Endothelin receptor alterations in equine airway hyperreactivity.

The purpose of this study was to evaluate the role of endothelin-1 (ET-1) and its receptors in the airway hyperreactivity of horses with obstructive pulmonary disease associated with summer pasture (SPAOPD). The right diaphragmatic lobe of the lung of 8 clinically healthy (unaffected) and 8 SPAOPD-affected horses was collected immediately after euthanasia. Bronchial rings (4 mm wide) were prepared and mounted in organ baths and attached to force transducers interfaced with a polygraph. Four rings were used to study each ET-1 receptor; 1 ring served as the control, and the other 3 were incubated with 10(-9), 10(-7), or 10(-5) M of either BQ-123, an ET(A)-receptor antagonist, or IRL-1038, an ET(B)-receptor antagonist. Cumulative concentrations (10(-8.5) to 10(-6) M) of ET-1 were applied to all rings. Using pooled pulmonary tissue from different regions of the lung, we performed a reverse-transcription polymerase chain reaction (RT-PCR) to determine ET(B)-receptor gene expression. Although ET-1 caused concentration-dependent bronchial ring contraction in both groups of horses, the rings of SPAOPD-affected horses had significantly greater contraction than the rings of unaffected horses. Whereas ET(A)-receptor blockade significantly increased the response to ET-1 in unaffected horses, ET(B)-receptor blockade significantly decreased the response in affected horses. The pA2 values showed a nonsignificant decrease in ET(A)-receptor affinity and a significant increase in ET(B)-receptor affinity in affected horses compared with unaffected horses. The ET(B)-receptor mRNA expression of the pooled pulmonary tissue showed a nonsignificant increase in affected horses compared with unaffected horses. The airway hyperreactivity to ET-1 observed in the bronchial rings from the affected horses appears to be due in part to activation of pulmonary ET(B) receptors, which appear to be inactive in unaffected horses.

The ribosomal protein rpL11 associates with and inhibits the transcriptional activity of peroxisome proliferator-activated receptor-alpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor superfamily whose ligands, the peroxisome proliferators (PPs), are liver tumor promoters in rodents. Interaction cloning was performed using bacterially expressed PPARalpha to identify proteins involved in PP signaling. The ribosomal protein L11 (rpL11), a component of the large 60S subunit, was identified as a PPARalpha-associated protein. Since rpL11 is a regulator of p53 and the cell cycle, the association between this protein and PPARalpha was examined in detail. PPARalpha-rpL11 interaction was confirmed using yeast and mammalian two-hybrid systems as well as in vitro pull-down assays. The association with rpL11 occurs within the D-domain (hinge-region) of PPARalpha. Unlike PPARalpha, the two closely related isoforms PPARbeta and gamma do not interact with rpL11. Cotransfection of mammalian cells with rpL11 resulted in ligand-dependent inhibition of transcriptional activity of PPARalpha. Ribosomal protein L11-mediated inhibition of gene expression is associated with decreased binding to the PPAR-response element (PPRE) DNA sequence. Release of rpL11 from the ribosome by serum deprivation or low-dose actinomycin D did not dramatically affect PPRE-driven luciferase activity when PPARalpha was overexpressed by cotransfection. However, when endogenous levels of PPARalpha are examined and rpL11 concentration is manipulated by expression by small interference RNA, the ability of peroxisome proliferator to induce PPRE-driven reporter activity and target gene mRNA is affected. These studies show that rpL11 inhibits PPARalpha activity and adds further evidence that ribosomal proteins play roles in the control of transcriptional regulation.

Regulation of peroxisome proliferator-activated receptor alpha by protein kinase C.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acids, hypolipidemic drugs, and peroxisome proliferators (PPs). Like other nuclear receptors, PPARalpha is a phosphoprotein whose activity is affected by a variety of growth factor signaling cascades. In this study, the effects of protein kinase C (PKC) on PPARalpha activity were explored. In vivo phosphorylation studies in COS-1 cells transfected with murine PPARalpha showed that the level of phosphorylated PPARalpha is increased by treatment with the PP Wy-14,643 as well as the PKC activator phorbol myristol acetate (PMA). In addition, inhibitors of PKC decreased Wy-14,643-induced PPARalpha activity in a variety of reporter assays. Overexpressing PKCalpha, -beta, -delta, and -zeta affected both basal and Wy-14,643-induced PPARalpha activity. Four consensus PKC phosphorylation sites are contained within the DNA binding (C-domain) and hinge (D-domain) regions of rat PPARalpha (S110, T129, S142, and S179), and their contribution to receptor function was examined. Mutation of T129 or S179 to alanine prevented heterodimerization of PPARalpha with RXRalpha, lowered the level of phosphorylation by PKCalpha and PKCdelta in vitro, and lowered the level of phosphorylation of transfected PPARalpha in transfected cells. In addition, the T129A mutation prevented PPARalpha from binding DNA in an electromobility shift assay. Together, these studies demonstrate a direct role for PKC in the regulation of PPARalpha, and suggest several PKCs can regulate PPARalpha activity through multiple phosphorylation sites.

Evidence of aryl hydrocarbon receptor ligands in Presque Isle Bay of Lake Erie.

The purpose of the present studies was to use a biomarker approach to examine xenobiotic exposure of brown bullhead in Presque Isle Bay, Lake Erie (USA). In particular, the presence of compounds that act through the aryl hydrocarbon receptor (AhR) was of interest due to its central role in gene regulation and carcinogenesis of dioxins and certain polycyclic aromatic hydrocarbons (PAHs). Initial screening of Presque Isle Bay sediment samples by gene expression microarray in mouse hepatocytes revealed prototypical dioxin-response genes such as cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1). The presence of AhR ligands in sediment samples was confirmed and quantified using an in vitro assay, the Chemical Activated Luciferase Expression (CALUX) assay. The CALUX assay system, by using different incubation times, allows for determination of total dioxin induction equivalents (IEQ) for less persistent compounds such as PAHs as well as for stable compounds such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and certain polychlorinated biphenyls (PCBs). Parts of Presque Isle Bay have significant concentrations of AhR ligands in sediment ranging from 200 to 1400 parts per trillion (ppt) dioxin IEQ equivalents (dry weight). This is much higher than levels of dioxin equivalents found in similar sediment samples (approximately 10 ppt). Cascade Creek appears to be a major source of dioxin-like contaminants as IEQs in sediments taken from various regions of this tributary ranged from 1300 to 42000 ppt IEQ. In addition, the CALUX assay indicated that the majority of the IEQs (>90%) in PIB samples were in fact derived from less stable compounds. To determine if brown bullhead are exposed and respond to these high levels of AhR ligands, CYP1A cDNA was cloned from this species and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine mRNA levels. The CYP1A mRNA concentration was lower and less variable in fish taken from Presque Isle Bay than from a body of water with much lower AhR ligand concentration. Taken together, these studies show that sediment in Presque Isle Bay is highly contaminated with AhR ligands including dioxins and PAHs, but the brown bullhead are either not exposed or are non-responsive to these carcinogenic compounds.