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P M Epstein

Publications and source records attributed to P M Epstein.

At least 37 records · Page 2Linked to original sources
PubMed1984

The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells.

Treatment of ROS 17/2.8 osteosarcoma-derived cells with dexamethasone potentiates the PTH stimulation of adenylate cyclase in these cells, yielding a detectable response to as little as 10 pM PTH. Isoproterenol stimulation was also enhanced. The dexamethasone effect is first apparent at 12 h and increases with time of treatment. The apparent EC50 for dexamethasone is 3 nM. Hydrocortisone and corticosterone act similarly to dexamethasone, but require 30-fold higher concentrations. Dexamethasone treatment produces no change in high affinity phosphodiesterase activity. Glucocorticoid-potentiating effects are much more pronounced in whole cells than in broken cells and do not influence forskolin stimulation. Particulate fractions of dexamethasone-treated cells have higher adenylate cyclase specific activity, but are stimulated by guanyl-5'-yl imidodiphosphate to the same extent as control cells. These findings suggest that the glucocorticoids potentiate hormone responsiveness through promotion of hormone receptor-adenylate cyclase coupling by a mechanism dependent on cellular integrity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Ketamine and ditran block end-plate ion conductance and [3H]phencyclidine binding to electric organ membrane.

Alterations by ketamine (10-100 microM) and ditran (50-100 microM) of end-plate currents were studied using transected cutaneous pectoris muscles. Both drugs reduced peak current and shortened the time constant for end-plate current decay (tau). Ketamine was more effective at pH 5.3 than at 7.4 or 9.1. Recovery from blockade was asymmetrical in that tau recovered more quickly than did peak current when the drugs were removed from the bath. By contrast, 4-aminopyridine antagonized the depression of peak current by ketamine, but not the reduction of tau. Both ketamine and ditran disrupted the voltage dependence of tau. The binding to microsacs prepared from electric organs of [3H]phencyclidine ([3H]PCP) was blocked by ketamine and ditran. In microsacs treated with carbachol, the IC50 for ketamine block of [3H]PCP binding was 6.6 X 10(-6) M. For ditran, the IC50 for block of [3H]PCP binding in the presence of carbachol was 1.7 X 10(-6) M. The binding of [alpha-125I]bungarotoxin to the microsacs or to the cultured chick myotubes was reduced only slightly by ketamine. Because ketamine has no effect on transmitter release and little effect on [alpha-125I]bungarotoxin binding, it is concluded that, like PCP, ketamine and ditran block open channels in the end-plate. In addition, the asymmetrical recovery of end-plate current parameters suggests that ketamine may block closed channels. The recovery from block of closed channels (caused by either a direct action on closed channels or a very slow channel unblocking rate) proceeds more slowly than does the block of open channels.

Animals↗
PubMed1981

Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase.

The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of approx. 3 microM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (Ki approx. 32 microM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (Ki approx 16 microM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (Ki approx 34 microM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 microgram/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc- and displays anomalous kinetics for cyclic AMP hydrolysis (app. Km of 2 and 20 microM) and linear kinetics for cyclic GMP (app. Km approx. 5 microM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc- and displays a higher apparent affinity for cyclic AMP (app. Km approx. 1.5 mu) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissues may be conversions from a single enzyme species.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Local anesthetics, mepacrine, and propranolol are antagonists of calmodulin.

Local anesthetics such as dibucaine, QX572, tetracaine, and phenacaine, as well as other drugs with local anesthetic-like properties (e.g., mepacrine, propranolol, and SKF 525A) inhibit the specific calmodulin-dependent stimulation of erythrocyte Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and cyclic nucleotide phosphodiesterases (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) from brain and heart. Basal activities of these enzymes in the absence of calmodulin are relatively unaffected by concentrations of local anesthetics that strongly inhibit the specific stimulation by calmodulin. Increasing calmodulin, but not Ca2+, overcomes the inhibitory action of the local anesthetics on brain phosphodiesterase. However, excess calmodulin does not fully restore activity of erythrocyte CA2+-stimulated ATPase. Although the mechanism(s) by which the local anesthetics act is unclear, they inhibit binding of 125I-labeled calmodulin to the erythrocyte membrane. Antagonism of calmodulin provides a molecular mechanism that may explain the inhibition of many Ca2+-dependent cellular processes by local anesthetics--e.g., Ca2+ transport, exocytosis, excitation-contraction coupling, non-muscle-cell motility, and aggregation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Postsynaptic inhibition of neuromuscular transmission by trifluoperazine.

The effect of trifluoperazine (TFP) on neuromuscular transmission was investigated on chick biventer cervicis and frog cutaneous pectoris and sartorius nerve-muscles. In the chick, TFP inhibited indirectly elicited twitches in a frequency-dependent manner. Inhibition was much more rapid at higher frequencies of stimulation. Directly elicited twitches, KCl contracture and action potentials of desheathed frog sciatic nerve and sartorius muscles were unaffected by TFP, suggesting an action of TFP on neuromuscular transmission. TFP depressed end plate potential amplitude and miniature end plate potential (MEPP) amplitude without affecting MEPP frequency. When MEPP frequency was increased by high Na+ Ringer, depression of MEPP amplitude was much more rapid. Similarly, at high frequencies of stimulation (100 Hz), TFP rapidly depressed end plate currents. TFP inhibited contractures induced by bath-applied acetylcholine (ACh); depressed ACh potentials produced by iontophoretically applied ACh; decreased ionic current and time constant of decay of end-plate currents of transected muscle; and inhibited [alpha-125I]bungarotoxin binding to ACh receptor. These data suggest that TFP acts postsynaptically in a frequency-dependent manner to inhibit neuromuscular transmission. Based on recent evidence that TFP is a potent antagonist of calmodulin and that calmodulin is localized mainly to postsynaptic regions, we postulate that the postsynaptic inhibitory actions of TFP may be mediated through antagonism of calmodulin, which in turn may regulate ACh receptor function.

Acetylcholine↗
PubMed1980

Activation of human lymphocyte high affinity cyclic AMP phosphodiesterase by culture with 1-methyl-3-isobutylxanthine.

When 1-methyl-3-isobutylxanthine (MIX), a potent inhibitor of human lymphocyte cyclic AMP phosphodiesterase in vitro, was added to intact lymphocyte cultures, MIX caused an activation of this enzyme. Activation by MIX which showed an EC50 = 0.2 mM, required 24 hr in culture, reached a maximum near 48 hr, and remained at maximum through 144 hr. The effect of MIX was to increase the maximum velocity of the 3.5-4S form but not the 5.5-6S form of the lymphocyte high affinity enzyme system. Activation required serum, occurred in calcium depleted media, and was unaffected by sodium azide, adenosine, cycliheximide, or actinomycin D. Further, the effect of MIX was not mimicked by cyclic AMP, cyclic GMP, or by a variety of cyclic nucleotide derivatives with the exception of N6-monobutyryl- and N6-2'0 dibutyryl cyclic AMP in the culture medium. No correlation was observed between the in vitro IC.50 value and the amount of intact cell activation with other phosphodiesterase inhibitors. Although MIX prevented the mitogenic response of lymphocytes to phytohemaglutinin (PHA), a synergistic effect to activate cyclic nucleotide phosphodiesterase was obtained when both agents were included in the culture medium. These data are discussed as an example of a novel modulation of a specific high affinity form of cyclic AMP phosphodiesterase by an apparent non-genetic regulatory mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Muscarinic antagonism of the effects of phosphodiesterase inhibitor (methylisobutylxanthine) in embryonic chick ventricle.

Methylisobutylxanthine (MIX) augmented contractions and Ca++-dependent action potentials in ventricles isolated from embryonic and hatched chicks. Acetylcholine (ACh) inhibited these effects of MIX. In ventricles from chicks on the 18th incubation day, cyclic AMP content was increased to about 150% of basal after 3 min in 3 X 10(-4) M MIX. ACh (10(-6) M) did not reduce the cyclic AMP accumulation caused by MIX, although the increase in twitch tension was abolished. However, 10(-4) M ACh blocked the MIX-induced increases in both twitch tension and cyclic AMP. We conclude, therefore, that ACh antagonizes the effects of MIX both by blocking the action of elevated cyclic AMP and in higher concentrations, also by inhibiting the accumulation of cyclic AMP. In homogenates of chick ventricles. ACh neither stimulated phosphodiesterase activity nor blocked the inhibition of phosphodiesterase by MIX. Therefore, inhibition of cyclic AMP accumulation by higher concentrations of ACh may result from an inhibition of adenylate cyclase activity. Since neither 10(-6) nor 10(-4) M ACh reduced basal cyclic AMP content, basal adenylate cyclase is presumably not affected by ACh. We speculate that MIX may indirectly increase adenylate cyclase activity by antagonizing an inhibitory effect of endogenous adenosine. Furthermore, ACh may interfere with this effect of MIX, thereby facilitating the inhibition of adenylate cyclase by adenosine.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report.

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.

Cyclic AMP↗
PubMed1979

Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts.

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.

3',5'-Cyclic-AMP Phosphodiesterases↗