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A Shainberg

Publications and source records attributed to A Shainberg.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of muscarinic cholinergic receptors in intact myocardial cells in vitro.

Muscarinic acetylcholine receptors (mAChR) were studied on heart cells grown in culture by the radioligand binding technique. We used [3H]n-methyl-scopolamine to monitor the level of receptors on intact cardiocytes. The number of mAChR was very low during the first days in culture (23 fmol/dish). It increased gradually until it reached a plateau on the 4th day (180 fmol/dish), where it remained for 1-2 weeks. To determine whether contractile activity affected the level or affinity of mAChR, the cardiocytes were exposed to agents that stimulate or arrest the heart beat. Treatment with triiodothyronine (T3, 10-90 nM) for 48 hr caused a reduction in the level of the receptors by 20-30% without changing significantly the affinity of the receptors. Similarly, electrical stimulation caused a reduction in the level of the receptors by 30-40%, without a significant influence on creatine kinase activity. When the myocardial cells were treated with Ca-channel blocker such as metoxyverapamil (D600) (10-30 micrograms/mL) or diltiazem (10-25 micrograms/mL) the level of the receptors was also reduced by 30-40%. The reduction in the receptor binding sites was accompanied by an increase in Kd from 0.8 to 3.2 nM in D600-treated cells, whereas there was no significant change in the radioligand affinity after application of diltiazem. Treatment with D600 or T3 together with cycloheximide showed that under these experimental conditions the rate of receptor degradation was accelerated. The half-life of the receptors in the control was 27 hr, whereas the half-lives of T3 and D600 were 15 and 18 hr, respectively. It is concluded that regulation of the amount of cholinergic receptors occurs at the level of receptor breakdown, and simple linkage does not exist between the rate of cardiac contractions and the number of mAChR.

Animals↗
PubMed1992

Effects of calcium and calcium-channel blocker methoxyverapamil on the beta-adrenoceptors in myocardial cells in vitro.

The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the beta-adrenoceptors was investigated on heart cells grown in culture, using [3H]CGP-12177 as a radioligand. Treatment with D600 (20 micrograms/mL) for 24 hr caused a decrease of 30% in the [3H]CGP-12177 binding sites. Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases. The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment. These results indicate that the action of D600 on the beta-adrenoceptors is a competitive inhibition of the [3H]CGP-12177 binding sites. We investigated the effect of Ca2+ in the growth medium on the level of beta-adrenoceptors. Heart cells grown for 24 hr in Ca(2+)-free medium showed a decrease of 36% in the [3H]CGP-12177 binding sites without changing the dissociation constant. This decrease is probably a result of reduction in synthesis of the receptors. The level of receptors returned to control values following replenishment with normal growth medium. These results show that calcium is essential for the development of the beta-adrenoceptors in heart cells in vitro.

Animals↗
PubMed1992

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers.

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulate in vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.

Animals↗
PubMed1991

Inhibition of Ca2+ accumulation in isolated sarcoplasmic reticulum by thyroid hormones.

Thyroid hormones inhibit Ca2+ accumulation and ATPase activity of isolated sarcoplasmic reticulum vesicles. Half-maximal inhibition was obtained by about 2.5 microM. The ATP hydrolysis activity of the purified (Ca2+ + Mg2+)-ATPase or of the SR vesicles, in the presence of the Ca2+ ionophore A23187, is not inhibited by T3 or T4. Modification of T3 or T4 in the ring portion, but not in the amino portion, of the molecules results in T4 and T3 analogues which are unable to inhibit Ca2+ accumulation. T3 and T4 have no significant effect on various partial reactions of the transport cycle such as: the binding of ATP and Ca2+, or ADP-ATP exchange and E-P formation from ATP, but they inhibit the E-P formation from inorganic phosphate (Pi) and ATP-Pi exchange. The inhibition of both Ca2+ accumulation and ATPase activity by T3 or T4 is increased in the presence of Pi. Binding sites for [125I]T3 and for [125I]T4 in SR proteins were demonstrated using either equilibrium dialysis or gel overlay techniques. The results suggest that the thyroid hormones inhibit the ATP-dependent Ca2+ accumulation, probably by inhibiting the transport of anions which act as the Ca2+ precipitating anion.

Animals↗
PubMed1991

Characterization of sarcoplasmic reticulum in skinned heart muscle cultures.

The plasma membranes of rat heart muscle, grown in cell culture, were made permeable with saponin in a Ca-free solution. The cells were then supplied with a medium resembling the cytosol, and the adenosine triphosphate (ATP)-dependent Ca2+ sequestration was measured in the presence of oxalate. The nonmitochondrial component accounts for about 50% of the total Ca2+ uptake. The nonmitochondrial accumulation of Ca2+ within myocardial cells was found to be reversible by addition of the Ca2+ ionophore A23187. On the other hand, the Ca2+ antagonist D-600 (50 microM) had almost no effect on Ca2+ accumulation. Caffeine reduced Ca2+ accumulation in the skinned cardiomyocytes in a concentration-dependent manner. In addition, the anticalmodulin drug trifluoperazine (TFP) reduced Ca2+ accumulation in the skinned cells. Because of the analogy between nonmitochondrial ATP-dependent Ca2+ accumulation and the sarcoplasmic reticulum (SR) function with regard to the influence of various agents, it is assumed that we actually measure Ca2+ accumulation in the SR. The rate of Ca2+ accumulation into the SR measured during the development of the cardiomyocytes in culture shows an almost linear increase as a function of culture age. Amiodarone, a potent antiarrhythmic agent, and its metabolite, desethylamiodarone, inhibited Ca2+ accumulation into SR, which may explain their therapeutic effect.

Amiodarone↗
PubMed1990

Characterization of beta-adrenoceptors on rat skeletal muscle cells grown in vitro.

The binding properties of an hydrophilic beta-adrenergic receptor radioligand, (-)[3H](4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazolo-2-one ); ([3H]CGP-12177), were investigated in rat skeletal muscle cells in culture. The binding of [3H]CGP-12177 at 25 degrees was saturable, reversible and of high affinity (Kd = 1.3 +/- 0.3 nM). The maximal number of [3H]CGP-12177 binding sites was 30.6 +/- 3.2 fmol/dish (34 +/- 3.5 fmol/mg protein). beta-Adrenergic agonists and antagonists inhibited [3H]CGP-12177 binding. The competing ligand inhibition binding is a typical one for beta 2-adrenoceptors. The increase in beta-adrenoceptors was independent of cell fusion. Amiodarone (10(-5) M) decreased the beta-adrenoceptor number in skeletal muscle cells differentiated in vitro by 48%, while the affinity for [3H]CGP-12177 was not affected.

Adrenergic beta-Agonists↗
PubMed1990

High endocytotic and lysosomal activities in segments of rat myotubes differentiated in vitro.

Endocytosis and the lysosome system have been studied in rat myotubes differentiated in vitro. Horseradish peroxidase was used as marker for endocytosis and was found to accumulate unevenly in the myotubes. Small segments of myotubes display very high endocytotic activity. Similar segments contained numerous lysosomes, as seen by the accumulation of neutral red or histochemical staining for acid phosphatase. The segments also contained accumulations of acetylcholine receptors as determined by binding of tetramethyl rhodamine-labelled alpha-bungarotoxin. Unstained segments in living cultures could be recognized by phase-contrast microscopy since they often appeared somewhat dilated and were not as well spread on the culture surface as the main parts of the myotubes. Ultrastructurally, the segments contained an intensely proliferating tubular system in communication with the extracellular space, which therefore probably represents the developing transverse tubular system. The segments also contained endocytosed marker within large phagosomes. Contractile filaments occurred in the segments but were frequently less well-organized than in other parts of the myotubes. The described characteristics of the segments in rat myotubes differentiated in vitro bear resemblance to some of the characteristics of the denervated endplate region of adult muscle.

Animals↗
PubMed1990

Activation of the interferon system during myogenesis in vitro.

Differentiation of skeletal muscle involves withdrawal of myoblasts from cell replication, fusion to form multinucleated myotubes, coordinate appearance of a variety of muscle-specific proteins and the disappearance of a set of other proteins responsible for cell growth. The possible activation of the interferon (IFN) system in this process was studied. Thus, the activity of two IFN-induced enzymes known to be part of the system-(2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA-activated protein kinase as well as the expression of 2-5A synthetase coding genes were examined during myogenesis. It is demonstrated that the activity of the enzymes is transiently increased in cultured myoblasts, reaching a peak activity on the 3rd day in culture and then declining to a basal level. This peak activity precedes both cell fusion and the appearance of muscle-specific proteins--acetylcholine receptors (AChR) and creatine kinase. The same kinetics of 2-5A synthetase activity was evident in myoblasts from chick, rat or mouse origin. The enzymatic product appears to be primarily the trimer form of 2-5A, rather than a set of oligomers observed in enzymatic reactions performed on IFN-treated cells, including muscle cultures. The kinetics of 2-5A synthetase gene expression revealed that the largest amount of specific RNA transcripts appeared on the 1st day after seeding, followed by a reduction thereafter. In addition, a decrease was also observed in expression of c-myc, a cell-growth-associated protooncogene. However, an increase towards the 2nd day of both AChR and myosin light chain gene expression was evident, indicating selective regulation of gene expression during myogenesis.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Thyroxine-induced redistribution of creatine kinase isoenzymes in rat cardiomyocyte cultures.

The present study demonstrates a change occurring in the creatine-kinase isoenzyme profile of cardiomyocyte cultures induced by a chronic administration of excessive amounts of thyroid hormones (TH). This change is manifested by an increased level of the CK-BB isoenzyme, generally at the expense of CK-MM isoenzyme. The elevation of CK-BB is probably a result of a specific effect of TH through activation of gene expression, rather than a contribution of an increased number of non-myocardial cells. The implications of these results in the diagnosis of heart failures are discussed.

Animals↗
PubMed1989

Characterization of sarcoplasmic reticulum in skinned muscle cultures.

The plasma membranes of chick or rat skeletal muscles, grown in cell culture, were made permeable with saponin in a solution lacking calcium. The cells were then supplied with a medium resembling the cytosol and the ATP-dependent Ca2+ sequestration was performed. Based on the low concentration of free Ca2+ in the medium (below 5 microM), the presence of mitochondrial inhibitors and the effect of drugs that interfere with sarcoplasmic reticulum (SR) function, we assume that the measured Ca2+ accumulation expresses SR function on the saponin-treated myotubes. The development of the SR in muscle cultures is augmented as myogenesis proceeds and depends on its occurrence. Whereas creatine kinase activity is elevated immediately following cell fusion, there is a delay of at least 1 day between myoblast fusion and the increase in Ca2+ accumulation in the SR. Thyroxine or triiodothyronine caused an inhibition of Ca2+ accumulation in rat or chick muscle cultures. This inhibition could explain some of the muscle abnormalities caused by excess of thyroid hormones. A comparison was made between a white-type (fast) and heterogeneous muscle, differentiated in cell culture. There was no significant difference in SR function, indicating the important role of innervation in specifying the properties of muscle fiber types.

Animals↗
PubMed1987

Pathways of adenine nucleotide catabolism in primary rat muscle cultures.

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2'-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined in cell extracts. The results demonstrate that under physiological conditions, there is a small but significant flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5'-nucleotidase (EC 3.1.3.5), reflecting the markedly higher Vmax/Km ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher Vmax/Km ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.

5'-Nucleotidase↗
PubMed1987

Synergistic inhibition by trifluoperazine and phencyclidine of carbamylcholine-induced cation influx in muscle cultures.

The effect of trifluoperazine (TFP) and phencyclidine (PCP) on acetylcholine receptor (AChR) function was studied in rat myotubes differentiated in vitro. While both drugs exerted an inhibitory effect on carbamylcholine (CCh)-induced Na+ or Ca2+ flux (I50 = 5-9 microM), alpha-bungarotoxin (alpha-Bgt) binding was not affected. The inhibitory effect of both drugs was independent of CCh concentration. The mutual inhibitory effect of TFP and PCP on Ca2+ influx was analyzed using three alternative models of interaction between the two drugs: competitive, additive and synergistic inhibition models. Our results are in accord with a synergistic interaction between the drugs probably not through desensitization. This synergistic interaction between the drugs provides a biochemical rationale to the phenothiazine contraindication in the treatment of PCP psychosis.

Animals↗
PubMed1987

Role of Na-K ATPase in regulation of resting membrane potential of cultured rat skeletal myotubes.

The role of Na-K ATPase in the determination of resting membrane potential (Em) as a function of extracellular K ion concentration was investigated in cultured rat myotubes. The Em of control myotubes at 37 degrees C varied as a function of (K+)0 with a slope of about 58-60 mV per ten-fold change in (K+)0. Inhibition of the Na-K pump with ouabain or by reduced temperature revealed that this relation consists of two components. One, between (K+)0 of 10 and 100 mM, remains unchanged by alterations in enzyme activity; The second, between (K+)0 of 1 and 10 mM, is related to the amount of Na-K pump activity, the slope decreasing as pump activity decreases. Indeed, with complete inhibition of the Na-K pump, Em does not change over the range of (K+)0 1 to 10 mM. Measurements of 86Rb efflux and input resistance of individual myotubes showed that membrane permeability does not change as (K+)0 increases from 1 to 10 mM but increases as (K+)0 increases further. Monensin, which increases Na ion permeability, increases Em at values of external K+ below 10 mM, and is without effect at higher values of K+ concentration. The effect of monensin is blocked by ouabain. Tetrodotoxin, which blocks voltage-dependent Na+ channels, decreases Em at low (2-10 mM) K+. We conclude that changes in Em as a function of extracellular K+ concentration in the physiological range are not adequately explained by the diffusion potential hypothesis of Em, and that other theories (electrogenic pump, surface-absorption) must be considered.

Animals↗
PubMed1987

Trifluoperazine and phencyclidine inhibit synergistically carbamylcholine-induced cation influx in muscle cultures.

The effect of trifluoperazine (TFP) and phencyclidine (PCP) on acetylcholine receptor (AChR) function was studied in rat muscles differentiated in cell culture. While both drugs exerted an inhibitory effect on carbamylcholine (CCh)-induced Na+ or Ca2+ influx (I50 = 5-7 microM), alpha-bungarotoxin binding was not affected. The inhibitory effect of both drugs was independent of CCh concentration, which deems it unlikely that these drugs enhanced desensitization. The mutual inhibitory effect of TFP and PCP on Ca2+ influx was analyzed using three alternative models of interaction between the two drugs: competitive, additive and synergistic inhibition models. Our results are in accordance with a synergistic interaction between the drugs. This synergistic interaction between the drugs provides a biochemical rationale to the phenothiazine contraindication in the treatment of PCP psychosis.

Animals↗
PubMed1987

Changes in the levels of acetylcholine receptors mediated by calcium concentration in the sarcoplasmic reticulum.

Regulation of the AChR level, assayed by 125I-alpha-bungarotoxin binding, was studied in chick skeletal muscles differentiated in cell culture. Variety of agents known to affect muscle contractions and calcium distribution were investigated. Synthesis of AChR is enhanced by treatment of myotubes with several drugs known to inhibit spontaneous activity, such as tetrodotoxin or D 600. In contrast, a reduction of AChR levels occurs due to prolonged treatment with caffeine or carbamylcholine. Sodium dantrolene which inhibits Ca2+ release from the sarcoplasmic reticulum, causes 90% elevation in the level of receptors when applied for 48 hr. Furthermore, in a combination of treatments with dantrolene and carbamylcholine, the effect of the last one is almost abolished. Similarly, in a combined treatment of D 600 together with caffeine, the last one is more dominant and reduces the level of the receptors, inspite of inactivity of the myotubes. These experiments support the idea that cytosolic Ca2+ acts as an information carrier to activate/inactivate the machinery of receptor synthesis. We conclude that muscle activity is not the main factor regulating AChR synthesis, but that intracellular Ca2+ released from the sarcoplasmic reticulum is a necessary mediator for the decline in AChR synthesis, whereas Ca2+ accumulation in SR exerts the opposite effect: enhancement of receptor synthesis.

Animals↗
PubMed1987

Human muscle-derived, tissue specific, myocytotoxic T cell lines in dermatomyositis.

Mononuclear cells were isolated from the inflamed muscle tissue of a patient suffering from dermatomyositis (DM). These were expanded in long-term culture and maintained in the presence of IL-2 containing culture medium. Two cell lines were established, one of the helper/inducer (OKT4+) and the other of the suppressor/cytotoxic phenotype (OKT8+). The OKT4+ cell line exhibited a non HLA-restricted, tissue-specific, myocytotoxic effect on rat muscle cell culture. Its lymphoproliferative response to human muscle antigen was HLA-restricted. The OKT8+ cell line exhibited a non HLA-restricted, tissue-specific response to muscle antigens and no myocytotoxic activity in in vitro rat muscle cell culture. It is likely that clones of OKT4+ lymphocytes in patients suffering from DM are associated with the pathogenesis of the disease--they probably mediate the diffuse damage to skeletal muscle through their myocytotoxic activity.

Aged↗