hypertrophy induced by pressure overload and makes this signaling process an interesting target for interventions to prevent myocardial hypertrophy.

Pleiotropic effects of Pasteurella multocida toxin are mediated by Gq-dependent and -independent mechanisms. involvement of Gq but not G11.

Pasteurella multocida toxin (PMT) is a highly potent mitogen for a variety of cell types. PMT has been shown to induce various cellular signaling processes, and it has been suggested to function through the heterotrimeric G-proteins G(q)/G(11). To analyze the role of G(q)/G(11) in the action of PMT, we have studied the effect of the toxin in Galpha(q)/Galpha(11) double-deficient fibroblasts as well as in fibroblasts lacking only Galpha(q) or Galpha(11). Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha(q) but not on the closely related Galpha(11). Although Galpha(q)/Galpha(11) double-deficient and Galpha(q)-deficient cells did not respond with any production of inositol phosphates to PMT, PMT was still able to induce various other cellular effects in these cells, including the activation of Rho, the Rho-dependent formation of actin stress fibers and focal adhesions, as well as the stimulation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. These data show that PMT leads to a variety of cellular effects that are mediated only in part by the heterotrimeric G-protein G(q).

Relationships between structure and induction of hyaline droplet accumulation in the renal cortex of male rats by aliphatic and alicyclic hydrocarbons.

A rapidly growing list of hydrocarbons has been reported to induce morphological changes in the kidney of adult male rats, beginning with hyaline droplet accumulation (HDA) followed by the development of granular casts, later on chronic nephrosis as sequela, and finally renal adenomas and carcinomas. The present study focuses on identifying structure-based properties common to HDA-inducing aliphatics and cycloaliphatics. On the basis of rank-ordered activities reported in the literature, a calculated n-octanol-water partition coefficient above 3.5 and the presence of an isopentyl structural moiety appear to be associated with HDA-inducing activity in aliphatics. A binding site model for highly active aliphatics has been derived by superimposing their minimum energy conformations along the common isopentyl substructure and calculating the union volume of their respective van der Waal (VDW) volumes. Generalization of this model to include cycloaliphatics has been achieved by maximizing the steric overlap of the VDW volumes of the compounds with their binding site union volume. HDA-inducing cycloaliphatics are correctly identified on the basis of their negligible excess volume. This approach has been used to predict the HDA-inducing activity of previously untested compounds. Eighteen aliphatic/cycloaliphatic hydrocarbons were screened in a study on adult male Wistar rats treated with 250 mg/kg per day for 5 days. Azan-stained kidney sections were semiquantitatively evaluated for the presence of HDA. The predicted and observed HDA activities were in very good agreement.