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J Abramowitz

Publications and source records attributed to J Abramowitz.

At least 19 recordsLinked to original sources
PubMed2007

Know thy neighbor: a survey of diseases and complex syndromes that map to chromosomal regions encoding TRP channels.

On the basis of their ever-expanding roles, not only in sensory signaling but also in a plethora of other, often Ca(2+)-mediated actions in cell and whole body homeostasis, it is suggested that mutations in TRP channel genes not only cause disease states but also contribute in more subtle ways to simple and complex diseases. A survey is therefore presented of diseases and syndromes that map to one or multiple chromosomal loci containing TRP channel genes. A visual map of the chromosomal locations of TRP channel genes in man and mouse is also presented.

Animals↗
PubMed2001

Ouabain-induced signaling and vascular smooth muscle cell proliferation.

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nm ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10(-9) m ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nm had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.

Animals↗
PubMed2000

Expression of plasma membrane calcium ATPases in phenotypically distinct canine vascular smooth muscle cells.

Our laboratory has identified at least two types of vascular smooth muscle cells (VSMCs) that exist in canine arteries and veins: type 1 cells, located in the media express muscle specific proteins but do not proliferate in culture; and type 2 cells, located in both media and adventitia, do not express muscle specific protein but proliferate in culture. Plasma membrane Ca(2+)-ATPases (PMCAs) have been implicated in proliferation control. The present study examines the expression of PMCA isoforms and calmodulin-binding domain splice variants in these two types of canine VSMCs. PMCA protein was found in both type 1 and type 2 cells. Reverse transcriptase-polymerase chain reaction assays were developed for canine PMCA calmodulin-binding domain splice variants. We cloned and sequenced isolates corresponding to PMCA1b, 4a and 4b from canine VSMCs. PMCA 2 and 3 were not detected. Freshly isolated type 1 cells expressed PMCA 1b, 4a and 4b, while freshly isolated type 2 cells expressed PMCA1b and 4b. Upon placement in culture, type 2 cells originating from either carotid artery or saphenous vein demonstrated a time-dependent upregulation of PMCA4a mRNA. Treatment with the phosphoinositide 3-kinase inhibitor wortmannin produced concentration-dependent inhibition of both PMCA4a upregulation and [(3)H]thymidine incorporation. These findings suggest a role for phosphoinositide 3-kinase in regulating PMCA expression, which may be important in the control of Ca(2+)-sensitive VSMC functions.

Alternative Splicing↗
PubMed1998

High glucose enhances growth factor-stimulated nitric oxide production by cultured rat mesangial cells.

Nitric oxide (NO) contributes to the alterations in glomerular hemodynamics and extracellular matrix accumulation observed in diabetic nephropathy. High glucose concentrations directly inhibit NO production by rat mesangial cells (RMC). However, the role of peptide growth factors and chemokines in regulating NO synthesis by RMC under normal and high glucose conditions has not been studied. Therefore, we examined the effect of IGF-I, EGF, TGF-beta and RANTES on NO production by RMC maintained in normal (5.6 mM) or high glucose (33.3 mM) for 48 h. No synthesis was determined by measuring nitrite accumulation in conditioned media with the Greiss reaction. In normal glucose media, IGF-I, EGF, and RANTES had no effect on nitrite accumulation while TGF-beta inhibited NO synthesis. In high glucose conditions, IGF-I and EGF significantly enhanced NO production. The effects of RANTES and TGF-beta were unchanged by an elevated glucose concentration. EGF-induced stimulation of NO production in high glucose media was associated with parallel alterations in iNOS gene and protein expression. The modest enhancement in nitrite accumulation provoked by IGF-I in high glucose conditions was not accompanied by demonstrable increases in iNOS mRNA abundance or protein content. In conclusion, peptide growth factors modulate the direct inhibitory effect of high glucose on NO production by cultured mesangial cells. These actions in vivo may limit the adverse consequences of reduced NO production in promoting diabetic nephropathy.

Animals↗
PubMed1997

Plasma membrane calcium ATPase isoform expression in cultured rat mesangial cells.

The maintenance of intracellular ionized calcium (iCa2+) in the submicromolar range is important for mesangial cell (MC) function, and, as in most mammalian cells, plasma membrane Ca(2+)-ATPases (PMCA) play an important role in the homeostatic process. Molecular studies have demonstrated four PMCA isoforms, each with multiple splice variants. The present study examines the expression of PMCA isoforms and calmodulin-binding region splice variants in cultured MC from Sprague-Dawley rats and from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats before and after the onset of hypertension in SHR. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, we have demonstrated PMCA1, -3, and -4, but not PMCA2, to be present in MC from these rat strains. Splice variant analysis revealed PMCA1a and -1b, PMCA3a, -3b, and -3c, and PMCA4a and -4b to be expressed in MC from all three strains. The relative quantities of PMCA1 and PMCA4 mRNA were not different in age-matched SHR vs. WKY rats, correlating with similar iCa2+ measurements. The expression of all three isoforms declined with age in SHR and WKY.

Alternative Splicing↗
PubMed1996

Ca-ATPase and bone cell mineralization.

The plasma membrane Ca-ATPases play an important role in the regulation of intracellular Ca2+ ion concentration by pumping Ca2+ out of the cell into the extracellular fluid at the expense of ATP. These pumps potentially play an important role in the delivery of Ca2+ during mineralization of hard tissues such as bone. The properties of the plasma membrane Ca2+ pump are compared with those of the sarco(endo)plasmic reticulum and the factors regulating pump function are presented. The different gene products for the plasma membrane Ca-ATPases are described as well as their known functional significance. Particular attention is paid on the plasma membrane Ca2+ pumps present in mineralizing tissues and evidence supporting a role for these pumps in the transcellular delivery of Ca2+ during the process of mineralization is also presented.

Animals↗
PubMed1995

Differential expression of plasma membrane calcium pump mRNA isoforms in rat osteoblast-like cells.

This study was conducted to identify the plasma membrane Ca2+ transport ATPase (PMCA) mRNA isoforms expressed in rat osteoblast-like cells as PMCAs are likely to participate in calcium deposition in bone. We designed oligonucleotide primers for each PMCA isoform based on rat cDNA sequences in order to develop reverse transcription polymerase chain reaction (RT-PCR) assays. Rat kidney total RNA was used to validate the assays as we have shown that each isoform is present in kidney. When used in RT-PCR assays each primer pair gave rise to a single major product of the appropriate size. Southern blot analysis of the PCR products with oligonucleotide probes specific for each isoform revealed that each probe hybridized only to the expected product. Reamplification of purified PCR products with probe and antisense primers gave rise to products of appropriate size, further confirming the identity of the products. Using these primers we have identified the presence of transcripts for PMCA1, 2 and 4 in RNA from UMR-106 osteoblasts and PMCA1 in RNA from ROS 17/2.8 osteoblasts. We conclude that the two major rat cell lines used as models to study osteoblast function differentially express PMCA mRNA isoforms.

Animals↗
PubMed1992

Evidence for a fourth rat isoform of the plasma membrane calcium pump in the kidney.

This study was conducted to identify plasma membrane Ca(2+)-transporting ATPases present in rat kidney. Characterization of the cDNAs of the plasma membrane Ca(2+)-ATPases revealed a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of the diversity of rat renal plasma membrane Ca(2+)-ATPases, we used the polymerase chain reaction to detect additional gene products in rat kidney mRNA that shared these conserved regions. Sequences corresponding to three previously known rat plasma membrane Ca(2+)-ATPases were obtained. In addition, we found sequence corresponding to a new putative plasma membrane Ca(2+)-ATPase. Our results demonstrate that the rat kidney contains at least four different plasma membrane Ca(2+)-ATPases and the complexity of this multigene family is greater than previously thought.

Amino Acid Sequence↗
PubMed1992

Microinjection of the alpha-subunit of the G protein Go2, but not Go1, reduces a voltage-sensitive calcium current.

Calcium currents can be modulated by receptor activation of the GTP-binding protein G(o). We have determined whether the two forms of G(o), Go1 and Go2, differentially regulate calcium current magnitude. Using identified neurons of the pond snail Helisoma, we demonstrate that a high-voltage-activated (HVA) calcium current is reduced by addition of the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and that this inhibition is mediated by a pertussis toxin (PTX)-sensitive G protein pathway. Using this calcium current as an assay for G protein activity, we microinjected GTP gamma S-activated alpha-subunits of G proteins into neuronal somata. We demonstrate that the calcium current is differentially regulated by the two forms of alpha o. Microinjection of alpha o2*, but not alpha o1*, reduces calcium current magnitude.

Amino Acid Sequence↗
PubMed1992

Reconstitution and partial purification of calcium transport activity from rat kidney cortex.

An ATP-dependent Ca2+ uptake system from rat renal cortical basolateral membranes was solubilized with Triton X-100 and reconstituted into liposomes with lecithin. In the presence of Mg2+, Ca2+ uptake in the reconstituted vesicles was time and ATP dependent and was inhibited by vanadate. Ca2+ uptake in basolateral membrane vesicles depleted of endogenous calmodulin was enhanced by exogenous calmodulin and depressed by R-24571. This sensitivity to calmodulin and R-24571 was lost upon reconstitution in the presence and absence of leupeptin. Vesicles containing Ca2+ uptake activity were separated by gradient centrifugation after Ca2+ was taken up and accumulated as calcium phosphate in the vesicles. This resulted in Ca2+ uptake activity that was enriched 25 times. However, Ca(2+)-dependent adenosinetriphosphatase (ATPase) activity was not enriched significantly. This Ca(2+)-ATPase had two kinetic forms for Ca2+: one was a high-affinity low-capacity form; the other had a low affinity and high capacity. The Ca(2+)-ATPase activity also had two kinetic forms for ATP. All kinetic forms were inhibited by Mg2+. Vanadate, calmodulin, and R-24571 had no effects on Ca(2+)-ATPase activity. A protein doublet of Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates was demonstrated at 125 and 136 kDa in the purified vesicles. This doublet was not altered by addition of leupeptin throughout the purification.

Adenosine Triphosphate↗
PubMed1991

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.

Adenylyl Cyclases↗
PubMed1991

TMB-8 prevents the hydroosmotic response to ADH in rabbit cortical collecting tubules.

Both AVP and dDAVP effect a transient increase in cytosolic free calcium (iCa2+) in cortical collecting tubule (CCT) cells. To investigate the physiological role of this increase in iCa2+, we examined the effect of TMB-8, a putative inhibitor of iCa2+ release, on the initial and sustained phase of AVP- and dDAVP-stimulated water permeability (Pf) in isolated, perfused CCTs. Pretreatment of tubules with TMB-8, 50 microM, suppressed the increase in osmotic water permeability (Pf) induced by 10 microU/ml AVP and dDAVP, but had no effect on the sustained phase of the response. When increased to 100 microM. TMB-8 inhibited the sustained phase of AVP action. A similar pattern was observed on AVP-stimulated adenyly cyclase activity in rabbit renal membranes. Pretreatment of tubules with 50 microM TMB-8 attenuated the initial increase in Pf in response to cholera toxin but not to 8-Br-cAMP or forskolin. There was no effect of this concentration of TMB-8 on the sustained phase of these agonists. These studies suggest that, in lower concentrations, TMB-8 inhibits the mobilization of iCa2+, which is important for the interaction of Gs with the catalytic unit of adenylyl cyclase and the initial increase in AVP-stimulated Pf. In higher concentrations, TMB-8 inhibits adenylyl cyclase activity directly.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Receptor-effector coupling by G proteins.

The primary structure of G proteins as deduced from purified proteins and cloned subunits is presented. When known, their functions are discussed, as are recent data on direct regulation of ionic channels by G proteins. Experiments on expression of alpha subunits, either in bacteria or by in vitro translation of mRNA synthesized from cDNA are presented as tools for definitive assignment of function to a given G protein. The dynamics of G protein-mediated signal transduction are discussed. Key points include the existence of two superimposed regulatory cycles in which upon activation by GTP, G proteins dissociate into alpha and beta gamma and their dissociated alpha subunits hydrolyze GTP. The action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP is emphasized, as is the role of subunit dissociation, without which receptors could not act as catalysts. To facilitate the reading of this review, we have presented the various subtopics of this rapidly expanding field in sections 1-1X, each of which is organized as a self-contained sub-chapter that can be read independently of the others.

Amino Acid Sequence↗
PubMed1990

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase.

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.

Adenosine Diphosphate Ribose↗
PubMed1990

Roles of G proteins in coupling of receptors to ionic channels and other effector systems.

Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels. The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels. One example of direct G protein gating is the atrial muscarinic K+ channel K+[ACh], an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV. Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS. A G protein, Gk, purified from human red blood cells (hRBC) activates K+ [ACh] channels at subpicomolar concentrations; its alpha subunit is equipotent. Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists. The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo. The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects. An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer. The techniques of molecular biology are now being used to specify G protein gating. A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k.

Animals↗
PubMed1989

Human chorionic gonadotropin-induced heterologous desensitization of rabbit luteal adenylyl cyclase is associated with altered receptor and G-protein function.

We injected hCG into pseudopregnant rabbits on day 7 of pseudopregnancy and analyzed changes in the components of luteal adenylyl cyclase system in order to determine which components are responsible for altered hormonal responsiveness upon desensitization. hCG-induced desensitization was homologous (loss of responsiveness to LH) early (first 6 h), then became heterologous (partial loss of responsiveness to catecholamines) later (12-48 h). The total number of LH receptors was reduced approximately 30% 3 h after treatment at a time when LH stimulation of adenylyl cyclase activity was not altered. Total LH receptors remained at this level until 24 h, when total receptors were reduced by 88%. While total LH receptor number remained constant, LH-stimulated adenylyl cyclase activity was declining to 57% of the control value at 12 h. Available unoccupied LH receptors were reduced by 96% at 12 h. The affinity of the occupied receptors was reduced 4-fold before down-regulation. The changes in beta-adrenergic receptor number paralleled the changes in catecholamine responsiveness. hCG treatment also altered luteal G-protein function, as assessed by reconstitution of adenylyl cyclase activity in S49 cyc- lymphoma membranes and ADP ribosylation by cholera and pertussis toxins. Isoproterenol (ISO)-reconstituting activities of luteal Gs (the stimulatory G-protein of adenylyl cyclase) were depressed by 65% 12-48 h after hCG treatment, the same time as reduced catecholamine responsiveness. In contrast, NaF-reconstituting activities were at control levels at 12 h and reduced by 55% at 24 and 48 h. Pertussis toxin's ability to ADP ribosylate alpha i 40 was increased 3 and 6 h after treatment, while cholera toxin's ability to ADP ribosylate alpha s 45 was reduced throughout the study period. These studies demonstrate that hCG-induced heterologous desensitization results in a complex series of changes in beta-adrenergic and LH receptors as well as G-protein function, which account for the altered hormonal responsiveness.

Adenylate Cyclase Toxin↗