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.     

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.     

Role of α1-adrenoceptor subtypes which mediate positive chronotropy in neonatal rat cardiac myocytes

We investigated the involvement of α₁-adrenoceptor subtypes in the positive chronotropic response to norepinephrine (NE) in neonatal rat cardiac myocytes at day 3 of culture. The cardiac myocytes at day 3 of culture exhibited a dose-dependent positive chronotropic response to NE in the presence of propranolol, a β-adrenoceptor antagonist. The positive chronotropic responses to NE were completely antagonized by the α₁-adrenoceptor antagonist prazosin. The NE-induced positive chronotropic response was inhibited 68% by the α_1B-adrenoceptor antagonist, chloroethylclonidine (CEC), but partially (41%) so by the α_1A-adrenoceptor antagonist, WB4101. In the membrane fraction derived from cardiac myocytes at day 3 of culture, pretreatment with CEC decreased the Bmax of the α₁-adrenoceptor to 22% of the control value. The NE-induced positive chronotropic response was inhibited 62 and 77% by the voltage-gated Ca²⁺ channel blocker such as nifedipine and verapamil, respectively. These findings indicate (1) that cultured neonatal rat cardiac myocytes possess both α₁-adrenoceptor subtypes, i.e., α_1A and α_1B, (2) that the predominant α₁-adrenoceptor subtypes mediating NE-induced positive chronotropy in neonatal rat cardiac myocytes at day 3 of culture are α_1B-subtypes, and (3) that NE-induced positive chronotropy may be caused via voltage-gated Ca²⁺ channel activation.     

The effect of tumor necrosis factor-α inhibitor soon after hypoxia-ischemia on heart in neonatal rats

AimsPerinatal hypoxic-ischemic insult has acute and long term deleterious effects on many organs including heart. Although tumor necrosis factor alpha (TNF-α) has been reported to increase soon after hypoxia, the inhibition of this mediator has not been documented. The aim of this study was to investigate the effects of a TNF-α inhibitor (etanercept) on contractility and ultrastructure of rat heart muscles exposed to hypoxia-ischemia during neonatal period.Main methodsForty-five seven-day old rats divided into three groups were included in this study. The right carotid arteries of Saline and Etanercept groups of rats were ligated and kept in a hypoxia chamber containing 8% oxygen for 2 h. Immediately after hypoxia, while Etanercept group was administered 10 mg/kg etanercept, Saline group had only saline intraperitoneally. The carotid arteries of rats in Sham group were located without ligation and hypoxia. Mechanical activity of heart was recorded and tissue samples were examined by electron microscopy in the sixteenth week following the hypoxia-ischemia.Key findingsWhile atrial contractile force in Etanercept group was similar to Sham group, there was significant decrease in Saline group (p < 0.001). However, there was only non-significant decrease in ventricular contractility of Saline group comparing to Sham group (p > 0.05). After hypoxia-ischemia, ultrastructural degenerative changes and mitochondrial damage in atriums of Etanercept group were significantly less severe than Saline group.SignificanceThis study demonstrated that neonatal hypoxia-ischemia caused long term cardiac dysfunction and ultrastructural degenerative changes in the heart of rats. TNF-α inhibitor administration soon after hypoxia-ischemia may have heart protective effect.     

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is 10-fold below the EC₅₀ values for most purinergic receptors expressed in the heart (1.5–20 µM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP. cardiomyocytes; luciferase     

Expression and redistribution of β-catenin in the cardiac myocytes of left ventricle of spontaneously hypertensive rat

Beta-catenin is not only an adhering junction protein, but also the central player of the canonical Wnt signalling pathway. In order to investigate the roles of β-catenin in the mechanism of myocardial hypertrophy, we determined the expression and distribution of β-catenin in the cardiomyocytes of spontaneously hypertensive heart failure (SHHF) rats and age-matched Wistar-Kyoto (WKY) rats. We identified the reducing of β-catenin expression in the membrane protein fraction but increasing in the nuclear protein in the 6 and 12 month-old SHHF rats as compared with the age-matched WKY rats by Western blotting. Immunolabeling of β-catenin colocalized with cadherin at the intercalated disc sites and showed nuclear accumulation in myocytes of SHHF rats. We also revealed that the association between glycogen synthase kinase-3β and β-catenin had weakened in the 6 month-old SHHF rats as compared with the age-matched WKY rats by immunoprecipitation. These findings suggested that nuclear translocation of β-catenin might play important roles in regulating signal transduction in the decompensated hypertrophy stage.     

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.     

α1-adrenoceptors in neonatal rat cardiac myocytes: Hypoxia alters the responsiveness of α1A and α1B subtypes

We investigated the contribution of α₁-adrenoceptor subtypes to the chronotropic response to norepinephrine (NE) in cultured neonatal rat cardiac myocytes under normoxia and hypoxia. A dose-dependent negative chronotropic response was induced by NE in the presence of propranolol. Hypoxic exposure inverted the negative chronotropic response to NE to a positive one. All of these chronotropic responses were completely antagonized by prazosin. In normoxic conditions, the NE-induced negative chronotropic response was completely antagonized by WB4101 but only partially (55%) so by chloroethylclonidine (CEC). After hypoxic exposure, WB4101 partially antagonized the positive chronotropic response to NE (54%), while CEC completely suppressed the action of NE. Hypoxic exposure did not alter the number of α_1A - and α_1B-adrenoceptor subtypes as measured by [³H]prazosin binding following CEC treatment. These results indicate (1) that cultured neonatal rat cardiac myocytes contain both α₁-adrenoceptor subtypes, i.e., α_1A and α_1B, and (2) that the predominant α₁-adrenoceptor subtypes mediating NE-induced chronotropy were altered by hypoxia.     

Hexokinase–mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

The effect of PGP on β-hexosaminidase and histamine secretion in rat peritoneal mast cells in vitro

The investigation of the effect of peptide prolyl-glycyl-proline (PGP) on β-hexosaminidase and histamine secretion by mast cells in primary culture has shown that incubation of mast cells with PGP (6 × 10⁻⁵ M) before their activation by synacten significantly decreased the amount of secreted histamine and β-hexosaminidase in comparison with the action of synacten only. The peptide in investigated concentration had no influence on the level of spontaneous secretion. Incubation of cells with PGP did not prevent their activation by compound 48/80. Therefore, PGP can have a direct effect on isolated rat mast cells in vitro and diminish their secretory activity under activation by synacten.