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W Rosenthal

Publications and source records attributed to W Rosenthal.

At least 91 records · Page 5Linked to original sources
PubMed1989

Secretion-stimulating and secretion-inhibiting hormones stimulate high-affinity pertussis-toxin-sensitive GTPases in membranes of a pituitary cell line.

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing-hormone-releasing hormone (LHRH) stimulates and somatostatin inhibits voltage-dependent Ca2+ channels of GH3 cells via pertussis-toxin-sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627-1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis-toxin-sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin-releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis-toxin-independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to elucidate which of the hormone receptors interact with pertussis-toxin-sensitive and -insensitive G-proteins, we measured the effects of LHRH, somatostatin and TRH on high-affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high-affinity GTPase by 20%, somatostatin by 25%. Maximal hormone effects were observed at a concentration of about 1 microM. Pretreatment of cells with pertussis toxin abolished pertussis-toxin-catalyzed [32P]ADP-ribosylation of 39-40-kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertussis toxin. Somatostatin but not LHRH and TRH decreased forskolin-stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis-toxin-sensitive and -insensitive G-proteins, whereas effects of somatostatin are exclusively mediated by pertussis-toxin-sensitive G-proteins.

Adenylate Cyclase Toxin↗
PubMed1989

Severe intrahepatic cholestasis in patients treated with recombinant interleukin-2 and lymphokine-activated killer cells.

Immunotherapy with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK) has become a new form of therapy that has been shown to induce remissions in patients with renal cell carcinoma and melanoma. Despite encouraging results, this form of therapy has been associated with increasing toxicity, often requiring therapy in an intensive-care unit. In this report severe intrahepatic cholestasis occurred in two patients receiving rIL-2 and LAK cells. This form of cholestasis appeared to be directly related to rIL-2 administration at a doses of 2 x 10(6) U/m2 and 3 x 10(6) U/m2 t.i.d. A liver biopsy showed moderate hepatocellular bile stasis, with lobular and portal inflammation. All other studies for potential cause of this cholestasis were negative, including studies for metastatic disease. When therapy was discontinued, evidence for cholestasis and bile stasis resolved. We conclude that rIL-2 is a drug with a potential to induce severe hepatic injury that is reversible upon cessation of therapy with rIL-2. Further care should be exercised when rIL-2 is administered to patients with abnormal liver function.

Adult↗
PubMed1989

G-protein alpha-subunits in cytosolic and membranous fractions of human neutrophils.

In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.

Cell Membrane↗
PubMed1989

[Bidirectional hormonal modulation of voltage dependent ca2+ channels].

The role of guanine nucleotide-binding proteins (G-proteins), acting as transducers between membranous receptors activated by extracellular signals and enzymatic effectors controlling the concentrations of intracellular signal molecules, is well established. G-proteins are also involved in the hormonal modulation of voltage-dependent Ca2+ channels. In various cell types, the increase in intracellular signal molecules via G-protein-coupled receptors causes activation of protein kinases which may stimulate or inhibit voltage-dependent Ca2+ channels. For example, voltage-dependent Ca2+ channels of cardiac and skeletal myocytes are stimulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase. Other protein kinases, i.e., cyclic guanosine monophosphate (cGMP)-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase C, also appear to be involved in the hormonal modulation of Ca2+ channels. According to this principle, G-proteins exert a distant control of ion channel activity. In addition, there appears to exist another mechanism which does not involve intracellular signal molecules or protein kinases stimulated by intracellular signal molecules. The only signal transduction components identified so far include receptors, G-protein and Ca2+ channels. Ca2+ channel modulations following this apparently membrane-confined mechanism have been described to occur in neuronal, endocrine and cardiac cells. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis-toxin-sensitive G-protein, possibly G0. The G-protein involved in the hormonal stimulation of Ca2+ channels in adrenocortical and pituitary cells may represent a pertussis-toxin-sensitive G-protein of the Gi-type. The choleratoxin-sensitive G-protein, Gs, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells.

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes.

Amino Acid Sequence↗
PubMed1988

Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin.

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.

ADP Ribose Transferases↗
PubMed1988

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go.

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.

Adipose Tissue↗
PubMed1988

Degradation of inositol 1,3,4,5-tetrakisphosphates by porcine brain cytosol yields inositol 1,3,4-trisphosphate and inositol 1,4,5-trisphosphate.

Inositol 1,3,4,5-tetrakisphosphates (Ins(1,3,4,5)P4), 32P-labelled in positions 4 and 5 were prepared enzymatically, using [4-32P]-phosphatidylinositol 4-phosphate (PtdInsP) and [5-32P]phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates, respectively. Degradation studies of Ins(1,3,4,5)P4, using an enriched phosphatase preparation from porcine brain cytosol, led to the formation of two inositol trisphosphate isomers which were identified as inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). This novel degradation pathway of Ins(1,3,4,5)P4 to Ins(1,4,5)P3 provides an additional source for the generation of Ins(1,4,5)P3, involving a 3-phosphatase.

Animals↗
PubMed1988

Activation of protein kinase C by cis- and trans-fatty acids and its potentiation by diacylglycerol.

Both cis- and trans-unsaturated but not saturated fatty acids activated protein kinase C purified to apparent homogeneity from rat brain. Fatty-acid-induced enzyme activation was not more than additive with that by phospholipids and was potentiated by diacylglycerol. Recently, we demonstrated that cis- and trans-unsaturated fatty acids induced platelet aggregation and phosphorylation of specific proteins. Both events were potentiated by a cell-permeable diacylglycerol [(1987) Biochem. Biophys. Res. Commun. 149, 762-768]. Thus, trans-unsaturated fatty acids may provide useful experimental tools for the study of protein kinase C activation in vitro and in vivo. Our results suggest that fatty acids and diacylglycerol may synergistically be involved in hormonal stimulation of protein kinase C, as certain hormonal stimuli cause release of diacylglycerol and fatty acids from phospholipids by parallel activation of phospholipases C and A2.

Animals↗
PubMed1988

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides.

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.

Adenine Nucleotides↗
PubMed1988

[Functional guanine nucleotide-binding proteins in receptor-mediated modulation of voltage-dependent ion channels].

G-proteins act as transducers between cell surface receptors activated by extracellular signals and enzymatic effectors which control the concentrations of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and calcium. The receptor/G-protein-induced changes of the intracellular concentration of such signal molecules correlates with activity changes of various voltage-dependent ion channels. In some instances, cytosolic signal molecules appear to interact directly with ion channels, thereby causing an alteration of ion channel activity. In other instances, signal molecules affect the function of ion channels by activating protein kinases which, in turn, phosphorylate either proteins constituting extracellular signal- and voltage-dependent ion channels or non-identified membranous regulatory components. Recent findings suggest a third, membrane-confined mechanism which does not involve cytosolic signal molecules but a close control of voltage-dependent ion channels by G-proteins. Ion channels that are modulated by extracellular signals according to this newly discovered principle include those for calcium and potassium in neuronal, cardiac and endocrine cells. G-proteins involved in the hormonal stimulation of potassium and calcium channels belong to the family of Gi-type G-proteins which are functionally uncoupled from activating receptors by pertussis toxin. In addition, the cholera toxin-sensitive G-protein, Gs, may directly stimulate cardiac calcium channels. Hormonal inhibition of calcium channels is possibly mediated by Go which, like G-proteins of the Gi-family, is functionally impaired by pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

[Guanidine nucleotide binding proteins as membrane signal transduction components and regulators of enzymatic effectors].

The vast majority of extracellular signals alters cell function by activating cell surface receptors. The transmembranous signalling process initiated by an activated receptor leads to the generation of an intracellular signal and eventually to a cellular response. In contrast to receptors that are permanently coupled to an enzyme or an ion channel representing the effector, a large number of surface receptors for hormones, neurotransmitters and receptors for exogenous chemical or physical stimuli reversibly interacts with membranous signal transduction components which, in turn, regulate intracellular messenger-generating effectors. The transducer molecules isolated so far form a family of guanine nucleotide-binding proteins (G- or N-proteins). All isolated G-proteins are composed of three different subunits (alpha, beta, gamma). The alpha-subunit, which is specific for the individual G-protein, binds and hydrolyzes GTP and is target of ADP-ribosylating bacterial toxins. Hormone-induced activation of a receptor causes interaction with the alpha-subunit of a G-protein and the exchange of bound GDP with GTP. The GTP-bound form of the alpha-subunit represents the active form of the G-protein, which is capable of stimulating or inhibiting the respective effector. The active state of the alpha-subunit is terminated by its inherent GTPase activity causing hydrolysis of bound GTP. The beta gamma-complexes of G-proteins are structurally very similar and functionally interchangeable; they appear to dissociate from the alpha-subunits during receptor activation of the G-protein. Possible functions of the beta gamma-complex are to anchor the non-activated G-protein in the membrane, to facilitate G-protein-receptor interaction, and to promote the inactive state of the alpha-subunit. G-protein-regulated effectors include enzymes, ion channels and probably transporters. The best studied G-protein-regulated enzyme is the retinal cyclic GMP-phosphodiesterase which is activated by bleached rhodopsin via the tissue-specific G-protein, termed transducin. The ubiquitously occurring membrane-bound adenylate cyclase is under dual control by families of stimulatory and inhibitory receptors, acting via G-proteins called Gs and Gi, respectively. Moreover, the receptor control of phospholipases A2 and C and probably of phospholipase D most likely involves G-proteins which have not yet been identified. Finally, the activity of NADPH oxidase of neutrophils and that of cyclic AMP phosphodiesterases in liver and fat cells may be regulated via G-proteins. Modulations of non-enzymatic effectors are reviewed elsewhere.

Animals↗
PubMed1988

Angiotensin II-induced stimulation of voltage-dependent Ca2+ currents in an adrenal cortical cell line.

Biochemical studies suggest that stimulation of aldosterone secretion by angiotensin II involves activation of voltage-dependent Ca2+ channels. We used an adrenocortical cell line (Y1) to study the effect of angiotensin II on transmembranous currents. The hormone (1 nM to 1 microM) caused depolarization of the plasma membrane (from -35 to 10 mV) and elicited repetitive action potentials. Using the whole-cell clamp technique, we identified two types of voltage-dependent Ca2+ currents which differed with respect to their threshold potential and time course of inactivation. Angiotensin II (1 nM to 1 microM) stimulated a slowly inactivating Ca2+ current on average up to 1.7-fold whereas a fast inactivating Ca2+ current remained almost unaffected by the hormone. Ca2+ currents were not influenced by forskolin (1 microM) or intracellularly applied cAMP (50 microM). Pretreatment of cells with pertussis toxin abolished the hormonal stimulation of the slowly inactivating Ca2+ current but was without effect on control currents. The toxin ADP-ribosylated a single membranous peptide of 40 kd Mr. An antiserum raised against a synthetic peptide corresponding to a region common to all sequenced alpha-subunits of guanine nucleotide-binding proteins (G-proteins) and an antiserum raised against a peptide corresponding to a region of alpha-subunits of Gi-like G-proteins reacted with membranous 40 kd peptides, whereas an antiserum raised against a synthetic peptide corresponding to a region specific for the alpha-subunit of the G-protein, G0, failed to recognize a peptide in the 39 to 40 kd region.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.

Animals↗
PubMed1988

A radiometric method for the determination of NADH in subpicomole amounts.

A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [32P]orthophosphate from [gamma-32P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-32P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [32P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-32P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10(4)-fold excess of NAD over NADH.

Adenosine Triphosphate↗
PubMed1988

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors.

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.

Animals↗