Protein kinase Cbeta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells

We studied the effects of protein kinase C (PKC) activation onendothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca²⁺ concentration inhuman dermal microvascular endothelial cells (HMEC) exposed to-thrombin or phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA).Immunofluorescence showed that thrombin and TPA reduced the cellsurface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1desensitization. In contrast, prior activation of PKC with TPA produceddesensitization to thrombin and histamine, indicatingheterologous PAR-1 desensitization. Treatment of cells withstaurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes(PKC_I andPKC_II) by transducing cellswith antisense cDNA of PKC_Iprevented the TPA-induced decrease in cell surface PAR-1 expression andrestored ~60% of the cytosolic Ca²⁺ signal in response tothrombin. In contrast, depletion of PKC isozymes did not affect theloss of cell surface PAR-1 and induction of homologous PAR-1desensitization by thrombin. Therefore, homologous PAR-1desensitization by thrombin occurs independently of PKC isozymes,whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

     

Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets

A large pool of folate exists in the large intestine of humans. Preliminary evidence, primarily in vitro, suggests that this folate may be bioavailable. The purpose of this study was to test the hypothesis that supplemental folic acid and bacterially synthesized folate are absorbed across the large intestine of piglets. The pig was used as an animal model because it resembles the human in terms of folate absorption, at least in the small intestine. A tracer of [3H]-folic acid or [3H]-para-aminobenzoic acid ([3H]-PABA), a precursor of bacterially synthesized folate, was injected into the cecum of 11-day-old piglets. Feces and urine were collected for 3 days. Thereafter, piglets were killed, and livers and kidneys harvested. [3H]-Folate was isolated from biological samples by affinity chromatography using immobilized milk folate binding proteins and counted using a scintillation counter. In piglets injected with [3H]-folic acid, the feces, liver, urine and kidneys accounted for 82.1%, 12.3%, 3.9% and 1.7% of recovered [3H]-folate, respectively. In piglets injected with [3H]-PABA, the amount of recovered bacterially synthesized folate in the feces, liver and urine was 85.1%, 0.4% and 14.6%, respectively. Twenty-three percent and 13% of tritium were recovered in samples examined (liver, kidney, fecal and urine) from piglets injected with [3H]-folic acid and [3H]-PABA, respectively. Using our estimates of [3H]-folic acid absorption and the total and percent monoglutamyl folate content of piglet feces, we predict that at least 18% of the dietary folate requirement for the piglet could be met by folate absorption across the large intestine.     

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.     

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1(-/-)) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1(-/-) mice than wild-type (WT), and increased PVR in Cav1(-/-) mice was attributed to precapillary sites. Treatment with NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1(-/-) mice, indicating greater NO-mediated pulmonary vasodilation in Cav1(-/-) mice compared with WT. Pulmonary vasculature of Cav1(-/-) mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle alpha-actin in lung parenchyma of Cav1(-/-) mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1(-/-) mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1(-/-) mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.     

Salinity induced alterations in photosynthetic and oxidative regulation are ameliorated as a function of salt secretion

Journal of Plant Research - Ion secretion facilitates recretohalophytes to tolerate saline and drought conditions but its relative contribution to the survival of many species remains poorly...     

Improvement of cardiac function in mouse myocardial infarction after transplantation of epigenetically-modified bone marrow progenitor cells

Objective

To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.

Methods and Results

We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.

Conclusion

Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.