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G Schultz

Publications and source records attributed to G Schultz.

At least 127 records · Page 7Linked to original sources
PubMed1994

Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells.

The alpha-subunits of the widely expressed G-proteins Gq and G11 indistinguishably activate beta-isoforms of phospholipase C. In this report we have tested whether differences exist in the activation of both G-proteins via phospholipase C-linked receptors. We found that bombesin and vasopressin, with very similar potencies and time dependencies, induce the activation of both Gq and G11 in Swiss 3T3 cells, suggesting that these G-proteins, at least in part, serve interchangable functions.

3T3 Cells↗
PubMed1994

Maitotoxin activates cation channels distinct from the receptor-activated non-selective cation channels of HL-60 cells.

We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493]. Stimulation of dibutyryl cyclic AMP-differentiated HL-60 cells with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 0.1 microM), the Ca(2+)-ATPase inhibitor thapsigargin (0.1 microM) or maitotoxin (25 ng/ml) resulted in an increase in cytoplasmic free calcium concentration ([Ca2+]i). Unlike fMLP and thapsigargin, maitotoxin produced no increase in [Ca2+]i in the absence of extracellular Ca2+. The increase in [Ca2+]i induced by fMLP was blocked by pretreatment with pertussis toxin (100 ng/ml for 24 h) but not that induced by maitotoxin. Similarly, the increase in [Ca2+]i produced by fMLP but not that produced by maitotoxin was inhibited by pretreatment with phorbol myristate acetate (100 ng/ml). Both fMLP- and maitotoxin-induced increases in [Ca2+]i were blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl)-1H-imid azole hydrochloride (SKF 96365) in a concentration-dependent manner. However, the maitotoxin-induced increase in [Ca2+]i was more sensitive to inhibition by SKF 96365 than the fMLP-induced increase. fMLP-induced increases in [Ca2+]i were blocked by cations with Gd3+ being more effective than Cd2+, whereas for maitotoxin Cd2+ was more effective than Gd3+. Both fMLP and thapsigargin stimulated quenching of Fura-2 fluorescence in the presence of extracellular Mn2+, whereas maitotoxin produced no Mn2+ quenching. Taken together these results suggest that maitotoxin does not stimulate the nonselective cation channel activated by fMLP, but instead activates Ca2+ influx by a different mechanism.

Bucladesine↗
PubMed1994

Purification of a novel G-protein alpha 0-subtype from mammalian brain.

Three distinct G-protein alpha o-subtypes, i.e. alpha o1, alpha o2 and a newly observed 'alpha o3', are present in membranes of mammalian brain, each appearing as two isoforms on SDS/PAGE. Only alpha o1 and alpha o2 appear to be substrates for pertussis toxin (PTX) when membranes or partially purified proteins are examined. In order to elucidate the apparent PTX-resistance of the third alpha o-subtype, we purified alpha o3 from porcine and bovine brain membranes. During the purification procedures, alpha o3 occurred in its dissociated monomeric form and, together with beta gamma-complexes, as a heterotrimer. In a first attempt, we used purified G-protein alpha i/alpha o-mixtures to obtain a final separation of alpha o3. By using f.p.l.c. anion-exchange chromatography on a Mono Q column, complete separation of alpha i1 and alpha o2 was achieved. Partial resolution of alpha o1, alpha i2 and alpha o3 was observed; alpha o3 was eluted between alpha o1 and alpha i2. If alpha o-subunits free from alpha i contaminants were loaded on to the Mono Q column, all three alpha o-subtypes were resolved. The identity of the third subtype as an alpha o-subtype was confirmed by sequence analysis of tryptic fragments. All three alpha o-subtypes bound GTP[S]. Purified alpha o3 was ADP-ribosylated when subjected to PTX treatment in the presence of beta gamma-subunits, and on SDS/PAGE the mobility of alpha o3 was similar to that of ADP-ribosylated alpha o1. On the basis of results obtained with subtype-specific antibodies, the third alpha o-subtype is immunologically more related to alpha o1 than to alpha o2. Purified alpha o3 failed to reconstitute carbachol-mediated inhibition of Ca2+ current in PTX-pretreated SH-SY5Y-cells, whereas alpha o1 and alpha o2 did successfully restore this effect. We conclude that the novel alpha o3 forms differs from alpha o1 and alpha o2 in its primary structure and may be involved in signal-transduction pathways other than those described for alpha o1 and alpha o2.

Adenosine Diphosphate Ribose↗
PubMed1994

The human thyrotropin receptor activates G-proteins Gs and Gq/11.

The human thyrotropin receptor leads upon activation to the stimulation of phospholipase C and adenylyl cyclase. It is presently not known whether this bifurcating signaling occurs via two different G-proteins (Gq/11 and Gs) or via one G-protein (Gs). Receptor-activated Gs releases beta gamma subunits and alpha s, which then could regulate phospholipase C and adenylyl cyclase, respectively. In order to elucidate the signaling pathways induced by the activated thyroid-stimulating hormone (TSH) receptor, we studied the coupling of the TSH receptor to Gs and Gq/11 in human thyroid membranes. TSH concentration dependently led to the activation of two forms of Gs (Gs short and Gs long) as well as of Gq and G11, demonstrating that signaling pathways induced by TSH already bifurcate in the course of the receptor-G-protein interaction. These data strongly suggest the concept that phospholipase C and adenylyl cyclase activation through the TSH receptor are mediated by Gq/11 and Gs, respectively.

Adenylyl Cyclases↗
PubMed1994

High-performance liquid chromatographic determination of 3-hydroxy-3-methylglutaryl coenzyme A as diphenacyl ester of 3-hydroxy-3-methylglutarate.

A method for derivatization of (S)-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) by phenacylation in tetrahexylammonium hydrogensulphate as phase-transfer reagent and high-performance liquid chromatography of the HMG-diphenacyl ester is described. To avoid interference with buffers and other ions used to dissolve biological material, the alkaline hydrolysate of CoA esters has to be neutralized with phosphoric rather than hydrochloric acid. The latter reacts with phenacyl bromide. The pH during phenacylation has to be maintained in the range 6-7 to provide sufficient dissociation of HMG.

Acyl Coenzyme A↗
PubMed1994

Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] is a hemoprotein that exists as a heterodimer; the heme moiety has been proposed to bind nitric oxide, resulting in a dramatic activation of the enzyme. Mutation of six conserved His residues reduced but did not abolish nitric oxide stimulation whereas a change of His-105 to Phe in the beta 1 subunit yielded a heterodimer that retained basal cyclase activity but failed to respond to nitric oxide. Heme was not detected as a component of the mutant heterodimer and protophorphyrin IX failed to stimulate enzyme activity. The activity of the His mutant was almost identical to that of the wild-type enzyme in the presence of KCN, suggesting that disruption of heme binding is the principal effect of the mutation. Thus, the mutation provides a means to inhibit the nitric oxide-sensitive guanylyl cyclase signaling pathway.

Amino Acid Sequence↗
PubMed1994

G12 and G13 alpha-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues.

The cDNAs of two putatively pertussis toxin-insensitive G-protein alpha-subunits, alpha 12 and alpha 13, were recently cloned. mRNA analyses based on the reverse transcriptase polymerase chain reaction indicated a widespread distribution of both mRNAs [Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. USA 88, 5582-5586]. Generating specific antibodies directed against internal and C-terminal peptide sequences, we identified alpha 12 protein in all and alpha 13 protein in most tissues and cell lines tested. No species differences were observed, indicating a high degree of identity between mammalian species. Strong immunoreactive signals of both proteins were obtained in neuronal cell membranes of various species. Our results support the hypothesis that G12 and G13 are involved in pertussis toxin-insensitive pathways of signal transduction common to most tissues.

Amino Acid Sequence↗
PubMed1994

The alpha-subunits of G-proteins G12 and G13 are palmitoylated, but not amidically myristoylated.

The alpha-subunits of the G-proteins G12 and G13 were expressed with a baculovirus system in insect cells and analysed for acylation. Both proteins incorporated tritiated palmitic and to a lesser extent also tritiated myristic acid. Radiolabel from both fatty acids was sensitive to treatment with neutral hydroxylamine. This result supports a thioester-type fatty acid bond and argues against amidical N-myristoylation. Fatty acid analysis after labeling with [3H]palmitic acid showed that palmitate represents the predominant fatty acid linked to G alpha 12 and G alpha 13. Separation of cells into cytosolic and membranous fractions revealed that palmitoylated alpha-subunits of G12 were exclusively membrane-bound, whereas [35S]methionine-labeled proteins were detected in soluble and particulate fractions. Inhibition of protein synthesis with cycloheximide did not block palmitoylation of the alpha-subunits, which indicates that palmitoylation occurs independently of protein synthesis.

Acylation↗
PubMed1994

Some taste substances are direct activators of G-proteins.

Amphiphilic substances may stimulate cellular events through direct activation of G-proteins. The present experiments indicate that several amphiphilic sweeteners and the bitter tastant, quinine, activate transducin and Gi/Go-proteins. Concentrations of taste substances required to activate G-proteins in vitro correlated with those used to elicit taste. These data support the hypothesis that amphiphilic taste substances may elicit taste through direct activation of G-proteins.

Animals↗
PubMed1994

G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.

Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Stimulation of mitogen-activated protein kinase activity by different secretory stimuli in rat basophilic leukemia cells.

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.

Animals↗
PubMed1994

The isoquinoline derivative LOE 908 selectively blocks vasopressin-activated nonselective cation currents in A7r5 aortic smooth muscle cells.

The effect of (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl- N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of -60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca(2+)-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC50 of 560 nM, whereas dihydropyridine-sensitive Ba2+ current through voltage-dependent Ca2+ channels was blocked with an IC50 of 28 microM. Another organic blocker of receptor-mediated Ca2+ entry, 1-beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba2+ current with similar IC50 values of 13 microM and 8 microM, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd3+ > La3+ > Cd2+. Vasopressin-induced non-selective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5'-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Ca(2+)-activated K(+)-current, which overlapped with the nonselective cation conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗
PubMed1994

Histamine H1-receptors in HL-60 monocytes are coupled to Gi-proteins and pertussis toxin-insensitive G-proteins and mediate activation of Ca2+ influx without concomitant Ca2+ mobilization from intracellular stores.

The results of binding studies suggest the presence of histamine H1-receptors in human monocytes, but it is not known whether these receptors are functionally active. This prompted us to study the effects of histamine (HA) on cytosolic Ca2+ concentration ([Ca2+]i) and superoxide anion (O2-) formation in HL-60 cells differentiated towards monocytes with 1 alpha,25-dihydroxycholecalciferol. In HL-60 monocytes, HA increased [Ca2+]i with a half-maximal effect at 8 microM and a maximum at 30-100 microM. Pertussis toxin (PTX) partially inhibited the stimulatory effects of HA on [Ca2+]i. Betahistine, a weak partial H1-receptor agonist, also increased [Ca2+]i, whereas H2- and H3-receptor agonists were ineffective. H1- but not H2- and H3-receptor antagonists inhibited HA-induced rises in [Ca2+]i. HA-induced rises in [Ca2+]i were desensitized in a homologous manner and were also inhibited by the activator of protein kinase C, 4 beta-phorbol 12-myristate 13-acetate. Various protein kinase C inhibitors did not interfere with homologous desensitization. The stimulatory effects of HA on [Ca2+]i were completely dependent on the presence of extracellular Ca2+ and were inhibited by the blocker of non-selective cation (NSC) channels, 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1 H-imidazole hydrochloride (SK & F 96365). HA was much less effective than the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), to induce rises in [Ca2+]i. Unlike fMLP, HA did not activate O2- formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1994

Complex information processing by the transmembrane signaling system involving G proteins.

Much of the information cells receive is transduced by a membranous signaling system that uses heterotrimeric guanine nucleotide binding proteins (G proteins) to functionally couple cell surface receptors to a variety of effectors. During recent years it has been shown that receptors, G protein alpha, beta and gamma subunits as well as effectors involved in this signaling system exhibit a remarkable structural diversity and that the interactions of these components display a bewildering complexity. Even though many questions remain to be answered, it is becoming obvious that G proteins form the basis of a complex membranous signaling network which allows the cell to coordinate and to process incoming signals already on the level of the plasma membrane.

Animals↗
PubMed1994

Transforming growth factor-alpha is a constant component of human tear fluid.

Growth factors are known as a family of polypeptides with powerful influences on angiogenesis, tumor growth and wound healing. Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are structurally related peptides which bind to the same receptor, EGF-R, and also exert similar effects. EGF is a natural component of human tears, and ocular disease leads to decreased concentrations in tear fluid. Using a sensitive radioimmunoassay we investigated whether TGF-alpha is also to be considered a natural component of tear fluid and in which concentrations it occurs. All of 46 tear fluid samples from 24 volunteers contained TGF-alpha. The mean concentration was 161.4 pg TGF-alpha/ml (SD 11.6 pg). No statistically significant correlation was found between tear fluid flow and TGF-alpha concentration in the sample. However, the concentration of TGF-alpha in tear fluid decreased significantly with increasing total time of tear fluid collection (P = 0.002). TGF-alpha levels in samples collected from males (n = 16) appeared to be higher (mean 247.0 pg/ml, SD 15.3 pg/ml) than in those from females (n = 30; mean 180.0 pg/ml, SD 8.5 pg/ml; P = 0.05). No correlation was found between the age of the individuals and the concentration of TGF-alpha in their tear fluid. The findings show that TGF-alpha is, like EGF, a constant component of human tear fluid. The dependence of TGF-alpha concentration on tear fluid flow and the physiological importance of its presence for corneal integrity and ocular surface physiology, however, require further investigation.

Adult↗