Decrease In Total Expression Research Articles

Role of protein kinase C β₂ in relaxin-mediated inhibition of cardiac fibrosis.

Relaxin is a pleiotropic hormone owing endogenous antifibrosis effect on numerous organs. We demonstrated relaxin's inhibitive effect on cardiac fibrosis previously. The aim of this study was to investigate the role of protein kinase C (PKC) β2 in relaxin's action under high glucose conditions. Cardiac fibroblasts (CFs) were isolated, exposed to high glucose and incubated with recombinant human relaxin (rhRLX). Western blot analysis revealed a relaxin-mediated decrease in total expression and translocation of PKCβ2, showing downregulation of PKCβ2 is involved in relaxin's action. Blocking PKCβ2 pathway with ruboxistaurin accelerated rhRLX-mediated inhibition in both proliferation of CFs and deposition of collagen. In conclusion, relaxin can inhibit high glucose-associated cardiac fibrosis partly through PKCβ2 pathway. Further work should be done to fully understand intracellular mechanisms of relaxin's action to accelerate its clinical use.

Cysteine 96 of Ntcp is responsible for NO-mediated inhibition of taurocholate uptake

The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

Effect of hypokalemia on renal expression of the ammonia transporter family members, Rh B Glycoprotein and Rh C Glycoprotein, in the rat kidney

Hypokalemia is a common electrolyte disorder that increases renal ammonia metabolism and can cause the development of an acid-base disorder, metabolic alkalosis. The ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are expressed in the distal nephron and collecting duct and mediate critical roles in acid-base homeostasis by facilitating ammonia secretion. In the current studies, the effect of hypokalemia on renal Rhbg and Rhcg expression was examined. Normal Sprague-Dawley rats received either K(+)-free or control diets for 2 wk. Rats receiving the K(+)-deficient diet developed hypokalemia and metabolic alkalosis associated with significant increases in both urinary ammonia excretion and urine pH. Rhcg expression increased in the outer medullary collecting duct (OMCD). In OMCD intercalated cells, hypokalemia resulted in more discrete apical Rhcg expression and a marked increase in apical plasma membrane immunolabel. In principal cells, in the OMCD, hypokalemia increased both apical and basolateral Rhcg immunolabel intensity. Cortical Rhcg expression was not detectably altered by immunohistochemistry, although there was a slight decrease in total expression by immunoblot analysis. Rhbg protein expression was decreased slightly in the cortex and not detectably altered in the outer medulla. We conclude that in rat OMCD, hypokalemia increases Rhcg expression, causes more polarized apical expression in intercalated cells, and increases both apical and basolateral expression in the principal cell. Increased plasma membrane Rhcg expression in response to hypokalemia in the rat, particularly in the OMCD, likely contributes to the increased ammonia excretion and thereby to the development of metabolic alkalosis.

Mutant (CCTG)n Expansion Causes Abnormal Expression of Zinc Finger Protein 9 (ZNF9) in Myotonic Dystrophy Type 2

The mutation that underlies myotonic dystrophy type 2 (DM2) is a (CCTG)n expansion in intron 1 of zinc finger protein 9 (ZNF9). It has been suggested that ZNF9 is of no consequence for disease pathogenesis. We determined the expression levels of ZNF9 during muscle cell differentiation and in DM2 muscle by microarray profiling, real-time RT-PCR, splice variant analysis, immunofluorescence, and Western blotting. Our results show that in differentiating myoblasts, ZNF9 protein was localized primarily to the nucleus, whereas in mature muscle fibers, it was cytoplasmic and organized in sarcomeric striations at the Z-disk. In patients with DM2, ZNF9 was abnormally expressed. First, there was an overall reduction in both the mRNA and protein levels. Second, the subcellular localization of the ZNF9 protein was somewhat less cytoplasmic and more membrane-bound. Third, our splice variant analysis revealed retention of intron 3 in an aberrant isoform, and fourth quantitative allele-specific expression analysis showed the persistence of intron 1 sequences from the abnormal allele, further suggesting that the mutant allele is incompletely spliced. Thus, the decrease in total expression appears to be due to impaired splicing of the mutant transcript. Our data indicate that ZNF9 expression in DM2 patients is altered at multiple levels. Although toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for the differences in DM1.

Na, K-ATPase isoform gene expression in normal and hypertrophied dog heart.

The catalytic alpha subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms; the expression of these isoforms is tissue-specific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression. hypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immunofluorescence, and 3H-ouabain binding. RNA blotting indicated that the alpha 3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed alpha 1 and alpha 3 protein, but no detectable alpha 2; immunoreactive alpha 1 and alpha 3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all alpha isoform mRNA's in hypertrophied dog LV, resulting in a greater proportion of alpha 1. The expression level of the alpha 3 isoform mRNA and protein was lower in hypertrophied hearts. These results indicate a greater proportion of alpha 1 isoform pumps in experimental canine hypertrophy. Thus, shifts in NA, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.