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Biomedical subjects

A Shainberg

Publications and source records attributed to A Shainberg.

At least 37 records · Page 2Linked to original sources

Open state block by fendiline of L-type Ca++ channels in ventricular myocytes from rat heart.

The effects of fendiline on L-type Ca++ currents [ICa(L)] were investigated in rat ventricular cardiomyocytes using the patch-clamp technique both in the whole-cell disrupted-patch and in the cell-attached configuration. For comparison, the effects of verapamil were also investigated. Both compounds depressed the magnitude of whole cell ICa(L), verapamil being about 15 times more potent than fendiline. Verapamil did not change the time course of the current, whereas fendiline accelerated its decay when either Ca++ or Ba++ ions were used as charge carriers. In the presence of the Ca++ agonist BayK8644 (10 microM), the potency ratio of fendiline/verapamil was inverted. BayK8644 (10 microM) also reversed the potency ratio of verapamil/fendiline in smooth muscle, with respect to changes in tension induced by K+ (48 mM). In single channel recordings at 0.1 Hz, in the presence of BayK8644 (1 microM) and using Ba++ ions as the charge carrier, fendiline (1 microM) reduced mean open time by 34% and channel availability by 8%; the ensemble average current of Ca++ channels was reduced by 43%. In the same experimental conditions, verapamil (1 microM) was ineffective. These results can be explained by the assumption that fendiline blocks Ca++ channels preferentially in the open state, in contrast to verapamil which blocks preferentially inactivated Ca++ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Regulation of A1 adenosine receptors by amiodarone and electrical stimulation in rat myocardial cells in vitro.

The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.

Amiodarone↗

Thiamine deficiency in cardiac cells in culture.

Rat heart cells in culture were found to be a unique model for studying biochemical and pharmacological aspects of thiamine deficiency. When thiamine was excluded from the growth medium, the following effects were observed: (1) Morphological examination did not show any difference between control and thiamine-deprived cells during the first 10 days. However, after 10-11 days spontaneous contractions ceased, accompanied by initiation of cell degeneration; (2) Intensive degeneration and cell death were observed after 14-16 days. (3) Thiamine pyrophosphate (TPP) concentration in thiamine-deprived cells was decreased gradually, with an elimination half-life of 4-5 days. (4) [3H]deoxyglucose uptake by the cells was increased, even after 1 day of thiamine deprivation. (5) ATP level decreased after 8 days and reached 50% of control cells after 10 days. (6) In thiamine-deprived cells, thiamine addition caused a 60% rise in contraction amplitude but contraction rate was not altered significantly. (7) All these effects were reversible if thiamine was supplied before the initiation of the degeneration processes.

Adenosine Triphosphate↗

Inhibition of calcium accumulation by the sarcoplasmic reticulum: a putative mechanism for the cardiotoxicity of adriamycin.

The aim of this study was to examine the effect of adriamycin (ADR) on calcium accumulation by the sarcoplasmic reticulum (SR). Chemical skinning of cultured rat myocardial cells compromised the barrier function of the cell membrane and thus permitted direct exposure of mitochondrial and non-mitochondrial sites to ADR. In the presence of ATP, and sodium azide, mitochondrial calcium accumulation was negligible. Furthermore, it has previously been shown that non-mitochondrial calcium accumulation is mediated mainly by the SR under these conditions. Incubation with 10 microM ADR for 2 hr reduced the level of calcium accumulation by the SR by 50%. A similar effect was obtained after 24 hr incubation with 1 microM ADR. The addition of ferric iron to the culture medium further reduced the level of calcium accumulation. Neither vitamin E nor beta-carotene affected calcium accumulation by the SR. These results suggest that ADR interferes with the calcium accumulation activity of the SR and that ferric iron potentiates this effect.

Animals↗

Inhibition of malignant cell proliferation by culture media conditioned by cardiac or skeletal muscle.

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS-PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.

Analysis of Variance↗

The protective effect of class III antiarrhythmic agents against calcium overload in cultured myocytes.

Calcium ions have been implicated in the mechanisms of ventricular arrhythmias. Impairment of intercellular coupling by calcium overload is considered to facilitate ventricular fibrillation (VF) and to sup-press its self termination. According to our hypothesis, any compound that decreases intracellular calcium concentration [Ca2+]i during VF can serve as defibrillating drug. In this study, we examined the effect of d-sotalol and tedisamil on calcium overload in cultured, spontaneously beating rat cardiomyocytes. The changes of [Ca2+]i were measured by indo-1 method and the intercellular synchronization by image analysis. The results showed that increase in [Ca2+]o from 1.9 mM to 3.9 mM increased [Ca2+]i from 100 nM to 320 nM and transformed the synchronized cell movement to an asynchronous one. Administration of 5 x 10(-6) M d-sotalol or 10(-6) M tedisamil, decreased the [Ca2+]i to its basic level and restored the synchronized activity. In summary: Our results showed that increase in [Ca2+]i known to cause inhibition of intercellular coupling, that could lead to arrhythmia and fibrillation while d-sotalol or tedisamil prevented this effect. These results support our hypothesis, that class III antiarrhythmic compounds with positive inotropic effect, increase intercellular synchronization, by decreasing free [Ca2+]i, most probably by increasing the Ca2+ uptake by the sarcoplasmic reticulum, and therefore act as a defibrillating compound.

Animals↗

Lipoxygenase activity in heart cells.

Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat cardiomyocytes was investigated using 1-[14C]AA. LOX activity was detected in the microsomal fraction, in the high speed supernatant prepared from rat hearts and in rat cardiomyocyte supernatant. LOX products from all fractions comigrated in thin layer chromatography as 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE. Enzyme linked immunosorbent assay for 12-HETE showed its formation by the microsomal fraction, the ammonium sulfate (AS) pellet, and by rat cardiomyocyte supernatant, while radioimmunoassay for 15-HETE showed its formation only by the AS pellet. The properties of LOX in each fraction are reported here.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

Effect of Leiurus quinquestriatus hebreus venom on calcium and deoxyglucose uptake in cultured cardiac cells.

The effects of scorpion venom Leiurus quinquestriatus hebreus were studied on cardiac cells grown in culture. The venom (30 micrograms/ml) increased significantly (P < 0.05) Ca2+ uptake into intact cardiocytes and to sarcoplasmic reticulum of skinned cells. [3H]Deoxyglucose uptake was also increased significantly (P < 0.05) in venom treated cardiocytes. It was found that fractions I and III of the venom, separated by gel filtration and ion exchange chromatography, are responsible for the increased Ca2+ uptake by the sarcoplasmic reticulum, whereas fraction IIb, III and IV are responsible for the accelerated rate of uptake of 45Ca and [3H]deoxyglucose by intact cells. Ca channel blockers prevented these effects and similar results were obtained by propranolol. Thus, it is concluded that the venom exerts its effect through activation of beta-adrenoceptors which causes the opening of L-type Ca channels.

Adrenergic beta-Antagonists↗

Effects of theophylline and dibutyryl-cAMP on adenosine receptors and heart rate in cultured cardiocytes.

The effects of chronic exposure to the adenosine antagonist theophylline (Theo) and dibutyryl cyclic-AMP, a membrane-permeant derivative of the second messenger 3', 5'-cyclic-AMP (cAMP), on contractions and adenosine receptor levels in cultured cardiocytes were studied. Binding of the A1-adenosine receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) was used to monitor the level of the receptors in intact cardiocytes. Both Theo and cAMP stimulated the rate of contraction and also increased the density of adenosine receptors. The Bmax value for [3H]CPX binding to intact cardiocytes was increased by 45-47% following 4 days of exposure to either 50 microM Theo or 100 microM cAMP. Scatchard analysis indicated that the affinity of the A1 receptors for [3H]CPX remained unchanged (Kd 0.1-0.2 nM). No significant differences were observed in protein content or in cell number. A linear correlation was achieved between the level of A1-adenosine receptors and heart rate at various Theo and dibutyryl-cAMP concentrations, although Theo was more efficient in elevation of the receptor density. Increases of 82, 78, 138 and 235% in A1 receptor density and increases of 63, 59, 66 and 150% in heart rate were obtained following 5 days of treatment with 1, 10, and 1000 microM of Theo, respectively. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, changes in the density of adenosine receptors may compensate for chronic drug-induced changes in cardiac contractile activity so as to restore conditions to the normal state.

Animals↗

Interruption of myogenesis by transforming growth factor beta 1 or EGTA inhibits expression and activity of the myogenic-associated (2'-5') oligoadenylate synthetase and PKR.

Interferon-induced proteins have been previously implicated in the regulation of cell growth. In an attempt to provide evidence for the involvement of these proteins in differentiation, the effect of transforming growth factor beta 1 (TGF-beta) and EGTA on the expression and activity of (2'-5') oligoadenylate synthetase (2-5A synthetase) and double-stranded RNA activated protein kinase (PKR) during myogenesis of rat primary skeletal muscle cultures or the myogenic cell line L8 was studied. Both TGF-beta and EGTA inhibited the fusion of myoblasts and reduced significantly the level of the muscle-specific proteins, acetylcholine receptors, and creatine kinase activity in rat primary muscle cultures. Likewise, TGF-beta exhibited a similar inhibitory effect on the fusion of L8 cells and the level of creatine kinase activity in these cells. The kinetics of 2-5A synthetase activity in both types of cells during differentiation was then established. In both types, a transient increase in activity was observed followed by a decrease thereafter. However, while the peak activity in primary muscle cultures appeared after 24 h in culture, it was observed only on the third day in L8 cells grown in differentiation medium (DM). Treatment of primary cultures with either TGF-beta or EGTA reduced the amount of 1.7-kb 2-5A synthetase-specific RNA transcripts and decreased significantly the level of 2-5A synthetase activity compared to that in untreated cultures. Western blot analysis of 2-5A synthetase proteins in untreated primary muscle cultures showed that the major species synthesized in these cells was the 43-kDa isoform of the enzyme. However, the 71-kDa isoform was clearly visible after 72 h in culture. Both TGF-beta and EGTA abrogated the appearance of all forms of 2-5A synthetase. Similarly, in L8 cells grown in DM, TGF-beta down-regulated the expression of 2-5A synthetase and reduced the level of enzymatic activity. Western blot analysis revealed the presence of the 71-kDa isoform as the major species of 2-5A synthetase in L8 cells; however, the 43-kDa isoform was also visible on the third day in DM. TGF-beta treatment resulted in a reduced amount of 2-5A synthetase proteins. The kinetics of PKR activity in L8 cells grown in DM was similar to that observed with 2-5A synthetase. Furthermore, TGF-beta strongly reduced the level of PKR activity in differentiating L8 cells.

2',5'-Oligoadenylate Synthetase↗

Regulation of adenosine receptors in cultured heart cells.

A1 adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. Treatments with agents that accelerated heart rate for 3-4 days, caused an increase in the level of adenosine receptors. Treatments that attenuated heart rate, reduced the level of the receptors. Thus, the cardiac cells respond to environmental conditions affecting heart contraction so as to restore the basal rate of heart activity.

Animals↗

A 28,000 mol. wt toxin from Bacillus thuringiensis israelensis induces cation transport in rat muscle cultures.

The mechanism by which the Bacillus thuringiensis israelensis (Bti) 28,000 mol. wt toxin exerts its effect on mature muscle cultures was examined. The toxin inhibited Na+/K(+)-ATPase activity as revealed by 86Rb influx. A 50% inhibition of Na+/K(+)-ATPase activity was obtained with 0.2 microgram/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 microgram/ml. A considerable release of 86Rb was obtained by doses greater than 0.2 microgram/ml. The 86Rb release was also time and dose dependent. This effect is probably non-specific, since 45Ca influx is also accelerated by toxin-treated cultures. Pre-incubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma.

Animals↗

Characterization of adenosine receptors in intact cultured heart cells.

Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A1 adenosine receptor radioligand [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX), to monitor the level of the receptors on intact cardiocytes. The binding showed high affinity (Kd = 0.13 nM) and the number of [3H]CPX binding sites (Bmax) was 23.1 fmol/dish (21 fmol/mg protein). The Ki of the agonists R-N6-(2-phenylisopropyl)-adenosine (R-PIA) and S-N6-(2-phenylisopropyl)-adenosine (S-PIA), and of the antagonists CPX and theophylline were 3.57, 49.0, 1.63 and 4880 nM, respectively. The number of adenosine receptors was very low during the first days in cultures (5 fmol/dish) and increased gradually until it reached a plateau on days 8-10. Treatment with norepinephrine or isoproterenol which accelerated the rate of contractions, induced up regulation of the receptors. Bmax increased 2-3-fold by application of norepinephrine for 4 days, while receptor affinity to the radioligand was unaffected. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity increased only by 22 and 38%, respectively. Similarly, 3 days treatment with triiodothyronine (T3, 10(-8) M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to [3H]CPX. Carbamylcholine (5 x 10(-6) M), which reduced the rate of heart contractions, caused 26% down regulation while the affinity of the receptors remained unchanged. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, the level of adenosine receptors may respond to drug-induced chronic changes in cardiac contractile activity so as to restore conditions to normal (basal) contractions.

Animals↗

Biochemical and morphological changes in rat muscle cultures caused by 28,000 mol. wt toxin of Bacillus thuringiensis israelensis.

The 28,000 mol. wt protein of Bacillus thuringiensis israelensis showed a high degree of toxicity to rat muscle in culture. Application of 1 microgram/ml to the culture medium completely inhibited cell fusion. Reversibility of this effect was demonstrated by replacement of the culture medium with fresh medium, and the consequence was that cell fusion was resumed. When differentiated myotubes were treated with 1 microgram/ml of the toxin, the spontaneous contractile activity was abolished within 20 min. Cytotoxic effects were observed 1 hr after treatment was initiated, as manifested by creatine kinase (CK) release to the medium. Two hours after toxin was applied to the muscle culture, the myotubes were deteriorated whereas the mononucleated cells were not affected. Six or 7-day-old cultures which were treated by 1 microgram/ml of 28,00 mol. wt toxin revealed a change in the levels of Na+ and K+ within the fibres as analysed by X-ray microanalysis (XRMA). Preincubation of the toxin for 20 min with phospholipids before application to the cells reduced the cytotoxic effect. Phosphatidylinositol and phosphatidylserine were the most efficient inhibitors, whereas phosphatidylcholine, sphingomyelin and phosphatidylethanolamine were less effective in protecting cultures from the cytotoxic effects of the 28,000 mol. wt protein.

Animals↗

Electric stimulation regulates the level of Ca-channels in chick muscle culture.

To examine the role of muscle activity in the expression of 1,4-dihydropyridine (DHP)-sensitive Ca-channels, we electrically stimulated chick skeletal muscle cells growing in culture. The number of Ca-channels was determined by a radioligand binding technique using [3H]PN200-110. The function of these channels was measured by depolarization-induced 45Ca uptake. We found that both the amount and the function of these channels were higher as a result of the increased muscle activity caused by electrical stimulation (ES).

Acetylcholinesterase↗

Infection with Moloney murine sarcoma virus inhibits myogenesis and alters the myogenic-associated (2'-5')oligoadenylate synthetase expression and activity.

Infection of rat skeletal muscle cultures on the first or second day in vitro with Moloney murine sarcoma virus (MSV) led to the arrest of myotube formation and to inhibition of both the synthesis of the muscle-specific proteins acetylcholine receptors and creatine kinase and the expression of the myosin light chain-2. Mos-specific RNA transcripts were readily detected at 1 day after infection indicating that viral genes were expressed in infected cells. In parallel, the expression of the cell growth-associated gene--c-myc--in uninfected muscle cultures was drastically reduced with time, while in MSV-infected myoblasts, the amount of c-myc-specific RNA transcripts gradually increased with time after infection. Under these conditions we could demonstrate that the interferon-induced gene (2'-5')oligoadenylate synthetase (2-5A synthetase) was transiently activated in uninfected muscle culture reaching a peak activity on the third day. Infection of myoblasts with murine leukemia virus did not alter the pattern of 2-5 synthetase activity observed in uninfected cells. However, infection with MSV on the second day led to a slight reduction in activity followed by a significant increase on the sixth and seventh day. Similarly, 2-5A synthetase gene expression was down-regulated with time in culture in uninfected myoblasts while re-expressed between the fourth and seventh days in MSV-infected cultures.

2',5'-Oligoadenylate Synthetase↗

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↗