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W Rosenthal

Publications and source records attributed to W Rosenthal.

At least 73 records · Page 4Linked to original sources
PubMed1991

Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents.

The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively.

Animals↗
PubMed1991

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate.

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.

Amino Acid Sequence↗
PubMed1991

Evidence for opioid receptor-mediated activation of the G-proteins, Go and Gi2, in membranes of neuroblastoma x glioma (NG108-15) hybrid cells.

In membranes of neuroblastoma x glioma (NG108-15) hybrid cells, the photoreactive GTP analog, [alpha-32P] GTP azidoanilide, was incorporated into 39-41-kDa proteins comigrating in urea-containing sodium dodecyl sulfate-polyacrylamide gels with immunologically identified G-protein alpha-subunits, i.e. a 39-kDa Go alpha-subunit, a 40-kDa Gi2 alpha-subunit, and a 41-kDa Gi alpha-subunit of an unknown subtype. The synthetic opioid, D-Ala2,D-Leu5-enkephalin (DADLE), stimulated photolabeling of the 39-41-kDa proteins. In the presence of GDP, which increased the ratio of agonist-stimulated to basal photolabeling, DADLE at a maximally effective concentration stimulated photolabeling of the 39- and the 40-kDa protein 2-3-fold. Somatostatin, adrenaline, and bradykinin were less potent than DADLE and, to varying degrees, stimulated photolabeling of the 40-kDa protein more than that of the 39-kDa protein. Prostaglandin E1 was inactive. The present data represent direct evidence for an activation of endogenous Go and Gi2 via opioid receptors and other receptors in the native membrane milieu.

Alprostadil↗
PubMed1991

Immunocytochemical localization of G-proteins (alpha subunits) in rat heart tissue.

The subcellular localization of peptide antibodies against G-protein alpha subunits was studied by the indirect immunogold technique with Lowicryl K4M embedded rat cardiac tissue. Two antibodies were used. The alpha common peptide antibody recognizes the alpha subunits of Gs, Gi and Go. The alpha i common peptide antibodies recognize the alpha subunits of all Gi alpha subtypes (G1-3). Immunoreactivity against alpha common and alpha i common antibodies was found along the cell surface of cardiomyocytes and endothelial cells. No immunoreactivity was seen on sarcoplasmic reticulum membranes. T-tubule membranes showed a little reactivity. Distribution patterns obtained with alpha common and alpha i common antibodies were identical. Immunogold was seen in cardiocytes from both atria and ventricles. In light-microscopical studies with peroxidase-conjugated secondary antibodies, heavy immunostaining was seen in SA and AV nodes.

Adenylyl Cyclases↗
PubMed1991

G protein function in the ischaemic myocardium.

The activity of adenylyl cyclase (AC) is controlled by its interaction with receptor-regulated G proteins. The efficiency to form cyclic AMP is strongly influenced by the amount, the subspecies and function of these regulatory proteins. An impairment of AC function has been shown to occur in sarcolemmal preparations (SL) of hearts exposed to either local or global ischaemia. To examine the contribution of G protein function to this phenomenon, cholera toxin (CT)-catalysed ADP-ribosylation of Gs and pertussis toxin (PT)-catalysed ADP-ribosylation of G proteins have been investigated in SL of porcine hearts exposed to global ischaemia for 15-45 min. ADP-ribosylation by CT of an approximately 45 kDa polypeptide was 0.46 +/- 0.06 and ADP-ribosylation by PT of three 39-41 kDa polypeptides was 4.77 +/- 0.77 pmol mg-1 protein in SL of non-ischaemic myocardium. Whereas no change was observed in CT-catalyzed ribosylation after 30 min of ischaemia, there was a reduction in PT-catalyzed ADP-ribosylation to 3.7 +/- 0.35 pmol mg-1 protein after 30 min of ischaemia. Prolongation of ischaemia to 45 min did not reduce further ADP-ribosylation capacity. Quantitative immunoblotting of PT-sensitive G proteins suggests that the diminution of ADP-ribosylation occurred because of a loss of alpha-subunits of G0, Gi-1, and Gi-2 from sarcolemmal membranes.

Adenosine Diphosphate Ribose↗
PubMed1991

Inhibition of Ca2+ channels via alpha 2-adrenergic and muscarinic receptors in pheochromocytoma (PC-12) cells.

Biochemical studies have suggested a voltage-dependent dihydropyridine-sensitive catecholamine release in adrenal chromaffin cells. This release is inhibited by activation of alpha 2-adrenergic and muscarinic receptors; the underlying molecular mechanism is not known. We used undifferentiated PC-12 cells to study the effect of epinephrine and carbachol on transmembranous currents. Applying the patch-clamp technique in the whole cell configuration and using Ba2+ as charge carrier, we identified a high voltage-activated Ca2+ channel current. Both epinephrine (10 microM, in the presence of 1 microM propranolol) and carbachol (10 microM) reversibly inhibited the Ca2+ channel current by 30-40%. Yohimbine abolished and clonidine mimicked the effect of epinephrine. Phenylephrine failed to inhibit the Ca2+ channel current. The effect of carbachol was abolished by atropine. Epinephrine and carbachol did not affect the Ca2+ channel current reduced by the dihydropyridine, PN 200-110 (1 microM), suggesting a selective inhibition of dihydropyridine-sensitive Ca2+ channels. The Ca2+ channel current and its inhibition by receptor agonists were not influenced by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM). Pretreatment of cells with pertussis toxin or intracellular infusion of the GDP analogue guanosine-5'-O-(2-thiodiphosphate) was without effects on the control Ca2+ channel current but abolished its hormonal inhibition. Four pertussis toxin-sensitive G proteins were identified in membranes of PC-12 cells: two members of the Gi family, Gi1 and Gi2, and two members of the Go family, Go2 and another Go subtype (possibly Go1). The present data indicate that activated alpha 2-adrenergic and muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ channels via pertussis toxin-sensitive G proteins without the involvement of a cAMP-dependent intermediate step.

Adrenal Gland Neoplasms↗
PubMed1991

Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.

Morphological, electrophysiological, and biochemical properties of H9c2 cells, a permanent cell line derived from rat cardiac tissue, were studied. Although the lectin binding pattern revealed similar sugar residues in the surface coat of H9c2 cells and isolated rat cardiocytes, heart-specific morphological structures could not be detected in H9c2 cells. Under physiological ionic conditions, H9c2 cells exhibited an outwardly rectifying, transient K+ current. When this current component was blocked by Ba2+ and Cs+, we observed an inward current through Ca2+ channels (15.8 +/- 2.2 pA/pF, n = 18, measured as Ba2+ current) that showed all characteristics of cardiac L-type currents. The activation kinetics were fast, and the current was stimulated by isoproterenol. The effect of isoproterenol was mimicked by forskolin or intracellularly applied cAMP. In radioligand binding experiments, we identified a specific, saturable, stereoselective and reversible high-affinity [3H]-(+)PN 200-110 binding with a dissociation constant Kd = 0.53 +/- 0.28 nM and a maximal specific binding of Bmax = 129.3 +/- 16.1 fmol/mg protein. There was an additional low-affinity/high-capacity binding site, which is unlikely to be related to a Ca2+ channel protein. Signal-transducing G proteins in membranes were characterized by [32P]ADP-ribosylation catalyzed by bacterial toxins and by the use of various antibodies. Cholera toxin substrates of 42 and 45 kd were identified that apparently correlated to Gs alpha-subunits. Pertussis toxin substrates of 40-41 kd were tentatively identified as Gi alpha-subunits. The G protein Go was absent or at least extremely low in concentration.

Adrenergic beta-Agonists↗
PubMed1991

Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells.

In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.

Affinity Labels↗
PubMed1991

Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.

A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.

Animals↗
PubMed1990

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family.

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.

Adenosine Diphosphate Ribose↗
PubMed1990

Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells.

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

Affinity Labels↗
PubMed1989

Phosphorylation of G-protein alpha-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects.

The phosphorylation of G-protein alpha-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [32P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein alpha-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as Gs and Gi alpha-subunit, respectively, by comparison with [32P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [32P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [32P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with G alpha s, since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the Gs alpha-subunit, the phosphoprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the Gs alpha-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein alpha-subunits in the action of insulin.

Adipose Tissue↗
PubMed1989

Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions.

The effects of GTP gamma S on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin-sensitive glucose transporter may be mediated by guanine nucleotide-dependent mechanisms. GTP gamma S and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin-treated cells in a concentration-dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATP gamma S and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter.

Adipose Tissue↗
PubMed1989

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins.

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.

Amino Acid Sequence↗
PubMed1989

The cytosolic G-protein alpha-subunit in human neutrophils responds to treatment with guanine nucleotides and magnesium.

The hydrodynamic properties of the Gi2 alpha-subunit in human neutrophil cytosol were analyzed. The S20,w value was 3.3 S at 30 degrees C and 4.1 S at 4 degrees C. Reconstitution of the cytosolic alpha-subunit with beta gamma-complex purified from porcine brain resulted in an S20,w value of 4.0 S at 30 degrees C and 4.2 S at 4 degrees C. Treatment of cytosol with G-protein-activating agents, GTP gamma S and MgCl2, decreased the S20,w value to 2.4 S at 30 degrees C and 2.9 S at 4 degrees C. Our results indicate that the cytosolic alpha-subunit of neutrophils is an inactive monomeric species at 30 degrees C capable of interacting with G-protein beta gamma-complex and responsive to treatment with activating agents leading to the two activated forms, alpha at 4 degrees C and alpha at 30 degrees C.

Animals↗