Biomedical subjects

S Sasson

Publications and source records attributed to S Sasson.

At least 55 records · Page 3Linked to original sources

PubMed1990

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction.

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.

Animals↗

PubMed1989

Failure of fenfluramine to affect basal and insulin-stimulated hexose transport in rat skeletal muscle.

Fenfluramine is an effective appetite suppressant that mediates its action via serotoninergic neurons. We studied the effect of pure d- and l-fenfluramine on in vitro hexose transport in isolated rat soleus muscles and skeletal muscle cells in culture. We found no evidence to suggest that the fenfluramine enantiomers affect the basal transport activity. Furthermore, the drugs did not interfere with the ability of glucose to regulate its own transport. Muscle responsiveness to insulin was not altered by the enantiomers, nor did insulin unmask any effect of fenfluramine on muscle hexose transport. These conclusions are based on experiments performed with a wide concentration range of drug and insulin, from the therapeutic to suprapharmacological levels. We discuss our results in view of published data on the effects of fenfluramine on peripheral glucose metabolism.

Animals↗

PubMed1989

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor.

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.

Animals↗

PubMed1988

The effects of dimethylformamide on the interaction of the estrogen receptor with estradiol.

The study of the mechanism of interaction of antiestrogens with the estrogen receptor is complicated by the limited solubility of these compounds and their nonspecific, hydrophobic interactions with proteins in estrogen receptor preparations and with the glass or plastic wall of the test tube. The organic solvent dimethylformamide increases the solubility of these compounds in aqueous solution and interferes with hydrophobic interactions with solid phases and thereby reduces their loss from the solution. For these reasons some investigators use dimethylformamide in the antiestrogen-estrogen receptor binding assay. In this study we report that dimethylformamide interferes with the estrogen receptor's binding kinetics and the estradiol-induced activation of the estrogen receptors, and inactivates the receptor.

Allosteric Regulation↗

PubMed1988

Mechanism of the estrogen receptor interaction with 4-hydroxytamoxifen.

The binding mechanism of the estrogen receptor with 4-[3H]hydroxytamoxifen was investigated. The equilibrium binding analysis with 4-[3H]hydroxytamoxifen indicated a positive cooperative interaction: the Scatchard plot was convex and the Hill coefficient was 1.4-1.5. This binding appears similar to the positively cooperative interaction of the estrogen receptor with [3H]estradiol. However, a competitive binding assay with a saturating concentration of [3H] estradiol and variable concentrations of 4-hydroxytamoxifen produced nonparallel displacement curves indicating that the binding mechanism of the receptor with these two ligands is different. The competitive binding assay with [3H]estradiol and 4-hydroxytamoxifen at constant molar ratios demonstrated that the receptor's affinity for estradiol was reduced and the receptor preferentially bound 4-hydroxytamoxifen. These data suggest that 4-hydroxytamoxifen interacts with the receptor differently than estradiol; it antagonizes the binding of estradiol when these two ligands are simultaneously present.

Animals↗

PubMed1987

Polymyxin B is an inhibitor of insulin-induced hypoglycemia in the whole animal model. Studies on the mode of inhibitory action.

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.

Animals↗

PubMed1987

Evaluation of the supraspinal analgesic activity and abuse liability of ethylketocyclazocine.

Rats were trained to escape from aversive electrical brain stimulation delivered to the midbrain reticular formation (MRF) or to obtain rewarding intracranial stimulation delivered to the medial forebrain bundle (MFB). The threshold for escape behavior was determined by a modification of the psychophysical method of limits. Likewise, reward thresholds were determined by a rate-free psychophysical method. Acute administration of ethylketocyclazocine (EKC) (0.06-1.0 mg/kg s.c.) raised escape threshold in a dose-dependent manner while having no effect on non-specific measures of sedation or motor impairment. This suggests that opioids with kappa receptor activity specifically modulate pain at a supraspinal level. Administration of EKC had no effect on the threshold for rewarding intracranial stimulation to the MFB suggesting that EKC has low potential for abuse.

Analgesics↗

PubMed1987

In vitro autoregulation of glucose utilization in rat soleus muscle.

The possibility that glucose may directly regulate its rate of utilization in skeletal muscle was investigated in vitro with the rat soleus muscle. Preincubation of the muscles with varying glucose concentrations modulated the rate of glucose utilization through the glycolytic pathway during a subsequent incubation. At glucose concentrations below approximately 3.0 mM, utilization was maximal (26.8 +/- 2.4 and 87.5 +/- 4.8 nmol X g-1 X min-1 when assayed in the presence of 0.5 and 5.0 mM glucose, respectively). Preincubation with higher glucose concentrations (up to 20 mM) reduced the utilization by 40-60% in a biphasic manner: a rapid fall between 1 and 4 mM, followed by a gradual approximately 2% decrease per each millimolar increase of glucose. The effect of preexposure to glucose was not due to substrate dilution by the remaining glucose in the extracellular or intracellular space of the muscle. The uptake of the nonmetabolizable glucose analogues 3-O-methylglucose and 2-deoxyglucose was also modulated by extracellular glucose in a similar manner. Thus, the regulatory site seems to reside in the transport function of the hexose. The ATP content was very similar in muscles preexposed to 1.0 and 15.0 mM glucose for 3 h and therefore does not seem to be the mediator of the glucose effect. This substrate regulation of the rate of glucose transport and of the glycolytic flux may be operative in the in vivo glucose homeostatic system and may contribute to the reduced peripheral glucose utilization observed in diabetes.

3-O-Methylglucose↗

PubMed1986

Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture.

A self-regulatory mechanism of the glucose transport in rat skeletal muscle cells is described. In isolated rat soleus muscles and rat skeletal myocytes and myotubes in culture, pre-exposure to varying glucose concentrations modulated the rate of 2-deoxyglucose uptake. Maximal uptake was observed at glucose concentrations below 3 mM. Between 2.5 and 4.0 mM glucose it was reduced by 25-35%; further elevation of the glucose concentration resulted in a gradual decrease of the transport rate by approximately 2% for each millimolar glucose. The effect of glucose was time-dependent and fully reversible. Insulin rapidly increased the 2-deoxyglucose uptake in the soleus muscle; however, the insulin effect depended on the glucose concentration of the preincubation. Insulin was totally ineffective in muscles pre-exposed to 1.0-3.0 mM glucose, whereas its stimulatory action increased with increasing glucose concentrations above 4 mM. The effect of low glucose and insulin were not additive, and the maximal 2-deoxyglucose uptake rates induced by both conditions were of identical magnitude. It is postulated that glucose may "up- and down-regulate" its transport by affecting the number of active glucose transporters in the plasma membrane, and that insulin exerts its stimulatory effect only when the extracellular glucose reaches a threshold concentration.

Aminoisobutyric Acids↗

PubMed1986

Evidence for a supraspinal analgesic effect with cyclazocine and pentazocine.

The antinociceptive effect of the benzomorphan class of opioid analgesics have been difficult to measure utilizing some of the standard animal pain models. This may be due, in part, to the sedative and/or motor effects associated with these drugs. In addition, it has been proposed that the major site of action for drugs with agonist activity at the kappa opiate receptor is exclusively at the spinal level opposed to both spinal and supraspinal as with the mu receptor agonists such as morphine. The present study examines the antinociceptive effect of the mixed agonist-antagonists cyclazocine and pentazocine utilizing electrical stimulation of the midbrain reticular formation (MRF) as the aversive stimulus in the rat. Animals were trained to escape MRF stimulation by turning a cylindrical manipulandum. An escape threshold was determined by varying the current intensity according to a modification of the psychophysical method of limits. In addition to the determination of the escape threshold the response latency and strength of response was also measured. Both cyclazocine (0.25-1.0 mg/kg) and pentazocine (2.5-12.5 mg/kg) raised the escape threshold in a dose-dependent manner without any concomitant change in the response latency or strength of response. These data suggest that the observed threshold elevation is due to a specific antinociceptive effect. Since the aversive stimulation was delivered supraspinally, the data also suggest that there are supraspinal mechanisms mediated by kappa receptors responsible for this analgesic effect.

Analgesia↗

PubMed1986

Potentiation of morphine analgesia by d-amphetamine.

Rats were trained to escape from aversive electrical brain stimulation delivered to the mesencephalic reticular formation (MRF). The threshold for this escape behavior was determined by a modification of the classic psychophysical method of limits. Escape thresholds were determined after the administration of morphine alone, d-amphetamine alone, and the combination of d-amphetamine and an ineffective dose of morphine. Morphine alone caused a dose-dependent raising of the escape threshold (1.0-16.0 mg/kg IP) while d-amphetamine alone (0.06-2.0 mg/kg IP) had no effect or caused a slight lowering of threshold. For each animal, a dose of morphine that produced no change in escape threshold was then selected to be administered concomitantly with various doses of d-amphetamine. The co-administration of morphine and d-amphetamine resulted in a significant, dose-dependent increase in the escape threshold, which was not seen with d-amphetamine alone and was as great or greater in magnitude than the increase seen with the highest dose of morphine tested. The results of this study clearly demonstrate that opiate analgesia is potentiated by concomitant d-amphetamine administration. The mechanisms involved in this potentiation warrant further investigation for the clinical management of pain.

Analgesics↗

PubMed1986

Plasminogen activator activity in differentiating rat myoblasts.

Primary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10(-7) M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creatine phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.

Animals↗

PubMed1984

The estriol-induced inhibition of the estrogen receptor's positive cooperativity.

The effect of estriol on the positive cooperativity of [3H]estradiol binding to the partially purified calf uterine estrogen receptor was investigated using the kinetic analysis of Sasson and Notides (J. biol. Chem. 257 1982, 11540). The receptor was titrated with variable concentrations of [3H]estradiol with or without estriol; the estriol was maintained in a constant molar ratio to the [3H]estradiol concentration. A 4-fold molar excess of estriol above the [3H]estradiol concentrations inhibited the receptor's cooperative [3H]estradiol binding. In the absence of estriol, the [3H]estradiol receptor interaction was highly cooperative, the Scatchard plot was convex and the Hill coefficient was 1.61 +/- 0.02. In the presence of sufficient estriol to reduce the maximally bound [3H]estradiol to 77%, the Scatchard plot was linear and the Hill coefficient was 1.04 +/- 0.04. The inhibition of the cooperative [3H]estradiol binding by estriol was not due to isotope dilution of the specifically bound [3H]estradiol by the unlabeled estriol. These data demonstrate that the cooperative binding of [3H]estradiol by the receptor that is characteristic of the equilibrium between the two states of the receptor (active and nonactive) is eliminated by the presence of estriol. This finding is consistent with the agonist/antagonist activity of estriol observed in vivo.

Animals↗

PubMed1984

Inability of [3H]estriol to induce maximal cooperativity of the estrogen receptor.

The interaction of [3H]estriol with the partially purified estrogen receptor from calf uterus shows positive cooperativity that is dependent upon receptor concentration and temperature. At a receptor concentration of 1 nM and 25 degrees C the [3H]estriol-receptor cooperativity was low, the Hill coefficient (nH) was 1.03 +/- 0.02 however, with increasing receptor concentrations the receptor's cooperativity increased until at approximately intracellular receptor concentration (20 nM) the nH = 1.20 +/- 0.04. At 0 degrees C and a receptor concentration of 10 nM the [3H]estriol-receptor interaction was highly cooperative, the Scatchard plot was convex and nH = 1.58 +/- 0.04 while at 30 degrees C the Scatchard plot approached linearity and nH = 1.03 +/- 0.02. In comparison, [3H]estradiol was capable of inducing, at 0 or 30 degrees C and at a receptor concentration of 1 nM or greater, maximal receptor cooperativity, nH = 1.63. These data demonstrate: (a) the receptor's conformation and binding mechanism change in a specific manner with temperature, so that receptor analysis at 0 degrees C does not necessarily reflect the receptor's properties at biologically relevant temperatures; (b) the dependence of the receptor's cooperativity on receptor concentration, which suggests interaction between dissociable subunits; and (c) the lower cooperativity induced by estriol, in comparison with estradiol, which indicates estriol is less efficient in shifting the receptor toward a higher affinity or the activated state of the receptor.

Animals↗

PubMed1983

Estriol and estrone interaction with the estrogen receptor. I. Temperature-induced modulation of the cooperative binding of [3H]estriol and [3H]estrone to the estrogen receptor.

We investigated the calf uterine estrogen receptor's interaction with [3H]estriol and [3H]estrone and found that the receptor's cooperative binding is markedly dependent upon temperature and receptor concentration. With increasing temperature, the intensity of the estrogen receptor's cooperativity decreases: at 0 degrees C the Scatchard plot was convex and the Hill coefficient was 1.58 +/- 0.04 (n = 5), while at 30 degrees C the Scatchard plot approached linearity and the Hill coefficient was 1.03 +/- 0.02 (n = 3) for the binding of [3H]estriol with a receptor concentration of 10 to 12 nM. With increasing receptor concentration, the receptor's cooperativity gradually increased. At a receptor concentration of 1 nM, at 25 degrees C, with [3H]estriol the Hill coefficient was low, 1.03 +/- 0.02 (n = 3), while at approximately intracellular receptor concentration (20 nM) the Hill coefficient increased to 1.20 +/- 0.04 (n = 4). Similar results were observed with [3H]estrone. The cooperative interaction of the estrogen receptor with [3H]estriol or [3H]estrone is reversible and dependent upon temperature. The van't Hoff analysis of the apparent dissociation constant of the [3H]estrone- and [3H]estriol-receptor complexes indicates that a transition in the receptor's binding mechanism occurs at 15 degrees C. Therefore, measurements of ligand interactions with the estrogen receptor at 0 to 15 degrees C, may not reflect the binding mechanism of the receptor at more biologically relevant temperatures. The reduced, positively cooperative interactions of [3H]estriol and [3H]estrone with the estrogen receptor at 30 degrees C, in comparison with estradiol, decrease the presence of the activated receptor, which correlates with the partial agonist-antagonist activities of estriol and estrone observed in vivo.

Animals↗

PubMed1983

Estriol and estrone interaction with the estrogen receptor. II. Estriol and estrone-induced inhibition of the cooperative binding of [3H]estradiol to the estrogen receptor.

Kinetic analysis of the estrogen receptor's cooperative equilibrium [3H]estradiol binding (Sasson, S., and Notides, A. C., (1982) J. Biol. Chem. 257, 11540-11545) provides a sensitive method for probing the binding of partial agonists to the estrogen receptor. We studied the effects of estriol and estrone on the positive cooperativity of [3H]estradiol binding to the partially purified, calf uterine estrogen receptor. The receptor was titrated with variable concentrations of [3H]estradiol in combination with estriol or estrone, while maintaining a constant molar ratio of the estriol or estrone to the [3H]estradiol. With either a 4-fold molar excess of estriol or a 25-fold molar excess of estrone above the [3H]estradiol concentrations, the receptor's positive cooperative [3H]estradiol binding was inhibited. The Scatchard plot showed a transition from a convex to a linear curve and a decrease in the Hill coefficient value from 1.61 +/- 0.02 (n = 7) in the absence of estriol or estrone to 1.04 +/- 0.04 (n = 4) in the presence of estriol and 0.99 +/- 0.03 (n = 4) in the presence of estrone. The inhibition of the positive cooperativity of [3H]estradiol binding by estriol or estrone was shown not to be due to isotope dilution of the specifically bound [3H]estradiol by the unlabeled estriol or estrone. These kinetic analyses demonstrate that the positively cooperative equilibrium binding of [3H]estradiol by the receptor, which is characteristic of the receptor's activation process, is eliminated by estriol and estrone and consistent with their partial agonist-antagonist activities observed in vivo.

Animals↗

PubMed1983

Naloxone lowers brain-stimulation escape thresholds.

Rats were trained to escape from aversive electrical brain stimulation to the mesencephalic reticular formation (MRF). The threshold for this escape behavior was determined by a modification of the psychophysical method of limits. The administration of naloxone (4-16 mg/kg, IP) produced a decrease in escape threshold from MRF stimulation. These findings implicate the involvement of the MRF in the modulation of pain and suggest that threshold changes to stimulation at this level of the nociceptive neuro-axis may represent a change in a motivational-attentional dimension of pain.

Animals↗

PubMed1982

The inhibition of the estrogen receptor's positive cooperative [3H]estradiol binding by the antagonist, clomiphene.

The calf uterine estrogen receptor showed positive cooperativity of [3H]estradiol equilibrium binding; the Scatchard plot was convex and the Hill coefficient was 1.69 +/- 0.021 (n = 14). The effects of the estrogenic antagonists, zuclomiphene (cis-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate) and enclomiphene (trans-23-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate), on the positive cooperativity of [3H]estradiol binding were measured by titrating the receptor with a variable concentration of [3H]estradiol and antagonist while maintaining a constant excess in a specific ratio of the antagonist to the [3H]estradiol. With a 45- to 55-fold molar excess of zuclomiphene or an 820- to 900-fold molar excess of enclomiphene above the [3H]estradiol concentration, the receptor's positive cooperative [3H]estradiol binding was inhibited. A transition from a convex to a linear Scatchard plot and a decrease in the Hill coefficient from 1.69 to 1.10 +/- 0.02 (n = 6) were induced. The specifically bound [3H]estradiol was inhibited 43 to 50% by the zuclomiphene and enclomiphene. The addition of unlabeled estradiol in a 1- or 2.3-fold molar excess above that of the [3H]estradiol concentration produced a 50 to 75% competitive displacement of the specifically bound [3H]estradiol; nevertheless, the Scatchard plot remained convex and the Hill coefficient was 1.74 and 1.80, respectively. Thus, inhibition of the positive cooperativity of [3H]estradiol binding by the clomiphene isomers was not due to dilution of the specifically bound [3H]estradiol by the antagonist. These data demonstrate that there are two molecular mechanisms by which an estrogen antagonist interferes with the function of the receptor: as a competitor, thus blocking the estrogen receptor's binding site to an agonist, and second by inducing conformational changes that inhibit site:site interactions and receptor activation.

Animals↗