Heart Membrane Preparations Research Articles

The specific binding of broxaterol, a new β 2-selective agonist, to β-adrenoceptors

The specific binding of broxaterol, a potent new orally active antiasthmatic drug. to β 1- and β 2-adrenoceptors was characterized by receptor binding studies with rat heart and lung membrane preparations. Broxaterol showed high affinity and selectivity for the β 2-component of [ 3H]dihydroalprenolol binding in both lung (58% β 2-sites. K i = 130 nM) and membranes (19% β 2-sites, K 1 = 98 nM), whereas the binding to the β 1-component was at lower affinity (42% β 1-sites K i = 4100 nM in the lung and 81% β-sites, K i = 8460 mM in the heart). The influence of temperature changes on the binding properties of broxaterol towards β-adrenoceptors was also investigated. A marked increase in the affinity of broxaterol for lung β-receptors was observed on lowering the assay temperature, whereas the affinity for heart β-receptors was little affected by temperature changes. Thermodynamic analysis of the binding data showed that the binding of broxaterol as well as isoproterenol to lung β-receptors was associated with a larga decrease in enthalpy, which correlates well with the full agonistic properties of this compound at β 2-receptors.

Parainfluenza virus type 1 reduces the affinity of agonists for muscarinic receptors in guinea-pig lung and heart

Membrane preparations of guinea-pig lung (containing multiple muscarinic receptor subtypes) and heart (containing M 2 receptors only) were incubated with either neuraminidase, parainfluenza virus (which contains neuraminidase), or virus plus 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, a neuraminidase inhibitor. None of these treatments affected [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding. In the lung and heart, carbachol displaced 0.2 nM [ 3H]QNB from two sites. After treatment with either neuraminidase or virus the high affinity site was shifted to the right, and carbachol displaced QNB from one site with low affinity in the lung. In contrast, neuraminidase or virus decreased the affinity of carbachol for both sites in the heart. The neuraminidase inhibitor completely blocked virus-induced changes in carbachol affinity in both tissues. These results suggest that parainfluenza virus decreases the affinity of agonists for some of the muscarinic receptors in the lung, and for all of the muscarinic receptors in the heart due to its neuraminidase activity, which results in removal of sialic acid. The decreased agonist affinity in the lung may be responsible for the increased vagally induced bronchoconstriction seen in viral respiratory infections.

A 180,000 molecular weight glycoprotein substrate of the insulin receptor tyrosine kinase is present in human placenta and in rat liver, muscle, heart and brain plasma membrane preparations

Cell signalling for insulin may include insulin receptor tyrosine kinase catalysing the phosphorylation of one or more cell proteins. Since temporally the insulin receptor will encounter plasma membrane protein first, we have studied the in vitro phosphorylation of purified plasma membrane preparations. Two proteins were immunoprecipitated with anti-phosphotyrosine antibody from rat liver, muscle, heart and brain membranes and from human placenta membranes: the insulin receptor (detected as a phosphorylated-β-subunit) and a 180,000 molecular weight protein (pp180). pp180 is a monomeric glycoprotein that in the absence of dithiothreitol migrated in denaturing gels like a 150,000 molecular weight protein. pp180 was a substrate for the insulin receptor: (i) receptor and pp180 phosphorylation followed a similar insulin dose-response, although fold-stimulation of autophosphorylation was greater; and (ii) removal of insulin receptors with monoclonal antibodies prevented subsequent pp180 phosphorylation. Insulin-activated receptors increased the extent, but not the rate, of pp180 phosphorylation; the increased phosphate was incorporated into tyrosine and appeared to do so in three or four of pp180's 12 tryptic phosphopeptides. Some data suggest that pp180 is the same protein in each of the tested tissues. The occurrence of pp180, an insulin receptor substrate, in plasma membranes of several insulin responsive tissues suggests that it has a role in insulin signalling.

Alterations of heart dynorphin-A in the development of spontaneously hypertensive rats

In order to investigate the pathophysiological role of heart dynorphin-A (Dyn-A) in genetic hypertension, immunoreactive (ir)-Dyn-A was measured in heart extracts of spontaneously hypertensive rats (SHR) and compared with that of age matched Wistar (WR) and Wistar Kyoto (WKY) rats. Heart ir-Dyn-A contents in 8 week-old WK (84 fmol/g tissue) were not significantly different from those of age matached WKY (109 fmol/g tissue). In control WKY, the levels of ir-Dyn-A did not significantly vary with the age (from 109 to 117 fmol/g) except in 16 week-old animals which displayed a significant increase (238 fmol/g tissue) compared to younger animals. In SHR, the heart content of ir-Dyn-A displayed a 6.5 fold increase at 8 weeks compared to age matched WKY. Older SHR showed a return of their heart ir-Dyn-A content to control (12 week-old) or below control values (16 week-old; 121 compared with 238 fmol/g tissue in WKY). Heart ir-Dyn-A in WKY and SHR eluted as a single peak on μ-vondapak HPLC, corresponding with the retention time of synthetic Dyn-A. A local function for cardiac ir-Dyn-A is suggested by the presence in heart membrane preparations of a high affinity binding site for the κ selective opioid ligand, [ 3H]U-69593, with K D of 6.4 (WKY) and 8.5 (SHR) nM and B max of 3.7 (WKY) and 3.6 (SHR) pmol/g protein. The alterations in the levels of cardiac ir-Dyn-A during the development of hypertension in SHR were analyzed in regard with the reported effects of Dyn-related peptides on heart natriuretic and sympathetic functions.

Effects of free radicals on the fluidity of myocardial membranes.

Free radicals, including superoxide anions (O2.-), hydroxyl radical (HO.), and hypohalite radical (OCl.), as well as oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO., H2O2, and OCl. + HOCl increased the fluorescence polarization (FP) and microviscosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2-fold increase in FP by O2.-. O2.- did not alter the fatty acid profiles of the membrane phospholipids. However, HO. and H2O2 reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O2.- was only insignificant. These results suggest that HO. and H2O2 decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE, and PI.

Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C

The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [ 3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg 2+ reduced MAChR number by 50%. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the efrect of ATP/Mg 2+ and increased MAChR number by 74%.

Increased content of immunoreactive Leu-enkephalin and alteration of delta-opioid receptor in hearts of spontaneously hypertensive rats.

The levels of immunoreactive Leu-enkephalin (ir-Leu-Enk) were measured in acid extract of hearts and lungs of adult normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Heart contents in ir-Leu-Enk were significantly higher in SHR rats (2.67 pmol/g) than in WKY rats (1.5 pmol/g). However, no significant change was observed in lung ir-Leu-Enk levels of these same animals (0.7-0.91 pmol/g). Reverse-phase high-performance liquid chromatography (RP-HPLC) of heart extracts indicated that the major form of ir-Leu-Enk in SHR rats coelutes with synthetic Leu-Enk and is increased by an approximate factor of 4.2-fold as compared with its eluting counterpart in the WKY chromatogram. The delta-selective opioid ligand, [3H][D-Pen2, D-Pen5]-Enk [( 3H]DPDPE) bound to two distinct receptor sites in membrane preparations of hearts from WKY rats with Kd of 1.33 and 22.8 nM and Bmax of 3.9 and 14.6 pmol/g protein, respectively. The binding with the heart membrane preparation of SHR rats displayed only the high affinity delta-binding site with a Kd of 1.53 nM and a Bmax of 6.5 pmol/g protein. These data indicate that the hypertensive state in SHR rats is manifested through a rise in the cardiac level of Leu-Enk and a selective down-regulation of its low-affinity delta-opioid receptor.

The role of zinc status in altered cardiac adenylate cyclase activity in diabetic rats.

Zinc deficiency and altered myocardial adenylate cyclase activity commonly occur in diabetes. To determine whether the zinc intake of the animal can account for the altered beta-adrenergic receptor activity in the diabetic heart, we determined the beta-adrenergic receptor number and isoproterenol-, NaF- and forskolin-stimulated adenylate cyclase activity in diabetic and control rats maintained on low, normal and high zinc diets for 3 weeks. Scatchard analysis of [125I]iodocyanopindolol binding to control heart membrane preparations revealed a binding capacity of 17.3 +/- 1.3 fmol/mg protein with a Kd of 35 +/- 1.0 pmol/l. Neither the diabetic state nor the zinc status altered these binding parameters. The isoproterenol-stimulated adenylate cyclase activity was significantly lower in diabetic rats on low zinc diets compared with controls. The NaF- (65.1 +/- 5.4 vs 60.8 +/- 6.4 pmol cAMP.mg protein-1.min-1) and forskolin-stimulated adenylate cyclase activities (161 +/- 9.3 vs 154 +/- 21.2 pmol cAMP.mg protein-1. min-1) were not significantly altered in diabetic rats. Low dietary zinc intake compared with high zinc diet significantly increased NaF- and forskolin-stimulated adenylate cyclase activity both in diabetic rats and controls. The effect of dietary zinc content on isoproterenol-stimulated adenylate cyclase was significant in control rats only. Thus zinc intake appears to be an important determinant of cardiac adenylate cyclase activity level. Additional factors peculiar to the diabetic state are involved in the modulation of beta-adrenergic responsiveness of the diabetic heart.

Effects of amiloride and its analogues on [ 3H]batrachotoxinin-A 20-α benzoate binding, [ 3H]tetracaine binding and 22Na influx

The ability of amiloride and its analogues to inhibit [ 3H]batrachotoxinin-A 20-α benzoate ([ 3H]BTX-B) and [ 3H]tetracaine binding to rat synaptosomes and to a rat heart membrane preparation was tested. Their ability to inhibit 22Na influx was determined with rat synaptosomes. 5-N-substituted analogues were generally more potent in inhibiting [ 3H]BTX-B and [ 3H]tetracaine binding than compounds substituted on the guanidine group. However, the inhibition was not competitive. Amiloride and some of its analogues were as active or more active in inhibiting [ 3H]tetracaine binding than they were in inhibiting [ 3H]BTX-B binding. 22Na influx was inhibited with the same relative potencies as [ 3H]BTX-B binding and a good correlation was found between the two inhibitions. These results show an effect of amiloride and its analogues on the voltage-sensitive Na + channels, which could partly explain the inotropic effects of these drugs.

.beta.-Adrenoceptor antagonist activity of bivalent ligands. 1. Diamide analogs of practolol.

Two series of bivalent ligands (P-X-P) containing the (R,S)-3-[(4-aminoaryl)oxy]-1-(isopropylamino)propan-2-ol pharmacophore and a connecting alpha,omega-dicarbonylpoly(methylene) [X = -OC(CH2)nCO-] or alpha,omega-N,N'-bis(carbonylmethylene) polymethylenediamine [X = -OCCH2NH(CH2)nNHCH2CO-] spanner were synthesized and evaluated for beta-adrenoceptor antagonist activity in rat heart and lung membrane preparations. The target compounds were obtained as a mixture of stereoisomers in modest yields by using a three to four step sequence beginning with N-benzylpractolol. The results from the competitive binding studies indicated that binding affinity increased by a factor of up to 160 by increasing the length of the group spanning the pharmacophore moieties. Modest increases in cardioselectivity were also obtained. The data suggest that further increases in spanner length and lipophilicity and optical resolution may improve the potential of a labeled bivalent beta 1-adrenoceptor antagonist to function as a myocardial imaging agent.

Effects of some antianginal and vasodilating drugs on sodium influx and on the binding of 3H-batrachotoxinin-A 20-alpha-benzoate and 3H-tetracaine.

The effects of antianginal drugs, especially arylalkylamines and structurally related derivatives, on 3H-batrachotoxinin-A 20-alpha-benzoate (3H-BTX-B) binding and on 3H-tetracaine binding were studied on rat synaptosomal and heart membrane preparations. The effect of the same drugs on the Na+ influx induced by protoveratrine B was studied on the rat synaptosomal preparation. Antianginal drugs tested inhibited 3H-BTX-B binding in rat synaptosomes, arylalkylamine derivatives being the most potent: IC50 values were 27 nM for flunarizine, 32 nM for prenylamine, 79 nM for cinnarizine. Similarly, these drugs were the most potent when tested in cardiac membrane preparations. All the drugs tested were very weak inhibitors of 3H-tetracaine binding (IC50 ranging from 0.01 mM to more than 1 mM) except for guanabenz, which was more potent (IC50:0.3 microM on the synaptosomal preparation). The various drugs tested inhibited the 22Na+ influx induced by protoveratrine B, with IC50 values ranging from 15 microM (prenylamine) to 110 microM (verapamil), with the exception of nifedipine which had an IC50 of more than 0.1 mM. The inhibition of 22Na+ influx correlated well with the inhibition of 3H-BTX-B binding. These findings suggest that some antianginal drugs, especially the arylalkylamines may have, in addition to their calcium antagonist activity, direct effects on sodium channels.

125Iodopindolol binding in postnatal primary myocardial cell cultures, myocyte suspensions, and whole heart membrane preparations

125Iodopindolol binding in postnatal primary myocardial cell cultures, myocyte suspensions, and whole heart membrane preparations

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. 1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. 2. [ 125I]HEAT and [ 125I]ICYP were used to determine respectively, the α- and β- adrenergic receptor binding in cardiac membrane fractions. 3. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. 4. In rat heart, the ratio of β- to α-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED 50 3.8 × 10 −7 M compared with 7.5 × 10 −7 M for the marmoset. 8. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.

Anti‐anginal arylalkylamines and sodium channels: [3H]‐batrachotoxinin‐A 20‐α‐benzoate and [3H]‐tetracaine binding

[3H]-batrachotoxinin-A 20-alpha-benzoate ([3H]-BTX-B) and [3H]-tetracaine are useful ligands for the study of sodium channels. Inhibition of their binding by various anti-anginal drugs was tested on a rat synaptosomal preparation and on a heart membrane preparation. Diphenylalkylamines and structurally related drugs inhibited [3H]-BTX-B binding in both the synaptosomal preparation and heart membrane preparation. They were almost inactive on [3H]-tetracaine binding. These results suggest that activity of arylalkylamines could be mediated by an interaction on the sodium channel.

Evidence for the presence of luteinizing hormone/human chorionic gonadotropin-binding sites in the porcine uterus.

LH/hCG-binding sites were measured in crude membrane fractions of porcine uteri. Specific high affinity and low capacity receptors for LH/hCG were found in all (n = 17) membrane preparations of myometrium but in only 5 of 17 crude membrane fractions of endometrium of porcine uteri. There was very little competition between hCG and porcine GH (pGH), bovine TSH, pFSH, and pPRL (0.5%, 0.3%, 0.2%, and less than 0.005%, respectively). Specificity of [125I]hCG binding to other tissues was determined by incubating crude membrane preparations of heart, skeletal muscle, liver, and kidney. Numbers and affinities of available LH/hCG-binding sites were characterized in all samples of myometrium and 5 endometrium membrane preparations that were positive for LH/hCG receptors. The results indicate that the number of uterine LH-binding sites in myometrium (0.66 +/- 0.17 fmol/mg) is 10 times less than the receptor capacity in porcine corpora lutea (7.46 +/- 0.54 fmol/mg) when expressed per mg protein of crude membrane preparation. However, it is approximately 60 times less when expressed per mg DNA equivalent of initial homogenate (1.31 +/- 0.28 vs. 81.18 +/- 3.64 fmol/mg, respectively). Receptor affinities of uterine LH/hCG-binding sites remained comparable to those of corpora lutea receptors (Ka = 7.8 X 10(10) M-1). Concentrations of LH/hCG-binding sites in myometrium taken from gilts in the late follicular phase of the estrous cycle (0.13 +/- 0.06 fmol/mg protein; n = 5) were significantly less (P less than 0.05 and P less than 0.01) compared to those in myometrium from luteal phase (0.85 +/- 0.22 fmol/mg protein; n = 6) or early pregnancy (1.03 +/- 0.15 fmol/mg protein; n = 6), respectively. This is probably the first evidence demonstrating specific binding of [125I]hCG by LH receptors in female uterine tissue.

Regulatory effect of calcium on 3H-dopamine binding to guinea-pig heart membrane preparations

The effects of various cations on the specific binding of dopamine to its recognition sites are described using guinea-pig heart membranes as a substrate. Only CaCl 2 provoked a dose-dependent facilitatory effect, while KCl, NaCl and MgCl 2 acted in the opposite sense. Moreover, Na 2EDTA also had an inhibitory effect on dopamine binding and completely prevented the facilitatory action of CaCl 2 on 3H-dopamine binding. This effect is partially counteracted by trifluoperazine but unaffected by verapamil. We can conclude from this that calcium seems to be essential to optimizing 3H-dopamine binding to cardiac membrane preparations.

Endogenous noradrenaline masks beta-adrenergic receptors in rat heart membranes via tight agonist binding

The tight binding of noradrenaline (NA) to β-adrenergic receptors was studied in membranes from the left ventricular myocardium of the rat. Addition of GTP (0.1 mM) to membrane preparations from control rats (NA concentration 8.5 ± 2.1 nM) caused a 4–35% (N = 8) increase (P < 0.01) in the number of specific binding sites of [ 125I](−)pindolol. In contrast, addition of GTP did not cause any changes in the number of β-adrenergic receptors in heart membranes from reserpinized animals (NA concentration < 0.1 nM). In heart membranes from reserpinized animals, preincubation with NA (followed by washing) revealed a time- and concentration-dependent decrease with a maximum of 35–40% in the [ 125I](−)pindolol-binding sites. This agonist-mediated decrease in the number of receptors was prevented if GTP was also present in the NA-preincubation medium. It is concluded that NA can undergo tight binding to β-adrenergic receptors in rat heart membranes. The heart-membrane preparations contain endogenous NA which, via tight agonist binding, is responsible for masking part of the β-adrenergic receptor population.

Calmodulin stimulates both adenosine 5'-triphosphate hydrolysis and synthesis catalyzed by a cardiac calcium ion dependent adenosinetriphosphatase.

A Ca2+-dependent ATPase purified from a rabbit heart membrane preparation was compared to the Ca2+-dependent ATPase purified from skeletal muscle sarcoplasmic reticulum. The two ATPases display an identical electrophoretic pattern and an identical Ca2+-concentration dependence. However, only the cardiac preparation exhibits a 2-3-fold activation by calmodulin. This effect is best observed when the molar concentrations of calmodulin and ATPase are equivalent and in the presence of high Ca2+ (approximately 10(-5) M) and ATP (approximately 10(-3) M) concentrations. It is demonstrated for the first time that calmodulin stimulates the rate of ATP synthesis, as revealed by an increased production of Pi and a faster ATP in equilibrium Pi exchange, as well as the rate of ATP hydrolysis. It is also demonstrated that calmodulin activation is expressed with purified and detergent-solubilized enzyme in addition to membrane-bound systems. These findings indicate that the effect of calmodulin is an acceleration of the enzyme turnover, due to direct interaction of calmodulin with the enzyme.

Early presence of phospholamban in developing chick heart

Phosphorylation of phospholamban and development of reticular Ca 2+ transport were studied in crude membrane preparations of embryonic, newborn and adult chick heart. Maximal phosphorylation of phospholamban by added catalytic subunit of cyclic AMP-dependent protein kinase increases from embryonic day 4–15. It decreases with further development. In the same membrane preparations active Ca 2+-uptake into vesicles of sarcoplasmic reticulum rises from day 4–7 and decreases then slightly until day 20. A several-fold increase in Ca 2+-transport activity occurs at the time of hatching. The data indicate separate genetic control for synthesis of phospholamban and sarcoplasmic reticulum Ca 2+-ATPase.

Interaction between thyroxine and tricyclic antidepressant drugs on cardiac neurotransmitter receptors.

The effect of chronic administration of desmethylimipramine (DMI) and iprindole on cardiac neurotransmitter receptors was studied. In addition, the influence of these drugs on the action of thyroxine (T4) on myocardial receptors was investigated. Administration of iprindole (10 mg/kg i.p., twice daily for 11 days) markedly increased the number of beta- adrenergic receptors in the heart and significantly reduced the affinity of these receptors for (-)[3H]dihydroalprenolol ([3H]DHA). Injection of T4 (500 mg/rat i.m., for 5 days) concomitantly with iprindole resulted in a lower beta-adrenergic receptor density that with iprindole treatment alone, but significantly higher than that measured in control rats. Affinity of the receptors for (-)[3H]quinuclidinyl benzilate ([3H]QNB) in heart membrane preparations of animals pretreated with T4 alone or with T4 plus iprindole was not different from control. Neither pretreatment with DMI nor with iprindole affected the density of cholinergic muscarinic receptors in the heart or their affinity for [3H]QNB. The decrease in cardiac cholinergic receptors induced by administration of T4 was not prevented by simultaneous injections of DMI. Combined administration of iprindole and T4 caused a reduction in the affinity of myocardial cholinergic muscarinic receptors, but restored the number of these receptors to normal. The results demonstrate distinct differences between DMI and iprindole in their effects on cardiac neurotransmitter receptors and in the influence of these drugs on the actions of T4 on receptors in the heart.