Biomedical subjects

H Shapira

Publications and source records attributed to H Shapira.

33 records · Page 2Linked to original sources

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↗

PubMed1988

Cellular immunity and suppressor T cell function in asthmatic children on prolonged ketotifen therapy.

This study was undertaken to investigate the effect of ketotifen on the immune system of asthmatic children receiving prophylactic treatment with ketotifen. E. rosettes, mitogenic response to phytohemagglutinin (PHA) and concanavalin A (Con A) as well as Con A induced suppression were measured in 15 asthmatic patients before and 1-3 months after treatment with ketotifen (1 mg x 2/day), and in 20 normal healthy controls. No significant difference in immunological parameters studied were found between the pre and post treatment periods in the asthmatic patients, neither was any difference demonstrated between the asthmatic and control group studied.

Adolescent↗

PubMed1988

Cellular immunity and suppressor T cell function in asthmatic children on prolonged theophylline therapy.

Measurements of cellular immunity including the percentage of T-cell mitogenic response of lymphocytes to phytohemagglutinin (PHA) and concanavalin A (Con A), as well as Con A-induced suppressor function, were determined in 20 asthmatic children (mean age 12.4 +/- 0.6 years) before and after 6 months of theophylline therapy. Only 9 of the 20 children patients that commenced the study completed the entire 6 months of therapy. A group of 37 healthy children (mean age 12.6 +/- 0.3 years) served as control. During the 6-month period of therapy, theophylline levels were maintained between 10 and 20 micrograms%. No significant difference in any of the parameters examined were found between the initial 20 asthmatic patients and the control group. Furthermore, in the 9 patients that were evaluated before and after 6 months of theophylline therapy no significant change in lymphocyte function including suppressor function was detected. We conclude that the beneficial effect of long-term theophylline therapy in asthmatic children is probably not related to an immuno-modulatory effect.

Asthma↗

PubMed1986

Double mitral valve orifice.

A two-day-old baby with a type-I truncus arteriosus and double mitral valve orifice is reported. The double mitral valve orifice and additional lesions of each subdivision of the mitral valve were clearly shown by two-dimensional echocardiography. The medial orifice had a cleft leaflet and the lateral one a parachute-like disposition of the tension apparatus. This is the first report of double mitral valve orifice associated with truncus arteriosus, and the first in which anomalies of the subdivisions of the mitral valve were detected by two-dimensional echocardiography.

Echocardiography↗

PubMedDate not supplied

The metabolism of microinjected inositol trisphosphate in Xenopus oocytes.

Microinjection of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) into Xenopus oocytes evokes a complex physiological response composed of a transient and a slow depolarizing chloride current. We investigated the relationship between intracellular levels of Ins(1,4,5)P3 and the kinetics of the physiological response. Microinjected Ins(1,4,5)P3 was slowly degraded following first order kinetics of disappearance (t1/2 = 10 min). The degradation products were inositol bisphosphate (InsP2), inositol monophosphate (InsP) and inositol, as well as inositol tetrakisphosphate (InsP4). The rate of degradation of injected 3[H]-Ins(1,4)P2 was much greater (t1/2 = 3 min), indicating that the conversion of InsP3 to InsP2 may be the rate-limiting step in the degradation process. The slow degradation of 3[H]-Ins(1,4,5)P3 was not a result of its conversion to Ins(1,3,4)P3 since no accumulation of InsP3 was observed within 10 min of microinjection of 3[H]-Ins(1,3,4,5)P4. Activation of protein kinase C (PK-C) with a phorbol ester transiently increased the rate of conversion of 3[H]-Ins(1,4,5)P3 to InsP2. This, however, did not significantly affect the overall kinetics of 3[H]-Ins(1,4,5)P3 disappearance. Our results indicate that the kinetics of Ins(1,4,5)P3 degradation do not correlate well with the termination of both the rapid and the slow components of the physiological response. The termination of the slow component of the response, however, may be related to the decay of Ins(1,4,5)P3-induced 45Ca efflux, which lasted about 10 min.

Animals↗