Xinfang Zhao

Ethanol metabolism is not required for inhibition of LPS-stimulated transcription of inducible nitric oxide synthase.

We examined the effect of inhibition of ethanol metabolism on ethanol-mediated suppression of Escherichia coli endotoxin (LPS-induced upregulation of transcription and release of inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFalpha) from rat alveolar macrophages (AM) in vivo. Ethanol (3.45 and 5.5 g/kg/IP) and t-butanol (3.7 g/kg, IP), given 30 min before intratracheal administration of LPS (1.0 mg/kg), inhibited the upregulation of iNOS mRNA and protein, determined by competitor equalized RT-PCR and Western immunoblot, respectively, but not TNFalpha mRNA in AM obtained 2 h after LPS administration by bronchoalveolar lavage (BAL). However, ethanol and t-butanol inhibited LPS-stimulated nitrate and nitrite (RNI) and TNFalpha protein in BAL fluid. Pretreatment of rats with 4-methylpyrazole (100 mg/kg, IP) 2 h before, or disulfiram 30 min before, administration of ethanol (3.45 g/kg, IP) failed to attenuate the inhibitory effect on iNOS mRNA or protein. t-Butyl hydroperoxide (100 mg/kg, IP) given to rats 30 min before administration of LPS enhanced LPS-mediated upregulation of iNOS mRNA and TNFalpha protein in AM and BAL fluid. The inhibitory effect of ethanol on iNOS mRNA was not mediated by an interaction with elevated levels of circulating corticosterone because pretreatment of rats with RU-38486 (100 mg/kg, IM), which inhibited prednisolone (50 mg/kg, IM), induced suppression of LPS-stimulated iNOS mRNA, and failed to attenuate ethanol-mediated inhibition of LPS-stimulated iNOS mRNA in AM. We conclude that metabolism of ethanol to acetaldehyde via alcohol dehydrogenase is not required for ethanol-mediated suppression of LPS-induced iNOS transcription and TNFalpha synthesis/release in AM. Moreover, an interaction of ethanol or acetaldehyde with circulating corticosterone is not involved in ethanol-mediated attenuation of LPS-stimulated iNOS mRNA or protein or TNFalpha protein in the lung. Speculatively, because oxidation of t-butanol to t-butylhydroperoxide results in activation, rather than inhibition, of iNOS and TNF-alpha, the reported ethanol-mediated enhancement of iNOS mRNA may result from the action of the hydroxyethyl radical.

PROTEIN KINASE C ISOZYMES IN SKELETAL MUSCLES DURING THE EARLY STAGE OF GENETIC AND STREPTOZOCIN DIABETES

Abstract This study examined the changes in PKC isozyme activity, content, and cellular distribution in rat gastrocnemius and soleus muscles prior to any evidence of neural degeneration or impaired skeletal muscle function, during the onset of streptozocin-induced (STZ) and genetic diabetes mellitus (DM). PKC activity was increased more in the particulate than in the soluble fractions of the soleus and gastrocnemius muscles obtained from rats treated with STZ and the gastrocnemius muscle obtained from BB-Wor diabetic rats (D rats). The predominant constitutive PKC isozymes in the skeletal muscles obtained from the STZ-treated and D rats were PKCα ≫ PKC∊ > PKCδ as determined by Western immunoblot assay. The content of each PKC isozyme did not differ between the soleus and gastrocnemius muscles of the control Sprague-Dawley rats for the STZ-treated rats and the BB Wor diabetic resistant (DR) rats. Moreover, the PKC isozyme content did not differ in the soluble fraction of D or STZ rats when compared to their corresponding control animals. PKCδ increased more than PKCα or PKC∊ in the particulate fraction of gastrocnemius and soleus muscles when obtained from either D or STZ rats. Since similar changes in skeletal muscle PKC isozyme profiles occurred independent of the duration of the diabetes and thereby the degree of nerve degeneration, insulin resistance, and the model of DM tested, we conclude that changes in skeletal muscle PKC precede the skeletal muscle myopathy of DM.

Neural Nitric Oxide Synthase and Neutrophil Function

Nitric oxide synthases (NOS, EC 1.14.13.39) are members of a family of cytochrome P450-like reductases linked to an NADPH oxidase enzyme. Nitric oxide synthase converts L-arginine to nitric oxide (NO) and L-citrulline utilizing the electrons generated by the reduction of oxygen to superoxide, hydrogen peroxide, and ultimately water (Wang and Marsden,1995,Xie and Nathan,1994; Forstermann and Kleinert,1995). At least three isozymes of NOS exist, each of which is derived from a different gene (Forstermann and Kleinert,1995). Endothelial NOS (ecNOS) and neural NOS (nNOS) are constitutive, calcium-activated isozymes found in endothelial and endocardial cells, airway epithelial cells, adrenal cortical and medullary cells, neurons, skeletal muscle, and many soft tissues (Forstermann and Kleinert,1995;Xie and Nathan,1994;Forstermann and Kleinert,1995). Inducible NOS (iNOS), primarily regulated by transcription, is elicited in inflammatory and immunocompetent cells; cardiac, striated, and smooth muscles; pancreatic beta cells; and a wide variety of tissues by bacterial endotoxins, cytokines, and autacoids (Wang and Marsden,1995; Xie and Nathan, 1994;Forstermann and Kleinert,1995;Greenberg et al.,1996,Greenberg et al.,1997;Nichols et al.,1988;Kwon et al.,1996).