Inotropic, electrophysiological and biochemical responses to histamine in rabbit papillary muscles: evidence for coexistence of H1- and H2-receptors.

This study was performed to determine the subtypes of histamine receptors that are involved in the electrophysiological, inotropic and biochemical responses to histamine in isolated rabbit papillary muscles. Histamine increased force of contraction and shortened action potential duration (APD) in a concentration-dependent manner. The former was antagonized by chlorpheniramine, a H1-antagonist, whereas the latter was blocked by cimetidine, a H2-antagonist. However, even when H1-receptors were blocked entirely by chlorpheniramine, histamine still produced a positive inotropic effect, an effect which was antagonized by cimetidine. On the other hand, when H2-receptors were eliminated by cimetidine, histamine caused a H1-receptor mediated APD prolongation. Carbachol attenuated the decrease in APD but not the increase in force of contraction caused by histamine. Cyclic AMP and cyclic GMP levels both were elevated significantly by histamine. The increase in cyclic AMP level induced by histamine was abolished by cimetidine, but not altered by chlorpheniramine, whereas the converse was true for the increase in cyclic GMP level. Additionally, histamine produced a significant stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation, although its extent was far less than that produced by carbachol. The phosphoinositide response to histamine was blocked by chlorpheniramine. These data suggest that H1- and H2-receptors coexist in rabbit ventricles. Stimulation of H1- and H2-receptors with histamine independently sets off the biochemical responses linked specifically to the respective subtypes of histamine receptors. On the other hand, the inotropic and electrophysiological responses to histamine are governed predominantly by H1- and H2-receptors, respectively, and this results in an apparent restriction of the expression of the responses mediated by another subtype.

Evidence against spare or uncoupled beta-adrenoceptors in the human heart.

It is well established that increasing degrees of heart failure are accompanied by a reduced density of myocardial beta-adrenoceptors. It is unclear, however, whether all beta-adrenoceptors in the cardiac cell membrane are coupled to the effector system or whether "spare receptors" or "uncoupled" beta-adrenoceptors also exist. To investigate this, we measured the density of beta-adrenoceptors and the positive inotropic response to isoprenaline in preparations from the same human hearts. The myocardium from nonfailing hearts had significantly (p less than 0.01) higher numbers of beta-adrenoceptors (104 +/- 7 fmol/mg protein) compared with tissue from moderately (mitral valve disease, New York Heart Association [NYHA] class II to III, 60 +/- 2.8 fmol/mg protein) and terminally (dilated cardiomyopathy, NYHA class IV, 35 +/- 2.7 fmol/mg protein) failing human hearts. The KD values of the drug-receptor complexes did not differ within the different patient groups. There was a linear relationship (r = 0.97) between the beta-adrenoceptor density measured and the maximally obtainable positive inotropic effect elicited by isoprenaline in the three groups tested. Thus there seem to be no spare beta-adrenoceptors, that is, receptors not required for the production of the maximal inotropic response in the left ventricular human myocardium, and there are no uncoupled beta-adrenoceptors. The beta-adrenoceptors associated with the plasma membrane (marker: 3H-ouabain binding sites) remained functionally active. In addition, these results indicate that either there is no amplifier system behind the receptor level or it remains unchanged in the failing left ventricular human myocardium under the conditions tested.

A comparative study of three methods for intracellular loading of the calcium indicator aequorin in ferret papillary muscles.

We compared the results of loading the bioluminescent Ca++ indicator aequorin by standard microinjection techniques to those obtained with two new chemical approaches to loading that utilize low concentrations of Ca++ chelator; i.e., 1) Immersion and 2) Macroinjection. After loading with the immersion and macroinjection methods, twitch tension returned to pre-load values indicating lack of damage to the muscles. The aequorin signals obtained with all three methods were similar and converted to similar quantitative values for [Ca++]i. Our data suggest that chemical loading (in particular macroinjection) may be preferable to microinjection, particularly in muscles with increased connective tissue content.

Inhalation anaesthetics decrease calcium content of cardiac sarcoplasmic reticulum.

In a Langendorff perfused rat heart model, caffeine was shown to have a negative inotropic effect similar to that of inhalation anaesthetics. Both inhalation anaesthetics and caffeine decreased calcium content of left ventricular muscle as measured by atomic absorption spectroscopy. Halothane 2.8% decreased left ventricular pressure by 75.4 (SEM 4.4)% and decreased the calcium content by 30%. Enflurane 5.1% decreased the pressure by 72.4 (6.0)% and decreased calcium content by 31%. Both halothane and enflurane decreased the calcium content of sarcoplasmic reticulum (SR) as measured by caffeine-induced calcium release. There was a high degree of correlation between the negative inotropic effect of inhalation anaesthetics and the decrease in calcium content of the SR (r = 0.95 for halothane, r = 0.91 for enflurane). These data suggest that inhalation anaesthetics increase the calcium permeability of cardiac SR, thereby decreasing the calcium content of SR, resulting in the negative inotropic effect.

Intracellular distribution of adrenoceptors in the failing human myocardium.

Although it is increasingly recognized that the density of cardiac membrane-bound beta adrenoceptors declines in heart failure, the mechanisms involved are unclear. Furthermore, it is not known whether cardiac alpha-1 adrenoceptors are similarly affected. Inasmuch as agonist-induced desensitization results in translocation of adrenoceptors from the plasma membrane to an intracellular vesicular fraction, we determined the intracellular distribution of cardiac adrenoceptors in two groups: group 1 (n = 9) consisted of papillary muscles from patients with mild-to-moderate heart failure undergoing valve replacement, and group 2 (n = 8) consisted of severely failing hearts removed during orthotopic cardiac transplantation. The density of cardiac beta adrenoceptors was lower in membranes from group 2 (17.8 +/- 3.3 fmol/mg protein vs 27.8 +/- 3.7 fmol/mg in group 1; (p less than 0.01), and the percentage of beta receptors recovered in the vesicular fraction was higher in group 2 (47.1 +/- 3.3% vs 36.8 +/- 5.0% in group 1; p less than 0.01). In group 1 but not group 2 there was a significant inverse correlation (r = -0.87; p less than 0.001) between the density of membrane-bound beta receptors and the percentage of beta receptors recovered in the vesicular fraction. Alpha-1 adrenoceptors were lower in both membrane and vesicular fraction of group 2 compared to group 1; in group 2 but not group 1 there was a significant negative correlation between the density of membrane-bound alpha-1 adrenoceptors and the percentage of alpha-1 receptors in the vesicular fraction (r = -0.8; p less than 0.01). These results suggest that the regulation of alpha-1 and beta adrenoceptors differs in the failing myocardium. Furthermore, agonist-induced desensitization may play a predominant role only in mild-to-moderate heart failure.

[Effect of liposomes on the adrenoreactivity of the papillary muscle]. / Vliianie liposom na adrenoreaktivnost' papilliarnoi myshtsy serdtsa.

Unloaded phosphatidylcholine-cholesterol liposomes enhanced the affinity of adrenoreactive system of the rat heart muscle with the ligands and altered the degree of adrenoreceptors' negative cooperativity. The radioligand analysis with 3H-dihydroalprenolol revealed that the liposomes altered kinetic properties of sarcolemma's receptors as revealed by the enhancement of affinity with the marker, decreasing of maximal specific binding and augmentation of the degree of receptors' negative cooperativity.

A comparative study of chicken ventricular and slow skeletal myosin light chains.

1. Comparison of ventricular and slow skeletal myosin light chains was performed using high performance liquid chromatography (HPLC), peptide mapping of electrophoretically purified proteins and immunoblots. 2. Both in rabbit and in chicken ventricular and slow skeletal myosin light chain composition was identical.

Immunocytochemical studies of endothelial cells in vivo. II. Chicken aortic and capillary endothelial cells exhibit different cell surface distributions of the integrin complex.

Frozen sections of chicken tissues containing aortic and capillary endothelial cells were immunolabeled with two mouse monoclonal antibodies directed to different epitopes of the chicken integrin beta-chain. Integrin is an integral membrane protein complex that is believed to mediate a transmembrane linkage between the extracellular matrix and the actin cytoskeleton. In immunofluorescence experiments with semi-thin frozen sections, the aortic endothelial cells were labeled for integrin all around their surfaces, whereas capillary endothelial cells of heart and kidney were labeled only on their basal surfaces. At the immunofluorescence level of resolution, the distribution of integrin appeared to be correlated with that of F-actin in double-labeling experiments with NBD-phallacidin. These different distributions of integrin on the two types of endothelial cells were definitively confirmed by immunoelectron microscopic labeling with the monoclonal antibodies on ultra-thin frozen sections. These results therefore indicate that the luminal surfaces, as well as the underlying cytoskeleton of capillary endothelial cells, are significantly different in structure from those of aortic endothelial cells. These differences may reflect the vastly different hemodynamic stress to which the two types of endothelial cells are subjected, and in addition may mediate different adhesion properties of the luminal surfaces of the two cell types.

Ceftizoxime level in the myocardium (right atrial muscle and mitral papillary muscle) during open heart surgery.

We determined the level of sodium ceftizoxime (CZX) in the right atrium and mitral papillary muscle of 22 adults and 6 children undergoing open-heart surgery, 60 and 120 minutes after intravenous administration of this drug at the dosages of 2 grams for adults and 1 gram for children. The CZX level in the right atrial muscle after 60 minutes was 37.0 micrograms/g in adults and 51.0 micrograms/g in children. The CZX level in the papillary muscle of the mitral valve, determined at 120 minutes was 16.9 micrograms/g. In the present study, we measured the level of the antibiotic CZX in the myocardial tissue during open-heart surgery. The purpose of this was to determine the quantity in which the antibiotic is taken into the myocardial tissue.

Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: decreased beta-adrenergic responsiveness and evidence of increased intracellular calcium.

The heart has not been regarded as a major target organ of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity notwithstanding that lethal cardiac dysfunction can occur in the absence of histopathological changes. To assess possible TCDD cardiotoxicity, we studied the effect of TCDD five days after treatment (10 micrograms/kg of body weight; single dose given i.p. in corn oil) on the contractility of guinea pig right ventricular papillary muscle. Controls were treated with corn oil. TCDD treatment significantly decreased beta-adrenergic responsiveness. In papillary muscles from TCDD-treated guinea pigs, the positive inotropic effect of isoproterenol (0.03-0.3 microM) was decreased by a mean of 65% (P less than 0.001), and the enhancement in the velocity of relaxation was 60% less than in the controls (P less than 0.05). On the other hand, TCDD treatment did not alter the positive inotropic effect of lower concentrations of isoproterenol (0.1-10 nM). After TCDD, responsiveness to low-frequency stimulation (0.1 and 0.25 Hz) was enhanced, responsiveness to increases in extracellular Ca2+ concentration was attenuated, and isoproterenol-elicited aftercontractions in K+-depolarized preparations were increased in magnitude. Collectively, the latter findings suggest that in addition to decreasing beta-adrenergic responsiveness, TCDD increases the intracellular Ca2+ concentration in papillary muscle. Finally, slow Ca2+ channels were not blocked after TCDD treatment, inasmuch as isoproterenol restored contractility equally effectively in K+-depolarized TCDD-treated and control papillary muscles. Our findings indicate that TCDD causes a specific pattern of cardiac dysfunction in a mammalian species, selectively augmenting or decreasing different cardiac responses. The cardiac changes are consistent with reported membrane effects of TCDD; further, they suggest that the heart may be a major target organ for TCDD toxicity.

Effects of enoximone and isobutylmethylxanthine on contractile tension and cyclic nucleotide levels in isolated blood-perfused dog papillary muscle.

The effects of enoximone (MDL 17,043) and 3-isobutyl-1-methylxanthine (IBMX) on developed tension, blood flow, and cyclic nucleotide levels were investigated in the isolated blood-perfused dog papillary muscle preparation. Intraarterial infusion of each agent, over 15 s, in doses ranging from 0.001 to 3 mg, produced a dose-dependent positive inotropic effect accompanied by increases in rate of contraction, rate of relaxation, and blood flow. The dose-response relationships for enoximone were always less steep than those for IBMX. Enoximone did not enhance automaticity at any dose, whereas the higher doses of IBMX (0.3, 1, and 3 mg) enhanced automaticity and produced arrhythmic preparations. Both agents produced increases in cyclic AMP during the peak positive inotropic effect (45 s); however, only IBMX produced an increase in cyclic GMP. The increase in cyclic AMP produced by enoximone lagged behind tension development by at least 15 s, whereas the increases in cyclic nucleotides produced by IBMX occurred concurrently with the development of the positive inotropic effect. The evaluation in cyclic AMP produced by enoximone is consistent with the reported property of this agent to inhibit specifically and selectively the particulate high-affinity cyclic AMP phosphodiesterase from dog heart, and supports a cyclic AMP-dependent mechanism of positive inotropism for enoximone.

Negative inotropic effects of halothane, enflurane, and isoflurane in papillary muscles from diabetic rats.

Myocardial contractile responses to halothane, enflurane, and isoflurane were studied in papillary muscles isolated from streptozotocin-induced diabetic rats and in those from age-matched control rats. The developed tension in diabetic papillary muscles electrically paced at a constant rate was not different from that in control muscles, but both the time to peak tension and the relaxation time of the isometric contraction were significantly prolonged in diabetic muscles. Halothane, enflurane, and isoflurane all produced negative inotropic effects in both diabetic and control muscles. However, the negative inotropic effects were significantly less in the muscles from diabetic rats than in the muscles from control rats throughout the range of concentrations. In muscles from both the diabetic and control rats, the negative inotropic effects of these anesthetics were accompanied by decreases in the time to peak tension and in relaxation time. The results suggest that the myocardium from the diabetic rat is less sensitive to the negative inotropic action of volatile anesthetics than is the myocardium of normal hearts.

Localization artefacts in ultracytochemical ion precipitation reactions.

The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca2+--ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg2+--ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers). As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.

Muscle fluorometry: a determination of the depth of penetration.

Fluorometric recordings of NADH (nicotinamide adenine dinucleotide) were made on rabbit papillary muscles. The specimens were placed between the UV light source and a small window of the detection stage. As the muscle was moved over the window in a transverse direction, simultaneous measurements could be taken of transmitted UV light and fluorescent light for various thicknesses of tissue. It is concluded that a muscle thickness of 0.65 mm is optimal before absorption of the incident light decreases the fluorescence signal.

Effects of non-toxic doses of ouabain on sodium, potassium, calcium distribution in guinea pig papillary muscle. Electronprobe microanalysis.

The mechanisms and the cellular structures which are definitely involved in the accumulation and release of calcium in heart muscle treated with cardiac glycosides are not yet known. The distribution of sodium, potassium and calcium in small papillary muscles of the guinea pig right ventricle was examined with the aid of energy dispersive x-ray microanalysis and cryotechniques. The primary aim of the present study was twofold: firstly, to determine whether an increase in intracellular sodium concentration is detectable in muscles showing positive inotropy resulting from treatment with non-toxic doses of ouabain; and secondly, whether at the end of diastole cellular stores are detectable accumulating Ca which could be responsible for the pronounced contraction which normally would follow. Analyses on interstitium, cell membrane, sarcomeres, Z-lines and mitochondria of 7 muscles strips treated with non-toxic doses of ouabain and frozen at the end of diastole showed the following: sodium concentration in the sarcoplasm was significantly higher than over the mitochondria; it was also higher than over the sarcoplasm of non-treated muscles frozen at the end of diastole. High calcium concentrations were also measured over the cell membrane. These calcium concentrations were higher than that detected in sarcomeres, Z-lines and mitochondria. Over the sarcomeres, the calcium concentration was higher than in experiments on non-treated muscles which were also frozen at the end of diastole. Mitochondria did not accumulate any detectable concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative x-ray microanalysis of the elemental composition of individual myocytes in hypoxic rabbit myocardium.

The purpose of this study was to use energy dispersive x-ray microanalysis to test the following hypotheses: (1) that individual myocytes may exhibit important variation in the severity of alterations in intracellular ionic homeostasis in response to hypoxia and (2) that hypoxic myocytes may accumulate certain elements in quantities sufficient to impair organellar function and structure. A rabbit interventricular septal preparation with attached small right ventricular papillary muscles was used to obtain control oxygenated myocardium (six papillary muscles) and myocardium rendered hypoxic for 1 to 1 1/2 hr (n = 8). Myocardium not perfused in vitro was also obtained (n = 4). Microanalysis was performed on freeze-dried thin sections of unfixed papillary muscles. Elemental concentrations were determined by suitable cryostandards of elements of interest. Sarcoplasm and mitochondria of most hypoxic myocytes exhibited significant alterations of diffusible elements, including increases in sodium and chloride and decreases in potassium, phosphorus, and magnesium, without major change in calcium. The most severely altered myocytes showed evidence of calcium overloading manifested by markedly increased levels of mitochondrial calcium and phosphorus associated with formation of electron-dense mitochondrial inclusions. Levels of mitochondrial calcium and phosphorus exceeded those previously found to markedly impair the function and structure of isolated mitochondria. Thus x-ray microanalysis of unfixed cryosections provides direct measurements of subcellular alterations in elemental composition of individual myocytes in injured myocardium and demonstrates that both calcium and phosphorus accumulate in mitochondria of severely injured myocytes in concentrations sufficient to exert deleterious effects on these organelles.

The time course and characterization of myocardial hemorrhage after coronary reperfusion in the anesthetized dog.

We quantitated hemorrhage associated with reperfusion after varying periods of myocardial ischemia and examined the flow characteristics that accompany reperfusion hemorrhage. Anesthetized dogs were reperfused after 2, 6 or 24 hours of circumflex occlusion. A control group underwent coronary occlusion without reperfusion. Radioactive microspheres were injected before and 5 minutes and 24 hours after reperfusion. The papillary muscles were analyzed for hemoglobin content, flow during myocardial ischemia and flow early and 24 hours after reperfusion. Myocardial creatine kinase activity was assayed to determine the severity of myocardial necrosis in the papillary muscles. Hemorrhage into the posterior papillary muscle was dependent upon the duration of coronary artery occlusion. Posterior papillary hemoglobin averaged 14 mg/g in the 2-hour group, 28 mg/g in the 6-hour group and 36 mg/g in the group reperfused 24 hours after occlusion, compared with 8.7 mg/g in the control group. Myocardial hemorrhage was associated with severe depression in myocardial CK and marked depression in flow to the ischemic area (i.e., collateral flow) during the occlusion. Early reflow averaged 112 ml/min/100 g in the 2-hour group, 61 ml/min/100 g in the 6-hour group and only 5.8 ml/min/100 g in the 24-hour group. Therefore, myocardial hemorrhage induced by reperfusion of the acutely ischemic myocardium is associated with severe ischemia during occlusion and severe myocardial necrosis, but does not depend upon the magnitude of early reflow. Myocardial hemorrhage may occur even though initial reflow values are markedly decreased.

Determination of adenine nucleotides and inosine in human myocard by ion-pair reversed-phase high-performance liquid chromatography.

An isocratic high-performance liquid chromatographic system for the quantitation of AMP, ADP and ATP is presented. The separations were achieved at room temperature by reversed-phase chromatography (Supelcosil LC-18). The standard solvent was 220 mM potassium phosphate, pH 6.9, 1% (v/v) methanol and 0.3 mM tetrabutylammonium hydrogen sulphate. A selective retention of the adenine nucleotides as a group relative to the mono-, di- and triphosphates of guanosine, uridine and cytidine was observed under these experimental conditions. The adopted procedure was applied to the separation of adenine nucleotides in biological extracts, i.e., human myocard. The adenine nucleotides in an extract of myocard were quantitated in less than 20 min. Only 5-10 mg (wet weight) of myocard were needed in order to determine the energy charge of a myocardial sample. Also inosine was easily quantitated in this liquid chromatographic system.