Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Cellular localization, expression and regulation of neuropeptide Y in kidneys of hypertensive rats.

Neuropeptide Y (NPY) is a key modulator of the autonomic nervous system playing pivotal roles in cardiovascular and neuronal functions. In this study, we assessed the cellular localization and gene expression of NPY in rat kidneys. We also examined the relationship between NPY gene expression and renin in two rat models of hypertension (two-kidney, one-clip renal hypertension (2K1C), and deoxycorticosterone-salt-induced hypertension (DOCA-salt)) characterized by a similar blood pressure elevation. In situ hybridization and immunohistochemistry, using anti-NPY or anti-C-flanking peptide of NPY (CPON) antibodies, showed that NPY transcript and protein were colocalized in the tubules of rat kidneys. During experimental hypertension, NPY mRNA was decreased in both kidneys of the 2K1C animals, but not in the kidney of DOCA-salt rats. In 2K1C rats, renal NPY content was also decreased. The difference in NPY gene expression between 2K1C rats (a high renin model of hypertension) and DOCA-salt rats (a low renin model of hypertension) suggests that circulating angiotensin II plays a role in local renal NPY gene expression and that the elevated blood pressure per se is not the primary factor responsible for the control of NPY gene expression in the kidney.

Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of Renin genes.

Several studies have demonstrated that mice are polymorphic for the number of renin genes, with some inbred strains harboring one gene (Ren-1(c)) and other strains containing two genes (Ren-1(d) and Ren-2). In this study, the effects of 1% salt and deoxycorticosterone acetate (DOCA)/salt were investigated in one- and two-renin gene mice, for elucidation of the role of renin in the modulation of BP, cardiac, and renal responses to salt and DOCA. The results demonstrated that, under baseline conditions, mice with two renin genes exhibited 10-fold higher plasma renin activity, 100-fold higher plasma renin concentrations, elevated BP (which was angiotensin II-dependent), and an increased cardiac weight index, compared with one-renin gene mice (all P < 0.01). The presence of two renin genes markedly increased the BP, cardiac, and renal responses to salt. The number of renin genes also modulated the responses to DOCA/salt. In one-renin gene mice, DOCA/salt induced significant renal and cardiac hypertrophy (P < 0.01) even in the absence of any increase in BP. Treatment with losartan, an angiotensin II AT(1) receptor antagonist, decreased BP in two-renin gene mice but not in one-renin gene mice. However, losartan prevented the development of cardiac hypertrophy in both groups of mice. In conclusion, these data demonstrate that renin genes are important determinants of BP and of the responses to salt and DOCA in mice. The results confirm that the Ren-2 gene, which controls renin production mainly in the submaxillary gland, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, these data provide further evidence that mineralocorticoids promote cardiac hypertrophy even in the absence of BP changes. This hypertrophic process is mediated in part by the activation of angiotensin II AT(1) receptors.

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension.

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PURPOSE: Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat.

OBJECTIVES: Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. METHODS: In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. RESULTS: In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. CONCLUSION: After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Immunocytochemical localisation of microtubule-associated proteins 1b and 2 in the developing rat spinal cord.

The straightforward anatomical organisation of the developing and mature rat spinal cord was used to determine and interpret the time of appearance and expression patterns of microtubule-associated proteins (MAP) 1b and 2. Immunoblots revealed the presence of MAP1b and 2 in the early embryonic rat spinal cord and confirmed the specificity of the used anti-MAP mouse monoclonal antibodies. The immunocytochemical data demonstrated a rostral-to-caudal and ventral-to-dorsal gradient in the expression of MAP1b/2 within the developing spinal cord. In the matrix layer, MAP1b was found in a distinct radial pattern distributed between the membrana limitans interna and externa between embryonal day (E)12 and E15. Immunostaining for vimentin revealed that this MAP1b pattern was morphologically and topographically different from the radial glial pattern which was present in the matrix layer between E13 and E19. The ventral-to-dorsal developmental gradient of the MAP1b staining in the spinal cord matrix layer indicates a close involvement of MAP1b either in the organisation of the microtubules in the cytoplasmatic extensions of the proliferating neuroblasts or neuroblast mitosis. MAP2 could not be detected in the developing matrix layer. In the mantle and marginal layer, MAP1b was abundantly present between E12 and postnatal day (P)0. After birth, the staining intensity for MAP1b gradually decreased in both layers towards a faint appearance at maturity. The distribution patterns suggest an involvement of MAP1b in the maturation of the motor neurons, the contralaterally and ipsilaterally projecting axons and the ascending and descending long axons of the rat spinal cord. MAP2 was present in the spinal cord grey matter between E12 and maturity, which reflects a role for MAP2 in the development as well as in the maintenance of microtubules. The present description of the expression patterns of MAP1b and 2 in the developing spinal cord suggests important roles of the two proteins in various morphogenetic events. The findings may serve as the basis for future studies on the function of MAP1b and 2 in the development of the central nervous system.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

Morphogenesis of the ureterovesical junction:a histologic and microanatomic study in the rat.

OBJECTIVES: To investigate the development of the ureterovesical junction in rats. METHODS: A total of 110 albino rats (50 prenatal and 60 newborn) with a gestation of 21 days were studied at the age of 17 days after conception until 5 days after birth. The lower urinary tract was microdissected. Microphotography (110 animals), histologic examination (44 animals), and scanning electron microscopy (66 animals) of the ureterovesical junction were performed. Urea and creatinine from the amniotic fluid of 20 fetuses and from the urine of 10 neonates were measured. RESULTS: At day 17 after conception, separate penetration of the mesonephric duct and ureter into the wall of the urogenital sinus was observed. Continuity between the lumen of the ureter and the urogenital sinus was established on day 19 after conception. The straight passage of the intramural ureter into the urogenital sinus at day 17 after conception changed to the definitive L-shape with a vertical entry into the bladder on day 5 after birth. In the distal ureter, the change of the mesenchymal tissue into immature smooth muscle was first observed at birth, and the muscle became mature on the fifth postnatal day. At birth, Waldeyer's sheath was recognized. The creatinine and urea levels were stable prenatally (average 22.4 micromol/L and 6.88 mmol/L, respectively) and rose significantly postnatally (average 133 micromol/L and 32.65 mmol/L, respectively). CONCLUSIONS: The attachment of the ureter to the urogenital sinus and later to the bladder, the modification of its passage, and its mobility within Waldeyer's sheath may be essential in preventing vesicoureteral reflux. The production of urine and its flow does not seem to be the trigger of ureteral smooth muscle formation.