Renal corticosterone 6β-hydroxylase in the spontaneously hypertensive rat

Excess 6β-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na⁺ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6β-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6β-hydroxulation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6β-hydroxylation by SHR renal microsomes. Increased renal 6β-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.     

Proteome analysis of the left ventricle in the vitamin D₃ and nicotine-induced rat vascular calcification model

Vitamin D? and nicotine (VDN) serve as an animal model of arterial calcification. The vascular calcification induced by the VDN model is always accompanied by compensatory left ventricular (LV) hypertrophy and impaired cardiac performance. To determine the possible mechanisms that are responsible for the effects of VDN on the LV, a 2-DE based proteomics approach was used to evaluate the changes in protein expression of the left ventricle in VDN rats, to our knowledge, for the first time. We identified sixteen proteins that were markedly altered and involved in mitochondrial function, heat shock protein activity, myocyte cytoskeleton composition and enzyme activity for energy metabolism. We describe, for the first time, a novel pathway (NDPK) that is involved in LV hypertrophy and enzyme activities of three of the sixteen clinical identified proteins: lactate dehydrogenase (LDH), SOD [Mn] and GST.     

Association between transforming growth factor β1 polymorphisms and left ventricle hypertrophy in essential hypertensive subjects

Left ventricular hypertrophy (LVH) increases the risk of cardiovascular morbid events in hypertension. TGF-β1 is involved in pathologic states such as cardiac hypertrophy and cardiac fibrosis; we thus postulate that the TGF-β1 polymorphism is related to LVH in hypertensives. Six hundred and eighty essential hypertensive patients were recruited. Biochemical variables and clinical data were obtained and the determination of LVH was performed by echocardiography. According to the presence of LVH, all subjects were divided into the LVH+ and LVH? group. DNA was obtained, and two coding region polymorphisms of the TGF-β1 gene (+869 Leu→Proat codon 10 and +915 ARG→Pro at codon 25) were analyzed by the polymerase chain reaction. The product was cleaved with the restriction endonucleases. For the polymorphisms of the +869 Leu→Pro at codon 10, there was no marked difference in the distributions of genotypes and the allele frequencies between the LVH+ and LVH? subjects. For +915 Arg→Pro at codon 25, a significant difference in the distributions of genotypes of TGF-β1 was observed. The left ventricular mass index (LVMI) in Arg-Pro genotype carriers was significantly higher than those in the Arg-Arg and Pro-Pro carriers. Multivariate analysis showed that the Arg-Pro genotype was an independent risk factor for LVH (OR 3.23, 95% CI [1.48–5.63, P = 0.002]). The codon 10 genotypes did not show a significant association to LVH. Our data revealed a genetic association of TGF-β1+915 Arg→Pro at codon 25 polymorphism with LVH in a Chinese hypertensive population.     

Cumulative indices of DNA synthesizing myocytes in different compartments of the working myocardium and conductive system of the rat’s heart muscle following extensive left ventricle infarction

Ten successive³H-thymidine injections at 12h intervals (which is a little shorter than the adult heart myocyte S phase) were performed for labeling of the majority of cardiac myocytes synthesizing DNA at any moment of such a 5 days experiment. In the hearts of control unoperated rats ten-fold repeated³H-thymidine administration results in labeling of 2–3% myocyte nuclei, in both atria, ca. 1% of the specialized muscle cell nuclei in the atrioventricular conductive system, only occasional muscle cells being labeled in the working ventricular myocardium. When ten successive³H-thymidine injections were made between the 5th and 10th days following extended left ventricle infarction, the percentage of labeled myocytes in left and right atria reaches, respectively, 51.4±4.4% and 34.7±3.6%. In the left ventricle labeled muscle nuclei are accumulated predominantly (9.3±2.1%) within the thin subepicardial layer of the surviving myofibers, while myofibers located in other perinecrotic areas contained only 1.3±0.5% labeled muscle nuclei. The number of these nuclei in the atrioventricular system remains at the level observed in control hearts (up to 2%), approaching closely the zero level in the working myocardium of both the ventricles and interventricular septum, located at the considerable distance from the infarcted region. When similar experiments with ten-fold repeated³H-thymidine injections were performed between 15th and 20th post-infarction days the number of labeled myocyte nuclei was found to be reduced 4–6 times in atria, being changed rather a little in the perinecrotic ventricular myocardium and in the specialized myocardium of the atrioventricular system. Some possible reasons of the observed differences in the proliferative behaviour of cardiac myocytes in terms of their topology and/or specialization are discussed     

Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

α1-And β-adrenergic and muscarinic-cholinergic regulation in the spontaneous beating and Ca2+ oscillations in cultured neonatal rat cardiac myocytes

α₁-and β-adrenergic and muscarinic-cholinergic regulation in spontaneous beating and Ca²⁺ oscillations in neonatal rat cardiac myocytes at day 6 of culture was investigated. The spontaneous beating in myocytes decreased in the presence of 10 μM norepinephrine (NE). This negative chronotropic action was antagonized by prazosin. Carbachol (CCh) also showed negative chronotropic action which was inhibited by atropine. On the other hand, isoproterenol (ISP) increased the beating rate which was antagonized by propranolol. NE increased inositol phosphate formation whereas CCh and ISP did not. NE and CCh suppressed the frequency of the spontaneous Ca²⁺ oscillations but ISP increased. The present results suggest that α₁-adrenergic and muscarinic receptors regulate chronotropism to be negative whereas β-adrenoceptor regulates chronotropism to be positive in cultured neonatal rat cardiac myocytes.     

Comparison of β-endorphin immunoreactivity in hypothalamic and brainstem nuclei of normotensive and age-matched hypertensive rat strains

The concentration of β-endorphin immunoreactivity was determined in 21 hypothalamic and brainstem nuclei of Sprague-Dawley (SD) and 6 and 14 week Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SH) rats. The concentrations of β-endorphin immunoreactivity were greater in the hypothalamic nuclei than in the brainstem nuclei approximately by a factor of 5. A significant strain-age interaction was observed in the β-endorphin immunoreactivity levels in the anterior hypothalamic area, paragigantocellular reticular nucleus and locus coeruleus of age-matched SH and WKY rats in that immunoreactivity levels fell in the age period studied (6–14 weeks) in WKY rats and rose in SH rats. These biochemical differences are related in time to a growth period during which there are large increases in blood pressure in the SH rat and may thus have a pathogenetic significance.     

Alterations of cardiac and lymphocyte β-adrenoceptors in rat with chronic heart failure

The alterations of cardiac and lymphocyte β-adrenoceptors were observed in the rats with chronic heart failure produced by constriction of both abdominal aorta and renal artery. The results showed that β1-adrenocep-tor density and mRNA levels were increased, whereas these levels remained unchanged for β2 The concentration-contractile response curve for isoproterenol was shifted to the right in cardiac atrium, whereas the concentration-cAMP accumulation response curve for isoproterenol in myocardium was not changed. The number of β-adrenoceptors in blood lymphocyte was markedly reduced. Thus in the heart-failure rats the density of cardiac β-adrenoceptor was increased accompanying reduced β-adrenoceptor-mediated positive inotropic response, suggesting a post adenylate cyclase dys-function or impaired contractile components. In contrast, the alteration of β-adrenoceptor in lymphocyte is consistent with the reduced β-adrenoceptor-mediated inotropic response in heart.     

Characterization of α1-adrenoceptors which mediate chronotropy in neonatal rat cardiac myocytes

1. In the present study, we investigated the effect of culture on α₁-adrenoceptors that mediate chronotropy and on α₁-adrenergic signal transduction in neonatal rat cardiac myocytes.2. The spontaneous beating rate of neonatal rat myocytes after 3 or 7 days in culture was 37.4 ± 4.2 or 102.0 ± 4.3 beats min⁻, respectively. The α₁-adrenoceptor-mediated chronotropic effect of norepinephrine was positive at day 3 of culture. In contrast to day 3 of culture, the neonatal myocytes exhibited a negative chronotropic response to norepinephrine on day 7 of culture. Both of these effects of norepinephrine were completely abolished by prazosin.3. The affinity (K_d) and/or density (B_max) of α₁-adrenoceptors labeled with [³H]prazosin in membranes from cultured myocytes were not significantly different between day 3 and day 7 of culture.4. The expression of G_s, G_i, G_q and G_o, α-subunits in membranes from cultured myocytes was found to be significantly increased with the passage of culture time by immunoblot analysis. In contrast, no significant differences in G_β-subunit expression were observed between day 3 and day 7 of culture.5. Norepinephrine-stimulated inositol 1,4,5-trisphosphate production by radio-binding protein in neonatal myocytes after 7 days of culture was significantly higher than that of the day 3 counterpart.6. No significant changes in phospholipid and cholesterol contents in membranes from neonatal myocytes were observed with longer culture times.7. These results suggest that changes in the responsiveness to α₁-adrenergic stimulation from positive to negative chronotropy during culture of cardiac myocytes are mediated, at least in part, by functional alterations in the α₁-adrenergic signal transduction systems, including both G-protein expression and inositol 1,4,5-trisphosphate production.     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Modulation of β-receptors as adult and neonatal cardiac myocytes progress into culture

Summary Modulation of β-adrenergic receptors and their ability to respond to β-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (¹²⁵I-CYP) was used to identify β-adrenoceptors on the intact cells.¹²⁵I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of β-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to β-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional β-receptors as they progress into culture, and the expression of β-receptors is not influenced by culture substrates. This investigation was supported by grants HL 24935 and HL 33656 from the National Institutes of Health, Bethesda, MD, and Swedish Medical Research Council grant 07466.     

Extracellular proton depression of peak and late Na⁺ current in the canine left ventricle

Cardiac ischemia reduces excitability in ventricular tissue. Acidosis (one component of ischemia) affects a number of ion currents. We examined the effects of extracellular acidosis (pH 6.6) on peak and late Na(+) current (I(Na)) in canine ventricular cells. Epicardial and endocardial myocytes were isolated, and patch-clamp techniques were used to record I(Na). Action potential recordings from left ventricular wedges exposed to acidic Tyrode solution showed a widening of the QRS complex, indicating slowing of transmural conduction. In myocytes, exposure to acidic conditions resulted in a 17.3 ± 0.9% reduction in upstroke velocity. Analysis of fast I(Na) showed that current density was similar in epicardial and endocardial cells at normal pH (68.1 ± 7.0 vs. 63.2 ± 7.1 pA/pF, respectively). Extracellular acidosis reduced the fast I(Na) magnitude by 22.7% in epicardial cells and 23.1% in endocardial cells. In addition, a significant slowing of the decay (time constant) of fast I(Na) was observed at pH 6.6. Acidosis did not affect steady-state inactivation of I(Na) or recovery from inactivation. Analysis of late I(Na) during a 500-ms pulse showed that the acidosis significantly reduced late I(Na) at 250 and 500 ms into the pulse. Using action potential clamp techniques, application of an epicardial waveform resulted in a larger late I(Na) compared with when an endocardial waveform was applied to the same cell. Acidosis caused a greater decrease in late I(Na) when an epicardial waveform was applied. These results suggest acidosis reduces both peak and late I(Na) in both cell types and contributes to the depression in cardiac excitability observed under ischemic conditions.     

Alterations of cardiac and lymphocyte β- adrenoceptors in rat with chronic heart failure

The alterations of cardiac and lymphocyte β-adrenoceptors were observed in the rats with chronic heart failure produced by constriction of both abdominal aorta and renal artery. The results showed that β₁-adrenoceptor density and mRNA levels were increased, whereas these levels remained unchanged for β₂. The concentrationcontractile response curve for isoproterenol was shifted m the right in cardiac atrium, whereas the concentration-CAMP accumulation response curve for isoproterenol in myocardium was not changed. The number of β-adrenoceptom in blood lymphocyte was markedly reduced. Thus in the heart-failure rats the density of cardiac β-adrenoceptor was increased accompanying reduced β-adrenoceptormediated positive inotropic response, suggesting a post adenylate cyclase dysfunction or impaired contractile components. In contrast, the alteration of β-adrenoceptor in lymphocyte is consistent with the reduced β-adrenoceptor-mediated inotropic response in heart.     

Immunohistological study of small Rho GTPases and β-catenin during regeneration of the rat submandibular gland

Our study immunohistochemically evaluated the localization patterns of small Rho GTPases and β-catenin during regeneration of the rat submandibular gland. After 7?days of obstruction, regenerating glands were collected at days 0, 3, 7, 11 and 14 after duct release to study regeneration. RhoA was detected strongly and RhoC was detected weakly in the cytoplasm of newly formed acinar cells from day 3 to 7, and both RhoA and RhoC at the basal site and cytoplasm were detected moderately from day 11 to 14. RhoB was detected strongly and moderately in the cytoplasm of newly formed and matured acinar cells, respectively, and detected strongly in duct-like structures (DLSs) and intercalated ducts (ICDs). Rac1 was detected at the cell–cell and subcellular region, but β-catenin was not observed in newly formed acinar cells. Rac1 immunolabeling gradually reduced, and the β-catenin staining pattern became stronger. p-Rac1, a phosphorylated form of Rac1, was observed in the cytoplasm of newly formed acinar cells. At apical and subcellular region of DLSs and ICDs, Rac1 and β-catenin were detected. These findings suggest that RhoA and RhoC might be involved in the actin cytoskeleton at the basolateral site of regenerating acinar cells, and RhoB might play a unique role in regenerating acinar cells and in DLSs and ICDs. Rac1 and β-catenin at the cell–cell region might play important roles in cell–cell adhesion and the differentiation of regenerating acinar cells, as well as actin reconstruction at apical and subcellular regions of DLSs and ICDs.