Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Regulation of β-adrenoceptor properties and the lipid milieu in heart muscle membranes during stress

The purpose of this study was to examine changes in fatty acyl chain composition of major cardiac phospholipids in relation to down-regulation of -adrenoceptors during various forms of stress or chronic adrenergic stimulation. Analysis of the fatty acid profile of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in sarcolemma or cardiac muscle membranes showed partial replacement of 18:2n-6 by 20:4n-6 in PC and replacement of both 18:2n-6 and 20:4n-6 by 22:6n-3 in PE during daily administration of epinephrine or norepinephrine for 7 or 15 days, respectively These changes in membrane PC and PE coincided with down-regulation or the decrease in Bmax of -adrenoceptors during adrenergic stimulation. Cardiac membrane response to other forms of stress or chronic adrenergic stimulation such as neonatal stress, restriction stress or restricted food intake was expressed in the same way, that is replacement of 18:2n-6 by 20:4n-6 in PC and replacement of 18:2n-6 and 20:4n-6 by 22:6n-3 in PE.Conclusion: Adaptation to stress includes a decrease in the density of binding sites or down-regulation of -adrenoceptors in sarcolemma synchronized with specific alterations in the fatty acyl chain composition within the membrane bilayer. The changes in the lipid milieu of the membrane may facilitate conformational changes in the transmembrane segment of the receptor forming the ligand binding sites of the -adrenoceptor.     

Chronic effects of β2 agonists on body composition and protein synthesis in the rat

Chronic treatment of rats with the ₂-adrenergic agonists clenbuterol and fenoterol over 16–19 d raised energy intake, expenditure, and body weight gain but did not affect fat or energy deposition, and body protein gain was increased by 50 and 18%, respectively. Both drugs increased the protein content and mitochondrial GDP-binding capacity of brown adipose tissue. Clenbuterol did not affect plasma insulin, growth hormone, or triiodothyronine levels, although insulin levels were reduced by fenoterol. Both drugs caused hypertrophy of skeletal muscle (gastrocnemius), and muscle protein synthesis in vivo (fractional rate) was elevated by 34 and 26% in clenbuterol and fenoteroltreated rats, respectively.     

The effects of theβ2-agonist drug clenbuterol on taurine levels in heart and other tissues in the rat

Summary The administration of a single subcutaneous dose of clenbuterol to rats altered the level of taurine in certain tissues. Taurine levels in cardiac tissue were significantly decreased 3 h after the administration of 250g/kg of clenbuterol and remained significantly depressed at 12h post-dose only returning to control values by 24h. The level of taurine in the liver increased 3 h after clenbuterol administration but was lower than the control value at 24 h post dose. Lung taurine levels were significantly lower than the control value at 12 hr post dose and remained depressed until 24h post dose. Clenbuterol caused a significant increase in taurine levels in serum and muscle at 3 and 6 hr postdosing respectively but not at other time points. Serum creatine kinase (CK), activity was slightly but significantly raised at the 12 and 24 h time point.The effects of clenbuterol on tissue taurine content were not dose-dependent over the range studied (63–500g/kg). However taurine levels in the lung were significantly reduced at all doses and in the heart were significantly lower in the treated groups at all except the lowest dose, 12h post dosing. Liver taurine levels were significantly increased at the highest dose of 500g/kg.The reduction of taurine concentrations in the heart, caused by clenbuterol, is of concern as taurine has been shown to have protective properties in many tissues especially the heart.     

Lusitropic effects of α- and β-adrenergic stimulation in amphibian heart

The effects of and -adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3×10^–8 M isoproterenol produced a positive inotropic effect, as detected by a 92±24% increase in the maximal rate of contraction and a positive lusitropic effect characterized by a decrease in both the ratio (23±5%) and the half relaxation time (t_1/2) (19±4%). The mechanical behavior induced by the -agonist was associated with an increase in the intracellular cAMP levels from control values of 173±19 to 329±28 nmol/mg wet tissue. Hearts superfused with³²P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151±13 to 240±44 pmol³²P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by -adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [y ³²P] ATP.The effect of -adrenergic stimulation on ventricular strips was studied at 30 and 22°C. At 30°C, the effects of 10^–5 to 10^–4M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22°C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action. These propranolol-resistant effects were abolished by prazosin. Our results suggest that in amphibian heart, both the inotropic and lusitropic responses to catecholamines are mainly due to a -adrenergic stimulation which predominates over the -adrenergic response. Phospholamban phosphorylation seems not to be involved in mediating the positive lusitropic effect of -adrenergic agents whereas phosphorylation of troponin 1 may play a critical role.     

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.     

Effects of β-alanine treatment on the taurine and DNA content of the rat heart and retina

Experimental evidence has suggested that the high endogenous levels of taurine found in the rat heart and retina are maintained to a large extent by transport processes out of the blood, rather than by endogenous biosynthesis. When these high levels are depleted, dysfunction ensues. In vitro studies have shown that -alanine is a good antagonist of these transport processes. The current studies were done to evaluate the feasibility of depleting heart and retinal taurine levels in vivo through treatment of adult rats either orally or with injections of -alanine. None of the treatments had significant effects on retinal taurine content; ventricular taurine was reduced in some situations, but the effects were not maintained, nor as large as with another transport antagonist. No functional changes were observed. Oral treatment with -alanine had fewer obvious side effects than injections, but all treated rats had body weights less than age-matched controls.     

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.     

Extracellular ATP activates both Ca2+- and cAMP-dependent Cl− conductances in rat epididymal cells

Activation of Ca²⁺ and cAMP-dependent Cl^– conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 m), activation of wholecell Cl^– current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl^– current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 m) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mm) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nm) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mm) or treatment of the cells with trifluoperazine (40 m), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl^– currents. No current activation by ATP was observed when cells were dialyzed with the IP₃ receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP₃-linked Ca²⁺-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca²⁺ and cAMP-dependent Cl^– conductances in epididymal cells.The authors wish to thank Mr. W.O. Fu for technical assistance. This work was supported by the Croucher Foundation, the University and Polytechnic Grants Committee.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Central effects of TNFα on thermogenesis and fever in the rat

Intracerebroventricular (icv) injection of purified recombinant human tumour necrosis factor (TNF , 4–8g) in conscious rats, produced increases in colonic temperature (1.0°C) and resting oxygen consumption (VO₂, 14%) which were maximal after 80–90 minutes. Pretreatment with propranolol (10mg/kg s.c) significantly inhibited the rise in VO₂, and prevented the increase in body temperature. Icv injection of an antagonist to corticotropin releasing factor (-helical CRF 9-41, 25 g), which prevents the pyrogenic and thermogenic actions of interleukin-1, did not influence the effects of TNF on temperature or VO₂. Injection of a fragment of TNF (113–130 amino acid sequence) did not affect body temperature or VO₂. TNF injection (icv) significantly increased brown adipose tissue (BAT)in vitro mitochondrial GDP binding, and this effect was slightly inhibited, but not prevented, by surgical denervation of the tissue, and was unaffected by pretreatment with -helical CRF 9-41. These data indicate that TNF can stimulate thermogenesis by a direct central action. The effects are largely, but not totally, dependent on the sympathetic nervous system but, unlike the thermogenic actions of interleukin they do not require release of CRF.     

Characterization of the α-cyanocinnamate binding site in rat heart mitochondria and in submitochondrial particles

The effect of pH and substrates on the binding of radiolabelled α-cyanocinnamate to mitochondria and submitochondrial particles has been investigated. It has been found that the binding is strongly influenced by the pH of the medium (it decreases on increasing the pH of the medium). The inhibition of pyruvate oxidation by this inhibitor follows the same pH dependence. The pH affects only the affinity of the α-cyanocinnamate binding site without changing their total number. A similar pH dependence has been found in inside-out submitochondrial particles where the binding sites are directly accessible. The quantitative parameters of the binding of α-cyanocinnamate in submitochondrial particles have been determined. The binding can be prevented or displaced by pyruvate and other substrates of the carrier. The turnover number for pyruvate transport in rat-heart mitochondria has been determined.     

L‐leucine transport in rat heart under normal conditions and effects of a simulated hypoxia

L-leucine plays a central role in the regulation of protein metabolism in heart and has been implicated in myocardial protection, but little is known about the relationship between these phenomena and leucine transport across the cardiac sarcolemma. In this study we used sarcolemmal vesicles and ventricular myocytes isolated from rat heart to characterise L-leucine transport under normal conditions and to investigate the effect of simulated hypoxia or inhibition of protein synthesis. The Km and Vmax of leucine uptake were 5.24+/-0.65 mM and 1.43+/-1.84 nmol min(-1) mg(-1) protein in vesicles compared to 2.17+/-0.13 mM and 1.7+/-0.76 nmol min(-1) microl(-1) intracellular space in cells. Transport was not dependent on Na+ or H+ gradients. In vesicles L-leucine uptake was increased by trans-stimulation, whilst inhibition was observed with classical system L substrates including 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) suggesting that this system mediated L-leucine transport in heart. L-Leucine uptake into isolated cardiac myocytes was inhibited after 20, 30 and 60 min of simulated hypoxia. This was not caused by reduced cell viability, although the cells underwent a rigor contracture. Inhibition of protein synthesis did not affect L-leucine transport.     

Ecto-5′-nucleotidase is expressed by pericytes and fibroblasts in the rat heart

Ecto-5-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

A quantitative cytochemical method for the measurement of β-hydroxyacyl CoA dehydrogenase activity in rat heart muscle

Summary Although cytochemical methods exist for measuring dehydrogenases that act on substrates involved in the production of chemical energy from sugars, virtually no methods exist for measuring the dehydrogenases that act on fatty acids. Yet the oxidation of fatty acids accounts for over 60% of the oxidative activity of cardiac muscle. Consequently a new quantitative cytochemical method, based on a new substrate (DL-S--hydroxybutyryl-N-acetyl cysteamine), has been developed for measuring the activity of hydroxy-acyl coenzyme A dehydrogenase, which is the penultimate step of the -oxidation of fatty acids to acetyl-coenzyme A that is used in the Krebs' cycle. Menadione or phenazine methosulphate is used as the intermediate hydrogen-acceptor, with neotetrazolium chloride as the final acceptor. The medium contains nitroprusside, ostensibly to react with any cysteamine liberated by hydrolysis of the substrate. As a control, cysteamine is substituted for the substrate. The concentrations of reactants have been optimized for cardiac muscle; the reaction is linear with thickness of the sections and with time of reaction from 15 to 60 min.     

Protective effects of exogenous β-hydroxybutyrate on paraquat toxicity in rat kidney

In this study, we demonstrated the protective effects of β-hydroxybutyrate (β-HB) against paraquat (PQ)-induced kidney injury and elucidated the underlying molecular mechanisms. By histological examination and renal dysfunction specific markers (serum BUN and creatinine) assay, β-HB could protect the PQ-induced kidney injury in rat. PQ-induced kidney injury is associated with oxidative stress, which was measured by increased lipid peroxidation (MDA) and decreased intracellular anti-oxidative abilities (SOD, CAT and GSH). β-HB pretreatment significantly attenuated that. Caspase-mediated apoptosis pathway contributed importantly to PQ toxicity, as revealed by the activation of caspase-9/-3, cleavage of PARP, and regulation of Bcl-2 and Bax, which were also effectively blocked by β-HB. Moreover, treatment of PQ strongly decreased the nuclear Nrf2 levels. However, pre-treatment with β-HB effectively suppressed this action of PQ. This may imply the important role of β-HB on Nrf2 pathway. Taken together, this study provides a novel finding that β-HB has a renoprotective ability against paraquat-induced kidney injury.