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G Schultz

Publications and source records attributed to G Schultz.

At least 163 records · Page 9Linked to original sources
PubMed1993

G-proteins involved in the calcium channel signalling system.

Various mechanisms have been identified by which hormones and neurotransmitters interacting with seven transmembrane alpha-helical spanning segments receptors modulate the activity of ion channels. All of the mechanisms involve heterotrimeric G-proteins; the best documented are hormonal modulations of voltage-dependent Ca2+ channels in cardiac, neuronal and endocrine cells. Recent studies using antisense oligonucleotide probes allow the exact identification of the G-proteins involved in these signal transduction pathways.

Animals↗
PubMed1993

Visual hallucinations and mental state. A study of 14 Charles Bonnet syndrome hallucinators.

Complex visual hallucinations are usually a sign of acute psychopathology or gross cognitive impairment, but may also occur in people with visual deficits--the Charles Bonnet Syndrome. The mental state of 14 Charles Bonnet hallucinators was assessed using four psychological tests: the Beck Depression Inventory, the State-Trait Anxiety Inventory, the Mini-Mult, and the Mini-Mental State Examination. Results are consistent with earlier reports suggesting that these hallucinations are not due to psychopathology or compromised cognitive functioning. It is proposed that these complex visual hallucinations represent ongoing neural activity in the visual system following eye damage.

Aged↗
PubMed1993

Novel potent inhibitor of receptor-activated nonselective cation currents in HL-60 cells.

A pharmacological classification of receptor-activated nonselective cation channels has not been possible because of the lack of specific and potent pharmacological blockers. In dibutyryl-cAMP-differentiated HL-60 cells, we recently identified ATP- and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulated cation currents that were blocked by an organic inhibitor of receptor-mediated Ca2+ entry, 1-beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), with an IC50 of about 3 microM. Here, we describe a new compound, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-gamma 1)-2-phenyl-N, N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]acetamide (LOE 908), that fully blocked these currents at 3 microM. Half-maximal inhibition of agonist-activated nonselective inward currents was seen at 40 nM LOE 908, whereas voltage-dependent K+ currents in undifferentiated HL-60 cells were blocked with an IC50 of 620 nM. fMLP-induced single-channel currents of 4-5-pS conductance were abolished when the excised inside-out patch was exposed to 3 microM LOE 908. The rank order of potency of cations blocking ATP- and fMLP-induced inward currents was Gd3+ > Ni2+ > Cd2+.

Acetamides↗
PubMed1993

Specific binding of EGF in connective tissue repair.

Epidermal growth factor (EGF) is known to stimulate wound repair, including connective tissue repair as observed in the perforated rat mesentery. In the present study we assessed changes in expression of EGF receptors during healing of connective tissue by measuring the binding of [125I]EGF to perforated and unperforated mesenteric membranes. Autoradiographic grain density was measured on flat mounted or sectioned mesenteric tissue. Laparotomy alone caused an inflammatory reaction in the abdominal cavity and significantly (p < 0.04) increased the binding of [125I]EGF to unperforated membranes by 70% on days 1 and 3 postoperatively. In perforated mesenteric membranes, the binding of EGF in a 1 mm wide zone around the incision was significantly higher (p < 0.05) than EGF binding in the adjacent tissue on days 3 through 7. Furthermore, the average grain density in a 100 microns wide segment around the incision was approximately twice as high (p < 0.008) as the grain density in the mesentery 100 to 500 microns around the incision or in adjacent tissue (p < 0.004). These results indicate that expression of EGF receptor increases in the region of regenerating connective tissue and supports the hypothesis that EGF receptor plays a key role in mesenteric wound healing.

Animals↗
PubMed1993

Hormonal modulations of calcium channel activity.

Hormones and neurotransmitters which modulate the membraneous influx of calcium into the cytoplasm interact with heptahelical receptors coupling to heterotrimeric G-proteins. Whereas early studies on cardiomyocytes demonstrated the involvement of a cAMP (cyclic adenosine monophosphate)-stimulated phosphorylation in the regulation of calcium channels, there is growing evidence for a direct coupling of G-proteins to these channels. Various approaches led to the identification of the subtypes of G-protein involved in these signal transduction processes. Intranuclear injections of antisense oligonucleotides hybridizing to the mRNA of G-protein subunits and, thereby, blocking the expression of these proteins showed a high specificity of the involved G-proteins, suggesting a selective processing of signals from receptors through G-proteins. Whereas hormonal inhibitions of voltage-dependent calcium channels are mediated by G-proteins of the G0-family, hormonal stimulations involve G-protein of the G1-family and protein kinase C.

Animals↗
PubMed1992

Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production.

In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and choline chloride respectively. In the absence of Ca2+ and Na+, fMLP was ineffective. SK&F 96365 inhibited fMLP-induced beta-glucuronidase release and O2- production in the presence of both Ca2+ and Na+, and in the presence of Ca2+ or Na+ alone. NaCl (25-50 mM) enhanced the basal and absolute extent of fMLP-stimulated GTP hydrolysis of heterotrimeric regulatory G-proteins in HL-60 membranes. The order of effectiveness of salts in enhancing GTP hydrolysis was LiCl > KCl > NaCl > choline chloride.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Expression, characterization and purification of soluble G-protein beta gamma dimers composed of defined subunits in baculovirus-infected insect cells.

Recombinant beta 1 gamma 2 dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the gamma 2 subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble beta gamma dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an S20,w value of 4.1 +/- 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus-infected insect cells can be employed for high level production of pure G-protein beta gamma dimers suitable for functional and structural characterization.

Amino Acid Sequence↗
PubMed1992

Tyrosine phosphorylation is an early and requisite signal induced by interferon-gamma in HL-60 cells.

Interferon-gamma (IFN gamma) is a potent immunomodulatory cytokine. However, the early mechanisms which mediate the pleiotropic effects of IFN gamma on different cells are as yet poorly understood. Therefore, we tested the role of tyrosine phosphorylation in signalling induced by IFN gamma. IFN gamma was found to induce rapid tyrosine phosphorylation of several proteins in HL-60 cells. This effect was detectable by 2 min, reached a maximum by about 4-16 min and thereafter declined. Tyrosine phosphorylation was dependent on receptor occupation and was maximally stimulated by 10 ng/ml IFN gamma. Treatment of HL-60 cells with the tyrosine kinase inhibitors, genistein and herbimycin A, inhibited both IFN gamma-stimulated tyrosine phosphorylation and IFN gamma-induced Fc receptor expression. Thus, increased tyrosine phosphorylation appears to be an obligatory early and proximal signal mediating at least some of the later cellular responses induced by IFN gamma in HL-60 cells.

Benzoquinones↗
PubMed1992

Different beta-subunits determine G-protein interaction with transmembrane receptors.

Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.

Animals↗
PubMed1992

Heterogeneity of three electrophoretically distinct Go alpha-subunits in mammalian brain.

So far three splice variants of the alpha o-gene coding for two alpha o proteins have been identified by molecular cloning, and the corresponding proteins purified. In the present study subtype-specific peptide antibodies revealed the existence of an electrophoretically distinct third form of alpha o in mammalian brain membranes which migrates more slowly on SDS-PAGE and shows a more acidic pI value than the other alpha o-subunits. Each of the three alpha o-subunits is detected as two isoforms when enriched from brain membranes. Rodent alpha o-subunits differ from non-rodent species in their electrophoretic mobilities. The results suggest that (i) there may exist a novel alpha o-subunit which reacts with an alpha o1-subunit-specific antibody, (ii) each alpha o-subunit may exist in more than one co- or posttranslationally modified isoform in brain membranes, and (iii) differences between alpha o-subunits from different species exist which are detectable by gel electrophoretic methods.

Amino Acid Sequence↗
PubMed1992

Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.

Amino Acid Sequence↗
PubMed1992

Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes.

We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the vesicles containing the glucose transporter.

Adipose Tissue↗
PubMed1992

Lipopeptides are effective stimulators of tyrosine phosphorylation in human myeloid cells.

Synthetic lipopeptide analogues of the N-terminus of bacterial lipoprotein are effective activators of macrophages, neutrophils and lymphocytes. We studied the effect of the lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]- (R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine [Pam3Cys-Ser-(Lys)4] on tyrosine phosphorylation in dibutyryl-cyclic-AMP-differentiated HL-60 cells, using anti-phosphotyrosine antibodies. Pam3Cys-Ser-(Lys)4 concentration-dependently stimulated tyrosine phosphorylation of 100/110 kDa and 60 kDa proteins and, to a lesser extent, of 55 kDa and 70/75 kDa proteins. Half-maximal and maximal effects were observed at concentrations of 1-6 and 5-50 micrograms/ml respectively. The lipopeptide-induced increase in phosphorylation was rapid and transient, with a peak response after 30-60 s. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-Ser-(Lys)4 [Pam3Ahh-Ser-(Lys)4] was as potent as Pam3Cys-Ser(Lys)4, whereas (2S,6S)-2-palmitoylamino-6,7-bis(palmitoyloxy)heptanoyl++ +-Ser-(Lys)4 [Pam3Adh-Ser-(Lys)4] and Pam3Cys-Ser-Gly did not induce tyrosine phosphorylation. Lipopeptide-induced tyrosine phosphorylation was not affected by treatment of cells with pertussis toxin. Neither phorbol 12-myristate 13-acetate nor A23187 induced tyrosine phosphorylation in dibutyryl-cyclic-AMP-differentiated HL-60 cells. In HL-60 promyelocytes, Pam3Cys-Ser-(Lys)4 had no effect on tyrosine phosphorylation, whereas the lipopeptide also induced tyrosine phosphorylation in 1,25-dihydroxyvitamin-D3-differentiated HL-60 cells and in human neutrophils. These results show that lipopeptides are effective stimulators of tyrosine phosphorylation in mature human myeloid cells.

Blotting, Western↗
PubMed1992

Receptor-mediated increases in cytosolic Ca2+ in the human erythroleukaemia cell line involve pertussis toxin-sensitive and -insensitive pathways.

The pluripotent human erythroleukaemia cell line, HEL, possesses erythrocytic, megakaryocytic and macrophage-like properties. With respect to signal transduction, HEL cells have been used as a model system for platelets, but little attention has been paid to their phagocytic properties. We studied the effects of various receptor agonists on the intracellular free Ca2+ concentration ([Ca2+]i) in HEL cells. Thrombin, platelet-activating factor (PAF), ATP, UTP, prostaglandins E1 and E2 (PGE1 and PGE2), the PGE2 analogue sulprostone and the stable PGI2 analogues iloprost and cicaprost increased [Ca2+]i. ADP was less effective than ATP, and UDP was unable to increase [Ca2+]i. The increases in [Ca2+]i induced by thrombin, PAF, ATP, UTP, iloprost and cicaprost were pertussis toxin-insensitive, whereas the increases induced by PGE2 and sulprostone were completely inhibited by the toxin. The increase in [Ca2+]i induced by PGE1 was partially inhibited by pertussis toxin. PGE2 did not desensitize the increase in [Ca2+]i induced by iloprost, and vice versa. PGE1 desensitized the response to PGE2 and iloprost but not vice versa. Adrenaline potentiated the iloprost- but not the PGE2-induced rise in [Ca2+]i. The phorbol ester phorbol 12-myristate 13-acetate completely blocked the rise in [Ca2+]i induced by ATP and PGE1, whereas the increases induced by thrombin and PAF were only partially inhibited. Agonists increased [Ca2+]i through release from internal stores and sustained Ca2+ influx. Thrombin stimulated Mn2+ influx, which was blocked by Ni2+. Diltiazem, isradipine, gramicidin and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) did not affect agonist-induced rises in [Ca2+]i. HEL cells contained substantial amounts of beta-glucuronidase which, however, could not be released, and they did not aggregate or generate superoxide. Our data suggest that: (1) HEL cells possess nucleotide receptors with properties similar to those of phagocytes; (2) they possess receptors for PGE2 and PGI2, and PGE1 is an agonist at both receptors; (3) agonist-induced increases in [Ca2+]i are mediated through pertussis toxin-sensitive as well as -insensitive signal transduction pathways; and (4) agonists increase [Ca2+]i by mobilization from internal stores and influx from the extracellular space through cation channels with properties similar to those of phagocytes and platelets.

Adenosine Triphosphate↗
PubMed1992

Five-minute treatments with fluorouracil, floxuridine, and mitomycin have long-term effects on human Tenon's capsule fibroblasts.

Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, floxuridine, and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications.

Cell Division↗
PubMed1992

Inhibition of voltage-dependent Ca2+ channels via alpha 2-adrenergic and opioid receptors in cultured bovine adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamines and opioids. The effects of these agents on whole-cell Ca2+ channel currents were studied, using bovine adrenal chromaffin cells kept in short term culture. Ca2+ channel currents recorded during voltage-clamp pulses from a holding potential of -80 mV to 0 mV were reversibly reduced by 10 microM epinephrine (in the presence of 1 microM propranolol) or 5 microM of the synthetic opioid, d-Ala2-d-Leu5-enkephalin (DADLE) by approximately 35% and 25%, respectively. The inhibitory action of epinephrine was mimicked by clonidine, reduced by yohimbine but not affected by prazosin. The DADLE-induced reduction of the Ca2+ channel current was antagonized by naloxone. The dihydropyridine (+)PN 200-110 (5 microM) reduced the Ca2+ channel current by approximately 40%; the Ca2+ channel current inhibited by (+)PN 200-110 was not further reduced by epinephrine. Intracellular infusion of guanosine-5'-O-(2-thiodiphosphate) and pretreatment of cells with pertussis toxin abolished the inhibitory effect of both epinephrine and DADLE. In membranes of adrenal chromaffin cells, four pertussis-toxin-sensitive G-proteins were identified, including Gi1, Gi2, Go1 and another Go subtype, possibly Go2. The data show that activation of alpha 2-adrenergic and opioid receptors causes an inhibition of dihydropyridine-sensitive Ca2+ channels in adrenal chromaffin cells. These inhibitory modulations are mediated by pertussis-toxin-sensitive G-proteins and may represent a mechanism for a negative feedback signal by agents released from the adrenal medulla.

Adrenal Glands↗