Biomedical subjects

H Shapira

Publications and source records attributed to H Shapira.

At least 19 recordsLinked to original sources

PubMed1992

Cloning and expression of a novel angiotensin II receptor subtype.

Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.

Amino Acid Sequence↗

PubMed1992

Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas.

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic cleavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence of the cDNA clone contained the five partial peptide sequences obtained from the purified protein. Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory protein-coupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.

Amino Acid Sequence↗

PubMed1992

Inositol trisphosphate may access calcium from stores not coupled to muscarinic receptors in Xenopus oocytes.

Oocytes of a large fraction of Xenopus females exhibit a complex response to acetylcholine (ACh) consisting of rapid, transient and prolonged, slow chloride currents. Frequent consecutive challenges or a single prolonged challenge with ACh result in a marked decrease in response amplitudes, i.e. refractoriness. In ACh-refractory oocytes, the response to injected inositol 1,4,5-trisphosphate (InsP3), the intracellular mediator of the ACh response, is not affected. Similarly, InsP3-evoked responses were obtained in oocytes that lacked muscarinic response or that lost their responsiveness as a result of progesterone-induced maturation. To investigate the mechanism of this phenomenon, we have depleted intracellular calcium stores by repeated challenges with ACh in calcium-free medium. Disappearance of the ACh response through depletion of the ACh-coupled calcium store did not prevent a subsequent response to InsP3. These results imply that InsP3 can mobilize calcium from other stores, not depleted by previous exposure to ACh. This finding is further reinforced by our results that demonstrate that ACh causes 45Ca efflux in responsive oocytes, while InsP3 in supramaximal concentrations does not induce 45Ca efflux. Indeed, InsP3 can induce 45Ca efflux only when more than 2 pmol/oocyte is injected. This is also the concentration of InsP3 that desensitizes the InsP3 response. These data suggest that InsP3 also releases cellular calcium from stores different from those mobilized by ACh.

Acetylcholine↗

PubMed1992

Molecular genetic analysis of two distinct receptors for mammalian bombesin-like peptides.

The mammalian bombesin-like peptides are known to be growth factors for certain cells with high-affinity bombesin receptors and have been implicated as autocrine growth factors influencing the pathogenesis and progression of a subset of human small-cell lung carcinomas. Thus, antagonists that interfere with bombesin receptor-ligand interaction might prove to be of value in treatment of gastrin-releasing peptide (GRP)-responsive tumors. A precise definition of the structure and properties of the bombesin receptors found on human lung cancer cells would provide important information for the design and rational application of such antagonists. Recently, we isolated cDNA clones encoding two distinct receptors for the mammalian bombesin-like peptides, GRP, and neuromedian B (NMB). The two receptors show 56% amino acid identity, encode seven putative transmembrane domains, and are members of the G-protein-coupled receptor superfamily. Ligand-binding studies show that while both receptors can be activated by either GRP or NMB, one receptor has a higher affinity for GRP than for NMB (GRP-R), while the other has a higher affinity for NMB than for GRP (NMB-R). A different spectrum of antagonists is needed to block responses from the two different receptors. These studies indicate that it will be critical in future studies to define which bombesin receptor subtypes are present on a given tumor to optimize the potential therapeutic benefit of antagonists in blocking growth.

Amino Acid Sequence↗

PubMed1991

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.

Amino Acid Sequence↗

PubMed1991

Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1.

Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

Amino Acid Sequence↗

PubMed1991

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.

Animals↗

PubMed1991

Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

Amino Acid Sequence↗

PubMed1991

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.

Amino Acid Sequence↗

PubMed1990

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes.

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.

Acetylcholine↗

PubMed1990

Two types of intrinsic muscarinic responses in Xenopus oocytes. I. Differences in latencies and 45Ca efflux kinetics.

Oocytes of 40% of Xenopus laevis frogs respond to acetylcholine (ACh). Oocytes of the majority of responders exhibit the common two-component depolarizing muscarinic response (mean amplitude of the rapid component, 54 nA). Oocytes of approximately 10% of the responders ("variant" donors) exhibit a muscarinic response characterized by a very large transient, rapid current (mean amplitude 1242 nA, reversal potential -33 mV). Responses in oocytes of variant donors exhibit further qualitative differences: pronounced desensitization (absent in oocytes of common donors), characteristic prolonged latency (5.4 vs 0.9 s in oocytes of common donors) and marked inhibition of the response by activators of protein kinase C. Rapid responses in oocytes of variant donors are usually increased by treatment with collagenase, which, in common oocytes, often results in a complete loss of the response that correlates with the loss of muscarinic ligand binding. The number of muscarinic receptors was similar in oocytes of both types of donors (2.2 vs 3.0 fmol/oocyte). Also, the responses of oocytes of variant donors to microinjections of CaCl2 or inositol 1,4,5-trisphosphate were similar to those found in cells of common donors. These findings imply that altered receptor number, calcium stores and/or chloride channel density are not responsible for the variant responses. However, ACh caused an sixteen-fold greater efflux of 45Ca in oocytes of variant donors (35 vs 2.2% of total label in oocytes of common donors). Hence, the characteristics of the variant response may be related to a more efficient coupling between receptor stimulation and the mobilization of cellular calcium.

Acetylcholine↗

PubMed1990

Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.

Acetylcholine↗

PubMed1989

Dual regulation by protein kinase C of the muscarinic response in Xenopus oocytes.

Muscarinic stimulation of follicle-enclosed oocytes of Xenopus laevis results in a complex response that involves both depolarizing and hyperpolarizing currents (Dascal and Landau 1980). We studied the involvement of protein kinase C (PK-C1) in the regulation of the acetylcholine-evoked rapid (D1) and of the slow (D2) depolarizing chloride (Cl-) currents. In oocytes maintained at -100 mV [the reversal potential of potassium (K+) ions] under two electrode voltage clamp, the PK-C activatory 4-beta-phorbol 12-myristate 13-acetate (beta-PMA, 0.1 microM) stimulated D1 by 99 +/- 17% and inhibited D2 by 67 +/- 6%, vs. untreated controls. The inactive isomer (alpha-PMA) or phorbol alone had no significant effect on the components of the muscarinic response. In order to identify the site of the regulation, we have microinjected the intracellular second messenger of calcium mobilization, inositol 1,4,5-trisphosphate (IP3). beta-PMA or the diacylglycerol analog, oleoylacetylglycerol (OAG) stimulated the rapid depolarizing current evoked by IP3 by 220 +/- 26% and 394 +/- 102%, respectively. alpha-PMA had little if any effect. The calcium-evoked Cl- current in oocytes pre-treated with the divalent cation ionophore A23187 was, on the other hand, inhibited by beta-PMA and OAG (by 82 +/- 6% and 54 +/- 6%, respectively). alpha-PMA and phorbol had a limited inhibitory effect. beta-PMA, but not alpha-PMA, also mildly inhibited the IP3-evoked increase in 45Ca efflux. The intracellular metabolism of IP3 was not affected by exposure to either beta-PMA or OAG.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1989

The immune status of uraemic children/adolescents with chronic renal failure and renal replacement therapy.

In adults with chronic renal failure (CRF) and/or renal replacement therapy (RRT) various immunological abnormalities have been described, but few data are available for the paediatric age group. We performed basic in vitro immunological studies in 26 patients 10 months-19 years of age with advanced renal failure, 11 with CRF (creatinine clearance 16.8 +/- 5.2 ml/min per 1.73 m2), 15 on RRT with haemodialysis (HD; n = 9) and continuous ambulatory peritoneal dialysis (CAPD; n = 6) as well as in 16 healthy controls. None had clinical evidence of deranged immune function. No significant differences were found in the percentages of B- and T-cells, T-cell subsets CD3, CD4, CD8 and mitogenic responses to phytohaemagglutinin and concanavalin A (Con A) between RRT patients (HD = CAPD) and control children. Most parameters in CRF patients were also normal, although they had a low percentage of B-cells (12.1 +/- 4.1; RRT: 19.7 +/- 6.5; controls: 18.5 +/- 7.1; P less than 0.01), relatively low levels of serum immunoglobulin G (948.4 +/- 209.4 mg/dl; HD: 1374.7 +/- 235.2 mg/dl; P less than 0.01; CAPD: 966.3 +/- 430.2 mg/dl, NS) and a high normal response to Con A (34.3 +/- 13.6 cpm x 10(-3); RRT: 24.5 +/- 11.3 cpm x 10(-3); controls: 23.4 +/- 9.9 cpm x 10(-3), P less than 0.01). All these values were, however, well within the normal accepted range. These data indicate that children/adolescents with CRF and/or RRT have no significant basic in vitro immunological defects. This study did not test the functional immune status of the young uraemic patients.

Adolescent↗

PubMed1988

Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes.

Shallow injection of inositol 1,4,5-trisphosphate (IP3) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl2 near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, beta-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP3 injections at either hemisphere but did not affect the amplitudes of the responses to CaCl2. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl2. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Subcostal cross-sectional echocardiographic imaging of patent ductus arteriosus.

A modified subcostal short-axis cross-sectional echocardiographic view for imaging the patent ductus arteriosus (PDA) was studied. A total of 22 newborns with PDA and various associated cardiac anomalies and 16 newborns without PDA were examined. The PDA was imaged in the subcostal view in 19 newborns. In the control group, the absence of the PDA was established in 13 newborns and considered probable in three. The PDA was optimally imaged in the subcostal view in patients with normal or enlarged pulmonary artery, mainly those with the hypoplastic left heart. Among the eight patients with small pulmonary artery (pulmonary atresia or severe tetralogy of Fallot), the PDA was imaged in the subcostal view in three and in the suprasternal view in eight. By combining the suprasternal and the subcostal views, the PDA was imaged in each instance.

Ductus Arteriosus, Patent↗

PubMed1988

Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes?

Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of 45Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

Acetylcholine↗

PubMed1988

Immunoregulation with levamisole in children with frequently relapsing steroid responsive nephrotic syndrome.

The immunological and clinical effects of levamisole were studied in 10 children with frequently relapsing steroid responsive nephrotic syndrome (SRNS). The efficacy of the drug was tested during remission of the disease with all patients on alternate day steroid therapy. The lymphocyte proliferative response to phytohemagglutinin (PHA), concanavalin-A (Con-A) and pokeweed mitogen (PWM) were normal. The Con-A induced suppressor T-lymphocyte activity of 7 patients was low before treatment with levamisole 8 +/- 3.7% and increased to normal values during therapy 34 +/- 6%; p less than 0.001 (control 32 +/- 5%). In these 7 children prednisolone dosage could be decreased significantly or discontinued altogether (44.1 +/- 5.3%). Patients without immunoregulatory abnormalities did not respond to levamisole. In 3 out of 4 children tested the percentage of OKT8+ cells rose during levamisole therapy from 19.7 +/- 2.1 to 37 +/- 2.3 (p less than 0.001), thus correcting the elevated pre-treatment OKT4+/OKT8+ ratio from 3.1 +/- 0.2 to 1.5 +/- 0.2; p less than 0.001 (control 1.47 +/- 0.2). These data support the hypothesis that abnormal immunoregulation may play a role in the pathogenesis of SRNS. Treatment with levamisole can be useful in some patients with the frequently relapsing form of the disease.

Adolescent↗