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We evaluated the effects of intracerebroventricular (icv) infusion of [Na.sup.+]-rich artificial cerebrospinal fluid (aCSF), with or without the mineralocorticoid receptor (MR) blocker spironolactone, on epithelial [Na.sup.+] channel (ENaC) subunits and regulators, such as MR, serum/glucocorticoid-inducible kinase 1, neural precursor cells expressed developmentally downregulated 4-like gene, 11[beta]-hydroxylase, and aldosterone synthase, in brain regions of Wistar rats. The effects of icv infusion of the amiloride analog benzamil on brain tissue and CSF [Na.sup.+] concentration ([[Na.sup.+]]) were also assessed. In the choroid plexus and ependyma of the anteroventral third ventricle, ENaC subunits are present in apical and basal membranes. [Na.sup.+]-rich aCSF increased [beta]-ENaC mRNA and immunoreactivity in the choroid plexus and increased [alpha]- and [beta]-ENaC immunoreactivities in the ependyma. [Na.sup.+]-rich aCSF increased [alpha]- and [beta]-ENaC-gold-labeled particles in the microvilli of the choroid plexus and in basolateral membranes of the ependyma. Spironolactone only prevented the increase in [beta]-ENaC immunoreactivity in the choroid plexus and ependyma. In the supraoptic nucleus, paraventricular nucleus, and subfornical organ, [Na.sup.+]-rich aCSF did not affect mRNA expression levels of the studied genes. Benzamil significantly increased CSF [[Na.sup.+]] in the control, but not [Na.sup.+]-rich, aCSF group. In contrast, benzamil prevented the increase in hypothalamic tissue [[Na.sup.+]] by [Na.sup.+]-rich aCSF. These results suggest that CSF [Na.sup.+] upregulates ENaC expression in the brain epithelia, but not in the neurons of hypothalamic nuclei. ENaC in the choroid plexus and ependyma appear to contribute to regulation of [Na.sup.+] homeostasis in the brain. sodium-rich artificial cerebrospinal fluid; benzamil; cerebrospinal fluid sodium concentration doi: 10.1152/ajpregu.00834.2009.

E2F6 is believed to repress E2F-responsive genes and therefore serve a role in cell cycle regulation. Analysis of the human E2F6 promoter region revealed the presence of two putative E2F binding sites, both of which were found to be functionally critical because deletion or mutations of these sites abolished promoter activity. Ectopic expression of E2F1 protein was found to increase E2F6 mRNA levels and significantly upregulate E2F6 promoter activity. Deletion or mutation of the putative E2F binding sites nullified the effects of E2F1 on the E2F6 promoter activity. Studies on the temporal induction of E2F family members demonstrated that the activating E2Fs, and most notably E2FI, were upregulated before E2F6 during cell cycle progression at the G1/S phase, and this coincided with the time course of induction experienced by the E2F6 promoter during the course of the cell cycle. EMSAs indicated the specific binding of nuclear complexes to the E2F6 promoter that contained E2F1-related species whose binding was specifically competed by the consensus E2F binding site. Chromatin immunoprecipitation assays with anti-E2Fs demonstrated the association of E2F family members with the E2F6 promoter in vivo. These data indicate that the expression of the E2F6 repressor is influenced at the transcriptional level by E2F family members and suggest that interplay among these transcriptional regulators, especially E2F1, may be critical for cell cycle regulation. cell cycle; transcriptional control

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for [alpha]-, [beta]-, and [gamma]-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of [gamma]-ENaC was undetectable, whereas [alpha]- and [beta]-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial [Na.sup.+] concentration as well as neuronal excitation. reverse transcriptase polymerase chain reaction; immunohistochemistry

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the [[alpha].sub.1]-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-[gamma] agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment hut elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the over-expression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes. mice; microvascular function; gene expression; sarcolemmal membrane-associated protein

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus. sarcoplasmic reticulum; transmembrane domain; sarcolemmal membrane-associated protein

Abstract Background Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

Food constituents can either promote cardiovascular health or serve in its demise. In view of the lack of more effective pharmacological interventions in cardiovascular disease (CVDs), attention has focused on the potential protective effects of diet. Food components and their metabolites are emerging as major regulators of the human epigenome, which is being linked to CVDs. In this review, we summarize data from studies that suggest an important role for bioactive food compounds in cardioprotection and the potential for harnessing the epigenome as a nutrient sensor target in CVDs. While clinical data strongly support a role for effective diet intervention in CVDs protection, studies linking changes to human epigenome are now warranted for mechanistic insight and development of personalized care. [ABSTRACT FROM AUTHOR]

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dtmax) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca22+-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function. [ABSTRACT FROM AUTHOR]