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J Abramowitz

Publications and source records attributed to J Abramowitz.

At least 37 records · Page 2Linked to original sources
PubMed1989

Catecholamine-induced heterologous desensitization of rabbit luteal adenylyl cyclase: loss of luteinizing hormone responsiveness is associated with impaired G-protein function.

The effects of injecting epinephrine into pseudopregnant rabbits on the luteal adenylyl cyclase system were analyzed. Epinephrine-induced desensitization was heterologous and associated with a reduced response to isoproterenol, LH, NaF, and forskolin. Epinephrine-induced desensitization was rapid in onset, with a maximum decrease in responsiveness 6 h after treatment and responsiveness returning to control levels within 24 h of treatment. The changes in beta-adrenergic receptor content paralleled changes in catecholamine responsiveness. The affinity of the beta-receptors from treated animals decreased 1.5- to 2-fold before down-regulation. LH receptor number was not altered by epinephrine treatment, although responsiveness to LH was depressed. LH receptor affinity, however, was reduced about 2-fold by epinephrine treatment. Epinephrine treatment also altered G-protein function in corpora lutea, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- membranes and ADP ribosylation by cholera and pertussis toxins. NaF- and isoproterenol-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed for the first 6 h after treatment. The ability of cholera toxin to ADP ribosylate alpha s 46 and alpha s 45 was reduced 1.5-6 h and 3-12 h, respectively, after epinephrine treatment. The reduced ability of cholera toxin to ADP ribosylate alpha s 45 was associated with the decrease in LH receptor affinity after treatment. This supports the contention that alpha s 45 preferentially interacts with the LH receptor. These studies demonstrate that the loss of LH responsiveness upon epinephrine-induced heterologous desensitization is associated with altered G-protein function.

Adenylate Cyclase Toxin↗
PubMed1988

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels.

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.

Amino Acid Sequence↗
PubMed1988

Screening of cDNA libraries with oligonucleotides as applied to signal transducing G proteins, receptors and effectors.

Screening of cDNA libraries constructed in phage or plasmids with oligonucleotide probes has become one of the preferred cloning techniques with the least number of false positive failures. In this article we present our current protocols for designing the procedure to detect cDNA inserts and isolate them. We illustrate with primary screens for G protein subunits and membrane receptors.

Bacteriophage lambda↗
PubMed1987

The human genome encodes at least three non-allellic G proteins with alpha i-type subunits.

The amino acid sequence and composition of alpha-subunits of signal transducing G proteins of the same kind appear to vary by no more than 2% from species to species. Here we isolated a human liver cDNA using an oligonucleotide complementary to the sequences encoding the pertussis toxin (PTX) ADP-ribosylation site of the alpha-subunit of the rat brain G protein called Gi. Its open reading frame characterizes it as an alpha i-type cDNA--as opposed to alpha o-type--but predicts an amino acid composition that differs by 7% and 14%, respectively, from two other human alpha i-type molecules. Together with human brain alpha i (type-1) and human monocyte alpha i (type-2), the new human liver alpha i cDNA (type-3) forms parts of a family of alpha i molecules. Type-3 alpha i cDNA hybridizes to a approximately 3.6 kilobase long mRNA and type-2 alpha i cDNA hybridizes to an mRNA species of approximately 2.7 kilobases. This indicates that the human genome has at least three non-allellic genes encoding non-alpha o-type PTX substrates and provides structural evidence for the hypothesis that distinct effector systems are regulated by similar but nevertheless distinct PTX substrates.

Amino Acid Sequence↗
PubMed1987

Comparison of the ability of seven gonadotropin preparations from different mammalian sources to interact with the adenylyl cyclase system in corpora lutea from rabbits and rats.

The effects of guanine nucleotides and magnesium (Mg2+) on the interaction of seven different gonadotropin preparations with their rabbit and rat luteal receptors were studied and compared to the ability of these gonadotropins to stimulate luteal adenylyl cyclase activity. In both the rabbit and rat, human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) were less efficacious than the other gonadotropin preparations in stimulating luteal adenylyl cyclase activity and thus behaved as partial agonists. Addition of 2 mM MgCl2 increased the affinity of the rat luteal receptors for all seven gonadotropins tested, while in the rabbit Mg2+ increased the affinities for porcine, bovine, ovine, rat and rabbit LH but did not significantly alter the affinities for hCG or hLH. In no instance did the addition of 100 microM GTP alter the affinity of the receptor from that observed in the absence or presence of Mg2+. A positive correlation existed for both species between the Kd values calculated from binding experiments and the Kact values obtained in adenylyl cyclase assays suggesting that the specific gonadotropin-binding sites present in rabbit and rat luteal membranes represent receptors which mediate the stimulatory effect of LH. The magnitude of the Mg2+-induced increase in affinity of a given gonadotropin preparation for its receptor was correlated with the efficacy with which that gonadotropin stimulated luteal adenylyl cyclase activity in both the rabbit and rat.

Adenylyl Cyclases↗
PubMed1986

Seasonal variations in the responses of luteal and follicular luteinizing hormone-stimulated adenylyl cyclases to estradiol implants and implant removal in pseudopregnant rabbits.

Summer rabbits appeared to be less sensitive to the presence of exogenous estradiol or to the withdrawal of exogenous estradiol than were Winter rabbits. Suppression of the luteinizing hormone(LH)-stimulated adenylyl cyclase of corpora lutea by estradiol-filled silastic capsules, which resulted in serum estradiol concentrations approximately 3.8 X control (high level), appeared to be greater in Winter rabbits than in Summer rabbits. Both high level and low level (2.1 X control) estradiol implants suppressed follicular LH-stimulated adenylyl cyclase in Winter rabbits but neither size capsule had an effect in Summer rabbits. Serum LH concentrations were equally low in Winter rabbits using either high or low level estradiol implants, while only the high level implants caused a decrease in serum LH in Summer rabbits. Withdrawal of the exogenous estradiol caused a precipitous fall in serum progesterone concentrations in Winter rabbits; estradiol withdrawal in Summer rabbits was without effect.

Adenylyl Cyclases↗
PubMed1986

Association of pheochromocytoma with nephrocarcinoma. Case report and literature review.

We describe an unusual case of an elderly male with mild hypertension, who underwent a right radical nephrectomy for a nephrocarcinoma and developed accelerated hypertension intraoperatively. Postoperative pathology of the excised specimen revealed poorly differentiated nephrocarcinoma and pheochromocytoma of the right adrenal gland. The limited published literature on the association of nephrocarcinoma and pheochromocytoma is reviewed, and suggestions for optimal preoperative evaluation of these patients are offered.

Adrenal Gland Neoplasms↗
PubMed1986

Effects of N-ethylmaleimide on gonadotropin and beta-adrenergic receptor function coupled to rabbit luteal adenylyl cyclase.

The effects of the sulfhydryl-reactive alkylating agent N-ethylmaleimide (NEM) on the rabbit luteal adenylyl cyclase system were studied. Treatment of luteal membranes with NEM revealed three activities with differing sensitivities to NEM treatment. When luteal membranes were treated with NEM on ice for 30 min, it was found that NaF-stimulated adenylyl cyclase activity in cholate extracts of these membranes was most sensitive to this treatment. Half-maximal inhibition was obtained at 0.09 mM NEM. The activity of the stimulatory guanine nucleotide- and Mg-binding regulatory component (Ns), as assessed by functional reconstitution of NaF-stimulated adenylyl cyclase activity into membranes from the cyc-variant of the S49 mouse lymphoma, was less sensitive to this treatment, with half-maximal inhibition occurring at 0.69 mM NEM. In contrast, high affinity gonadotropin and beta-adrenergic binding, as assessed by competitive displacement of [125I]iodo-hCG by bovine LH and (-)3-[125I]iodocyanopindolol by isoproterenol, was unaffected by NEM concentrations up to 50 mM when membranes were treated on ice. However, when membranes were treated with NEM at 25 C for 30 min, high affinity gonadotropin and beta-adrenergic binding demonstrated similar sensitivities to NEM treatment, such that 50 mM NEM completely inhibited high affinity binding to both receptors. Under either of the conditions described above, neither the number of receptors nor the affinities of the labeled probes for their receptors were altered by NEM treatment. Thus, there appears to be at least three NEM-sensitive sites necessary for the functioning of the rabbit luteal adenylyl cyclase system, one associated with the catalytic component, one on Ns which interacts with the catalytic component, and one involved in high affinity agonist binding. Furthermore, it appears that formation of the high affinity binding state is regulated similarly for gonadotropin and beta-adrenergic receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1985

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component.

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.

Adenosine Diphosphate Ribose↗
PubMed1985

Invasive pulmonary aspergillosis complicating influenza A pneumonia in a previously healthy patient.

A rare occurrence of invasive pulmonary aspergillosis complicates influenza pneumonia in a previously healthy adult. Five other similar cases are reported in the literature. Both transient depression of cell-mediated immunity and loss of ciliary function in the tracheobronchial tree occurs during acute influenzal illness and may predispose to fungal superinfection. Early diagnosis and treatment of opportunistic Aspergillus infection complicating influenza is mandatory in view of the high mortality associated with this complication.

Adult↗
PubMed1984

Effects of guanine nucleotides and divalent cations on forskolin activation of rabbit luteal adenylyl cyclase: evidence for the existence of an inhibitory guanine nucleotide-binding regulatory component.

The effects of guanine nucleotides and divalent cations on the activation of rabbit luteal adenylyl cyclase by the diterpene forskolin were investigated. Saturating concentrations of forskolin elicited 10- to 15-fold stimulation of adenylyl cyclase activity in the absence of added guanine nucleotide. No lag was observed in the time course of forskolin-induced activation. Addition of 10 microM guanosine triphosphate (GTP) and guanyl-5'-yl imidodiphosphate [GMP-P(NH)P] inhibited forskolin activation by 10-15% and 30-40%, respectively, in the presence of 3.0 mM MgCl2. GMP-P(NH)P was more potent than GTP in inhibiting forskolin activation of adenylyl cyclase having an IC50 of 36 nM compared to 610 nM for GTP. In contrast, the Kact for stimulating adenylyl cyclase activity by both GMP-P(NH)P and GTP were similar, 1.00 and 0.86 microM, respectively. GMP-P(NH)P-induced inhibition of the forskolin-activated enzyme was not due to the hysteretic nature of GMP-P(NH)P activation of luteal adenylyl cyclase, as addition of GMP-P(NH)P to an enzyme that had been treated 5 min earlier with forskolin resulted in the immediate inhibition of enzymatic activity. Addition of GMP-P(NH)P to a concentration-effect curve for forskolin increased the Kact value for forskolin form 7.18 to 26.8 microM. There was a different MgCl2 concentration requirement for maximal stimulation of luteal cyclase by GMP-P(NH)P (8 mM MgCl2) and maximal inhibition of forskolin-stimulated activity by GMP-P(NH)P (0.5-0.6 mM MgCl2). Further, MnCl2 concentrations above 1.0 mM completely abolished the inhibitory action of GMP-P(NH)P on forskolin activation of luteal cyclase. In fact, at 2.0 mM MnCl2, adenylyl cyclase activity in the presence of GMP-P(NH)P plus forskolin was greater than that of forskolin alone. Thus taken together, these findings suggest that the rabbit corpus luteum contains an inhibitory guanine nucleotide-binding regulatory component in addition to a stimulatory regulatory component. Further, these components demonstrate differing requirements for both guanine nucleotides and divalent cations in order to interact with the catalytic moiety of adenylyl cyclase. The existence of such an inhibitory component suggests the presence of an inhibitory receptor in the corpus luteum which could negatively regulate adenylyl cyclase resulting in the inhibition of cAMP production and reduced progesterone output from the corpus luteum under normal physiological conditions.

Adenylyl Cyclases↗
PubMed1983

Enkephalin-mediated inhibition of forskolin-stimulated rabbit luteal adenylyl cyclase activity.

Forskolin at 25-100 microM elicited 10- to 15-fold stimulation of rabbit luteal adenylyl cyclase activity in the absence of guanine nucleotides. Addition of saturating concentrations of GTP or guanyl-5'-yl imidodiphosphate [GMP-P(NH)P] inhibited forskolin stimulation by 15-25% and 35-45%, respectively, in Na+-free media. The further addition of 8 microM [D-Ala2, Met5] enkephalin amide (Da-ENK) caused an additional 16-24% inhibition of activity in the presence of GTP plus forskolin, but did not alter enzymatic activity in the presence of forskolin alone or forskolin plus GMP-P(NH)P. Inhibition by guanine nucleotide alone or Da-ENK plus GTP was only observed in the presence of forskolin. Maximal inhibition by Da-ENK was observed at 25 microM forskolin. Da-ENK reduced the IC50 for GTP by 2.3-fold but did not alter the IC50 for GMP-P(NH)P. Addition of Na+ above 3 mM attenuated the inhibitory responses to GTP and GTP plus Da-ENK, but not to GMP-P(NH)P or GMP-P(NH)P plus Da-ENK. Above 100 mM, Na+ inhibited enzymatic activity in the presence of forskolin, forskolin plus GTP and forskolin plus GMP-P(NH)P in the absence and presence of Da-ENK. These findings suggest that the rabbit corpus luteum contains an inhibitory receptor for opiate peptides that couples to adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1982

Temporal characteristics of gonadotropin interaction with rabbit luteal receptors and activation of adenylyl cyclase: comparison to the mode of action of catecholamine receptors.

The temporal characteristics of gonadotropin (LH and hCG) and catecholamine interaction with their luteal receptors and activation of adenylyl cyclase were studied in rabbit luteal membranes. studies on hormone-receptor interaction showed that, once bound, [125I]iodo-hCG dissociated from its receptor very slowly. If excess LH was added 30 min after the initiation of [125I]iodo-hCG binding, 85% of the [125I]iodo-hCG bound at 30 min was still bound to the luteal receptors 4.5 h later. The rate of dissociation of [125I]iodo-hCG from its receptor was not altered by 100 microM GTP, 2 mM MgCl2, or GTP plus MgCl2. The slow rate of [125I]iodo-hCG dissociation observed at 30 min was not due to a time-dependent change in the hormone-receptor complex, as the dissociation of [125I]iodo-hCG was equally slow 5 min after the initiation of the binding reaction. Studies on the activation of luteal adenylyl cyclase by LH showed that stimulation by 1 microgram/ml ovine LH (oLH) could be prevented but, once initiated, could not be reversed by antiserum to oLH. This indicates that once bound to the rabbit luteal LH receptor, oLH causes persistent activation of rabbit luteal adenylyl cyclase. In contrast, the activation of luteal adenylyl cyclase by 1 microM isoproterenol could be completely reversed by the addition of 50 nM propranolol 5 min after the initiation of the reaction. The inhibitory effect of the propranolol could be completely overcome by the addition of excess isoproterenol, indicating that catecholamine binding to its luteal beta-receptor is readily reversible. Thus, there appears to be a basic difference in the mechanism by which the gonadotropins and catecholamines interact with their receptors and activate the rabbit luteal adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1980

Regulation of hormone-receptor coupling to adenylyl cyclase. Effects of GTP and GDP.

GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations giving 50% inhibition were determined for GTP- and GDP-mediated inhibition of guanyl-5'-yl imidodiphosphate stimulation in the absence and presence of stimulatory hormones. In all four systems studied, GTP and GDP interacted with about equal potency and hormonal stimulation was not accompanied by a selective decrease in affinity for GDP. One way to explain all of the results obtained is to view hormonally sensitive adenylyl cyclase systems as two-state enzymes whose activities are regulated by GTP and GDP through an allosteric site related to the catalytic moiety, and receptors as entities that are inactive and hence unable to couple unless occupied by hormones and activated by any guanine nucleotide through a distinct receptor-related process.

Adenylyl Cyclases↗