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G Schultz

Publications and source records attributed to G Schultz.

At least 91 records · Page 5Linked to original sources
PubMed1996

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.

Animals↗
PubMed1996

Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of G alpha 12 purified from rat brain.

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

A functional heme-binding site of soluble guanylyl cyclase requires intact N-termini of alpha 1 and beta 1 subunits.

Soluble guanylyl cyclase, a heterodimeric enzyme, is the most important intracellular target for the signalling molecule nitric oxide (NO). NO stimulates the enzyme by binding to a prosthetic heme group. The identity of the axial heme ligand, however, is still unknown. Here we show that guanylyl cyclase mutated at the residue His 105 on the beta 1 subunit, a mutant that we have shown before to contain no heme after purification [Wedel, B., Humbert, P., Harteneck, C., Foerster, J., Malkewitz, J., Böhme, E., Schultz, G. & Koesling, D. (1994) Proc. Natl Acad. Sci. USA 91, 2592-2596] can be reconstituted with heme. The reconstituted mutant remains NO-insensitive and displays an ultraviolet absorption spectrum consistent with an altered axial coordination. Thus, this residue is a strong candidate for the axial heme-ligating residue and appears to be necessary for NO stimulation. Apart from the axial heme ligand, the role of the enzyme's two subunits, alpha 1 and beta 1, in heme binding has not been clarified to date. To address this question, we purified mutant heterodimers in which the non-conserved amino termini of either alpha 1 (131 residues deleted), or beta 1 (64 residues) have been deleted. These deletion mutants had previously been found to be marginally (alpha 1 truncated) or not at all NO sensitive (beta 1 truncated) in cytosolic fractions [Wedel, B., Harteneck, C., Foerster, J., Friebe, A., Schultz, G. & Koesling, D. (1995) J. Biol. Chem. 270, 24871-24875]. Here, we show that the purified enzyme truncated on alpha 1 has a significantly reduced capacity to bind heme which explains the reduced NO sensitivity. By contrast, the beta 1-truncated enzyme binds an amount of heme comparable to the wild type but is only marginally NO-responsive and displays a shift in the heme ultraviolet absorption maximum indicative of altered heme coordination. In conclusion, the heme binding site of soluble guanylyl cyclase requires the presence of both subunits in full length to be able to bind wild-type quantities of heme and to be capable of mediating the NO-heme-induced stimulation. Despite some structural similarity, both subunits appear to participate differently in NO-heme-mediated enzyme regulation.

Animals↗
PubMed1996

Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C.

Binding of lutropin/choriogonadotropin to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. The mechanism underlying the generation of this bifurcating signal is presently not known. To analyze the coupling mechanism of the LH receptor, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In membranes of bovine corpora lutea and of L cells stably expressing the murine LH receptor (LHR cells), human chorionic gonadotropin (hCG) led to incorporation of the label into alphas and alphai2. Stimulation of LHR cells or of L cells expressing the M5 muscarinic receptor (LM5 cells) with the respective agonist resulted in activation of phospholipase C in both cell lines. However, alphaq and alpha11 were only labeled upon stimulation of the M5 muscarinic receptor. Agonist-induced Ca2+ mobilization and inositol phosphate accumulation were partially sensitive to pertussis toxin, and the expression of the betagamma-stimulable phospholipase C isoforms beta2 and beta3 could be demonstrated in LHR cells. Overexpression of phospholipase C-beta2 led to increased hCG-stimulated inositol phosphate accumulation, and expression of a beta-ARK1 C-terminal polypeptide effectively suppressed hCG-mediated phosphatidylinositol hydrolysis. Thus, the LH receptor couples to both Gs and Gi, and betagamma-subunits released from either G protein contribute to the stimulation of phospholipase C-beta isoforms.

Adenylyl Cyclases↗
PubMed1996

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Animals↗
PubMed1996

A specific G(o) heterotrimer couples somatostatin receptors to voltage-gated calcium channels in RINm5F cells.

The peptide hormone somatostatin inhibits glucose-induced insulin secretion in the rat insulinoma RINm5F cells by inhibition of voltage-gated calcium channels. Here we used micro-injection of antisense oligonucleotides directed against subtypes of G-protein subunits to determine the subunit composition involved in somatostatin-induced inhibition of voltage-gated calcium channels in RINmF5 cells. Injection of antisense oligonucleotides annealing to the respective mRNA of G alpha o2, G beta 1 and G gamma 3 reduced the somatostatin-induced inhibition of calcium channels in these cells. Injection of antisense oligonucleotides directed against other G-protein subunits did not, suggesting that in RINm5F cells the somatostatin receptor couples to a G protein of G alpha o2 beta 1 gamma 3 composition. By using a selective agonist of type 2 somatostatin receptors (SSTR 2) NC 8-12, we identified this receptor as the subtype coupling to calcium channels in RINm5F cells.

Animals↗
PubMed1996

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

Amino Acid Sequence↗
PubMed1996

Transforming growth factor beta 1 improves wound healing and random flap survival in normal and irradiated rats.

OBJECTIVES: To evaluate the effect of chronic irradiation on wound healing and random flap survival (FV), and the effect of transforming growth factor beta 1 (TGF-beta 1) in this setting using an animal model. DESIGN: A randomized, controlled study with four groups of rats to study the effect of irradiation 4 months before surgical intervention. The effect of TGF-beta 1 on FV and wound healing also was evaluated in the irradiated and nonirradiated groups. SUBJECTS: Ninety-five rats were available for evaluation. Group 1 (n = 10) was the control; group 2 (n = 28) received TGF-beta 1; group 3 (n = 28) received radiation therapy; and group 4 (n = 29) received radiation therapy and TGF-beta 1. INTERVENTION: The irradiated groups received 15 Gy to their dorsal skin. Four months later all received McFarlane skin flaps. Groups 2 and 4 received topical TGF-beta 1, 4 micrograms, to the bed of the flap; groups 1 and 3 received saline. On postoperative day 7 all rats were evaluated for tensile strength and FV, and histologic staining with hematoxylin-eosin for collagen and TGF-beta 1 was done. The slides were evaluated in a "blinded" fashion. RESULTS: Irradiation decreased tensile strength and FV, but not to a notable degree. Transforming growth factor beta 1 improved tensile strength in the irradiated (P = .04, Student's t test) and nonirradiated groups (P = .05, Student's t test). Transforming growth factor beta 1 also improved FV in all groups, but significantly in the irradiation plus TGF-beta 1 group (P = .001, Student's t test). The TGF-beta 1 group had the most mature collagen present at the wound edge. No qualitative difference was seen in the immunohistochemical staining for the four groups. CONCLUSIONS: Transforming growth factor beta 1 improves wound healing and random FV in radiated and nonirradiated rat skin. Further study is needed to determine the radiation dose necessary to create an "impaired wound-healing model" in rats, and the optimum dose of TGF-beta 1 in this setting.

Administration, Topical↗
PubMed1996

Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion.

Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.

Amino Acid Sequence↗
PubMed1996

gamma-Aminobutyric acid secretion from pancreatic neuroendocrine cells.

BACKGROUND & AIMS: Neuroendocrine cells and tumors derived therefrom contain hormone-storing large dense core vesicles and neuron-like small synaptic vesicle analogues with unknown function. The aim of this study was to characterize the small synaptic vesicle pathway in detail. METHODS: In human pancreatic neuroendocrine tumors and corresponding mammalian cell lines, the expression of key proteins if regulated secretion were detected by immunofluorescence microscopy. Using 3H-gamma-aminobutyric acid (GABA), uptake and release by small synaptic vesicle analogues were studied. RESULTS: Tumor tissues obtained from 14 patients expressed key proteins of neurosecretion such as synaptobrevin, syntaxins, and SNAP 25. These proteins were also found in the cell lines AR42J, BON, RIN, and INR. The cell lines specifically transported GABA by low-affinity plasma membrane transporter and showed an adenosine triphosphate-sensitive GABA uptake into an intracellular compartment. Stored GABA was released upon stimulation by regulated exocytosis. Electrophysiological analyses suggested that calcium-dependent secretion was mediated by activation of voltage-dependent calcium channels of mainly the L type, but also of the N and probably the T type. CONCLUSIONS: Small synaptic vesicle analogues in neuroendocrine cells and tumors can store and secrete GABA and probably other amino acid transmitters by regulated exocytosis comparable with neurons.

Adult↗
PubMed1996

[Initial experiences with dilatation of dialysis shunts with color-coded duplex ultrasonography monitoring].

PURPOSE: Aim of the study was to evaluate the technical aspects of colour coded duplex sonography guided interventions of peripheral vessels. METHODS: During 15 months 39 stenoses of shunt veins in 24 patients were dilated guided by colour coded duplex sonography. RESULTS: 38 stenoses were dilated without complications. The blood flow volume was increased from 361.9 +/- 83.5 to 718.9 +/- 189.2 ml/min. In one case it was not possible to dilate the stenosis because of a vasospasm. CONCLUSION: PTA guided by colour coded duplex sonography is an alternative to DSA guided interventions of superficial vessels without x-ray exposure or contrast agent application.

Adult↗
PubMed1996

Properties of complex hallucinations associated with deficits in vision.

Complex visual hallucinations are known to occur in individuals with impaired vision yet whose emotional and intellectual functions are within the normal range. These hallucinations, which were first reported by Charles Bonnet in 1760, have been described in many case studies, but have not been analyzed empirically to determine their major properties. In the present study, sixty complex hallucinators labeled as Charles Bonnet hallucinators were administered a questionnaire to determine the properties of their hallucinations. Combined use of multiple-correspondence analysis and hierarchical cluster analysis reveals a set of features that characterize the 'typical' Charles Bonnet hallucinatory experience: the hallucinators' experience occurs while they are alert and with the eyelids open; a sharply focused image appears suddenly, without any apparent trigger or voluntary control; the hallucination is present for seconds, does not move during this time, then suddenly vanishes. These features are discussed in terms of a 'dimension' of hallucinatory/perceptual experience, which ranges from discrete perceptual experiences to multiple, changing experiences. Possible mechanisms that underlie the Charles Bonnet hallucinations are discussed.

Blindness↗
PubMed1996

Diversity and selectivity of receptor-G protein interaction.

Cells in a living organism communicate with each other through extracellular molecules such as hormones, neurotransmitters, and growth factors. The majority of these molecules transmit their signal by interacting with a three-protein transmembrane signal transduction system whose single components interact sequentially and reversibly. Agonist binding to a heptahelical receptor results in activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) that modulate the activity of one or more effector systems. Considering that hundreds of G protein-coupled receptors transduce signals by interacting with a limited repertoire of G proteins, the question of specificity governing the coupling of receptors to G proteins arises. The conceptualization of signal transduction pathways in a linear fashion (one receptor coupling to one G protein that activates one effector) is inadequate to explain experimental results. In the present review, G protein-mediated signal transduction is depicted as a complex signaling network with divergent and convergent pathways at each transduction level, i.e. receptor, G protein, and effector. The recent realization that "classical" signaling pathways appear to be activated in parallel with signaling cascades primarily described for growth factors and cytokines adds an additional level of intriguing complexity.

Alternative Splicing↗
PubMed1996

Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor.

The involvement of growth factors such as epidermal growth factor (EGF) in regenerative processes of corneal endothelium and lens epithelium has recently been suggested. However, knowledge on the presence of growth factors in anterior chamber fluid (ACF) is still very restricted. Although we have previously shown that EGF is undetectable in the ACF of normal eyes undergoing cataract surgery even by the use of high-sensitivity assays, this does not exclude the possible presence of other, EGF-like proteins in ACF such as transforming growth factor alpha (TGF-alpha). In the present study, we have hence determined in ACF samples of 70 human eyes the concentrations of both EGF and TGF-alpha. As assays served ELISA techniques and RIA. In none of all the samples investigated could detectable amounts of EGF, i.e. above 0.2 pg/ml (detection limit of the assay), be found, confirming earlier results. Interestingly, however, also no TGF-alpha could be detected in ACF. If present at all, the level of any TGF-alpha concentration in ACF was hence below the detection limit, i.e. less than 20 pg/ml. Based on the results of this study, it seems therefore that under physiological conditions there is no measurable presence of free EGF or TGF-alpha in human ACF. Existing receptors in the structures of the anterior segment must hence have ample binding capacity which could explain the effect of externally applied growth factors. The physiological and clinical importance of this result is briefly outlined.

Adult↗
PubMed1996

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C.

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.

Adenylate Cyclase Toxin↗
PubMed1996

Detection of transforming growth factor-alpha mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-alpha in human tears.

PURPOSE: To assess whether the lacrimal gland is a possible site of synthesis of transforming growth factor-alpha (TGF-alpha) and to characterize TGF-alpha biochemically in human tears. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to analyze rat lacrimal glands for the presence of TGF-alpha mRNA. Specific monoclonal antibodies were used to localize TGF-alpha immunohistochemically in lacrimal gland tissue of rats. Human tears were analyzed for immunoreactive TGF-alpha protein using a specific radioimmunoassay, and the molecular weight of TGF-alpha in tears was characterized by Western blot analysis. RESULTS: RT-PCR amplification of rat lacrimal gland RNA generated a band of the predicted 492 base pairs for TGF-alpha mRNA. Immunohistochemical staining of rat lacrimal gland localized TGF-alpha protein to lacrymocytes constituting acini but not to interacinar and intraacinar ducts of lacrimal glands. Western blot analysis of human tears detected a single band at MWt 16,000. Logit transformation of radioimmunoassay data for tears and TGF-alpha standard generated parallel displacement lines, indicating the presence of immunoreactive TGF-alpha levels in human tears with an average concentration of 100 +/- 20 pg/ml (mean +/- SEM). CONCLUSIONS: Rat lacrymocytes synthesize TGF-alpha mRNA and protein, and human tears contain immunoreactive TGF-alpha, suggesting that the lacrimal gland may be an exocrine source for TGF-alpha in tears. The single MWt 16,000 form of TGF-alpha in human tears appears to be generated by an unusual proteolytically processing of the pro-TGF-alpha transmembrane precursor protein.

Adult↗