Biomedical subjects

G Schultz

Publications and source records attributed to G Schultz.

At least 37 records · Page 2Linked to original sources

PubMed2000

Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.

Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.

Animals↗

PubMed2000

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins.

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.

Adenylyl Cyclases↗

PubMed2000

Structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors.

Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.

Amino Acid Sequence↗

PubMed2000

Molecular responses of human dermal fibroblasts to dual cues: contact guidance and mechanical load.

Fibroblast contraction in wound healing involves the interaction of several cell types, cytokines, and extracellular matrix molecules. We have previously developed fibroblast alignment models using precise uniaxial mechanical loads in 3D culture and using contact guidance on fibronectin strands. Our aim here was to use contact guidance to place fibroblasts in their potentially most sensitive configuration, i.e., perpendicular to the axis of loading, to present cells with conflicting guidance cues. Gene expression at the mRNA level of cells recovered from different zones of the 3D collagen gel (with distinct orientation) was determined by quantitative RT-PCR for the matrix proteases MMP1, 2, and 3, and inhibitors TIMP1 and 2. Our results show a 2-, 4-, and 3-fold increase in MMP1, 2, and 3, respectively, in the non-aligned strain zone, relative to the aligned strain zone. These results suggest that cells unable to align to applied loads remodel their matrix far more rapidly than orientated cells. Where fibroblasts were held in an alignment perpendicular to the applied load by contact guidance, the fall in MMP mRNA expression was largely abolished, indicating that these cells remained in a mechano-activated state. The protease inhibitors TIMP1 and 2 were poorly mechano-responsive, further suggesting that changes in MMP expression result in functional matrix remodelling. These results indicate how mechanical loading in tissues may influence matrix remodelling, particularly under conflicting guidance cues.

Cell Adhesion↗

PubMed2000

Transient receptor potential channels as molecular substrates of receptor-mediated cation entry.

Calcium is a versatile multitarget intracellular second messenger in eukaryotic cells. In addition to calcium release from intracellular stores and influx via voltage- or ligand-operated channels, agonist-induced calcium entry constitutes one of the main pathways by which cytosolic calcium is elevated. Receptor-stimulated currents are initiated in response to agonist binding to G-protein-coupled receptors and to receptor tyrosine kinases. Within the past few years our knowledge about the molecular identity of receptor-stimulated channels has expanded substantially. Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant visual phenotypes were the first members of this category of channels to be identified at the molecular level. Since then an entire mammalian gene family of TRP homologues has been discovered by homology cloning. Only now are we beginning to fully understand the functional roles of TRP channels in mammalian cells. We review recent findings in TRP channel research and discuss the role of these proteins for receptor-activated cation entry.

Animals↗

PubMed2000

Contribution of receptor/G protein signaling to cell growth and transformation.

While the effects of receptor/G protein systems on intermediary metabolism have been intensively studied, it has only recently been appreciated that G protein-coupled receptors and G proteins (heterotrimeric GTP-binding proteins) play important roles in the regulation of cell growth, differentiation and even transformation. Naturally occurring mutations both in G protein-coupled receptors and in G protein alpha-subunits lead to autonomous cell growth resulting in human disease. One mechanism to transduce mitogenic signals from the cell membrane to the nucleus is the engagement of the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase (MAPK) cascade. Multiple distinct signal transduction pathways have been characterized which link G proteins with the ERK cascade. Receptor and non-receptor tyrosine kinases play central roles in these pathways. Mitogenic signaling by receptor/G protein systems is realized as a complex interplay between signals emanating from different classes of cell surface receptors. The characterization of receptor-, G protein- and tyrosine kinase-specific contributions to mitogenic signaling in a particular cell may ultimately allow for the rational design and application of pharmaceuticals to treat diseases involving uncontrolled cell proliferation.

Animals↗

PubMed2000

From worm to man: three subfamilies of TRP channels.

A steadily increasing number of cDNAs for proteins that are structurally related to the TRP ion channels have been cloned in recent years. All these proteins display a topology of six transmembrane segments that is shared with some voltage-gated channels and the cyclic-nucleotide-gated channels. The TRP channels can be divided, on the basis of their homology, into three TRP channel (TRPC) subfamilies: short (S), long (L) and osm (O). From the evidence available to date, this subdivision can also be made according to channel function. Thus, the STRPC family, which includes Drosophila TRP and TRPL and the mammalian homologues, TRPC1-7, is a family of Ca2+-permeable cation channels that are activated subsequent to receptor-mediated stimulation of different isoforms of phospholipase C. Members of the OTRPC family are Ca2+-permeable channels involved in pain transduction (vanilloid and vanilloid-like receptors), epithelial Ca2+ transport and, at least in Caenorhabditis elegans, in chemo-, mechano- and osmoregulation. The LTRPC family is less well characterized.

Amino Acid Sequence↗

PubMed2000

OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity.

Ca2+-permeable channels that are involved in the responses of mammalian cells to changes in extracellular osmolarity have not been characterized at the molecular level. Here we identify a new TRP (transient receptor potential)-like channel protein, OTRPC4, that is expressed at high levels in the kidney, liver and heart. OTRPC4 forms Ca2+-permeable, nonselective cation channels that exhibit spontaneous activity in isotonic media and are rapidly activated by decreases in, and are inhibited by increases in, extracellular osmolarity. Changes in osmolarity of as little as 10% result in significant changes in intracellular Ca2+ concentration. We propose that OTRPC4 is a candidate for a molecular sensor that confers osmosensitivity on mammalian cells.

Amino Acid Sequence↗

PubMed2000

Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats after PRK.

PURPOSE: To assess the role of the transforming growth factor (TGF)ss system in formation of corneal haze after excimer laser photorefractive keratectomy (PRK), levels of mRNAs for three TGFss isoforms (TGFss1, TGFss2, and TGFss3), the TGFss type II receptor (TssRII), and extracellular matrix (ECM) genes including fibronectin (FN), collagen I, collagen III, and collagen IV were measured in rat corneas. METHODS: Corneas were graded for corneal haze at 0, 1.5, 7, 21, 42, and 91 days after PRK. Total RNA was isolated from pooled corneas, and the levels of mRNAs were measured using competition-based quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Severe corneal haze developed by day 42 and persisted to day 91. Levels of TGFss1 mRNA were high in rat corneas before PRK and remained relatively constant. In contrast, levels of TGFss2 and TGFss3 mRNAs were very low in normal corneas, increased 300-fold and 25-fold, respectively, on day 21, and remained elevated on day 91. Levels of mRNA for TssRII increased, with a peak elevation of 50-fold on day 42 after PRK. Levels of mRNAs for ECM proteins also increased. Fibronectin mRNA was nondetectable in normal corneas but rapidly increased to 675 copies/cell on day 7 and remained elevated to day 91. Collagen III mRNA levels peaked on day 21 with a 700-fold increase compared with a very low level of expression in normal cornea, and then decreased on day 91. Expression of collagen I mRNA lagged expression of collagen III mRNA and peaked at day 42 after PRK with a 1200-fold increase over normal cornea. In contrast, mRNA for collagen alpha(1)IV, a major component in basement membranes, remained relatively stable through day 21 and then increased slightly on days 42 and 91. CONCLUSIONS: The synchronized increase in mRNA synthesis for both the TGFss system and key ECM genes supports the hypothesis that TGFss is a key growth factor promoting stromal haze formation in corneas after PRK and suggests that limiting TGFss system may reduce corneal scarring after excimer laser ablation.

Animals↗

PubMed1999

A novel subgroup of class I G-protein-coupled receptors.

Based on structural similarities of an expressed sequence tag with the platelet-activating factor (PAF) receptor a cDNA clone encoding a novel G-protein-coupled receptor (GPCR), named GPR34, was isolated from a human fetal brain cDNA library. Genomic DNA analyses revealed the receptor to be encoded by an intronless single-copy gene at Xp11. 3-11.4. The predicted 381-amino-acid protein disclosed all structural features characteristic of a member of the class I GPCR family. Except an obvious sequence homology in transmembrane domain 6, no further similarities to the PAF receptor or any other known GPCR were found. The corresponding mouse receptor DNA was isolated from a genomic P1 library displaying a 90% amino acid identity compared to the human receptor. Phylogenetic studies showed that GPR34 is preserved among vertebrates, and the existence of GPR34 subtypes was demonstrated. The receptor mRNA is abundantly expressed in human and mouse tissues. In addition to the major 2-kb transcript, a 4-kb transcript was found only in mouse liver and testis. Expression of the human GPR34 in COS-7 cells followed by Western blot studies revealed specific bands of a highly glycosylated protein between 75 and 90 kDa. A number of potential ligands including phospholipids, leukotrienes, hydroxy-eicosatetraenoic acids, nucleotides and peptides were tested in functional assays. Since none of the applied substances led to significant changes in second messenger levels (cAMP and inositol phosphates), the natural ligand and coupling profile of this novel GPCR subgroup remains unknown.

Amino Acid Sequence↗

PubMed1999

Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.

Actins↗

PubMed1999

Structural basis of G protein-coupled receptor function.

The vast majority of extracellular signaling molecules, like hormones and neurotransmitters, interact with a class of membranous receptors characterized by a uniform molecular architecture of seven transmembrane alpha-helices linked by extra- and intracelluar peptide loops. In a reversible manner, binding of diverse agonists to heptahelical receptors leads to activation of a limited repertoire of heterotrimeric guanine nucleotide-binding proteins (G proteins) forwarding the signal to intracellular effectors such as enzymes and ion channels. Proper functioning of a G protein-coupled receptor is based on a complex interplay of structural determinants which are ultimately responsible for receptor folding, trafficking and transmembrane signaling. Applying novel biochemical and molecular biological methods interesting insights into receptor structure/function relationships became available. These studies have a significant impact on our understanding of the molecular basis of human diseases and may eventually lead to novel therapeutic strategies.

Animals↗

PubMed1999

Adenoviral gene transfer of the human V2 vasopressin receptor improves contractile force of rat cardiomyocytes.

BACKGROUND: In congestive heart failure, high systemic levels of the hormone arginine vasopressin (AVP) result in vasoconstriction and reduced cardiac contractility. These effects are mediated by the V1 vasopressin receptor (V1R) coupled to phospholipase C beta-isoforms. The V2 vasopressin receptor (V2R), which promotes activation of the Gs/adenylyl cyclase system, is physiologically expressed in the kidney but not in the myocardium. Expression of a recombinant V2R (rV2R) in the myocardium could result in a positive inotropic effect via the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: A recombinant adenovirus encoding the human V2R (Ad-V2R) was tested for its ability to modulate the cardiac Gs/adenylyl cyclase system and to potentiate contractile force in rat ventricular cardiomyocytes and in H9c2 cardiomyoblasts. Ad-V2R infection resulted in a virus concentration-dependent expression of the transgene and led to a marked increase in cAMP formation in rV2R-expressing cardiomyocytes after exposure to AVP. Single-cell shortening measurements showed a significant agonist-induced contraction amplitude enhancement, which was blocked by the V2R antagonist, SR 121463A. Pretreatment of Ad-V2R-infected cardiomyocytes with AVP led to desensitization of the rV2R after short-term agonist exposure but did not lead to further loss of receptor function or density after long-term agonist incubation, thus demonstrating resistance of the rV2R to downregulation. CONCLUSIONS: Adenoviral gene transfer of the V2R in cardiomyocytes can modulate the endogenous adenylyl cyclase-signal transduction cascade and can potentiate contraction amplitude in cardiomyocytes. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in congestive heart failure by bypassing the desensitized beta-adrenergic receptor signaling.

Adenoviridae↗

PubMed1999

Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets.

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

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

PubMed1999

Selective activation of G protein subtypes in the vomeronasal organ upon stimulation with urine-derived compounds.

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Galpha-subunits, activation dependently labeled with [alpha-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by alpha2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gi may be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature.

Alpha-Globulins↗

PubMed1999

Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol.

Eukaryotic cells respond to many hormones and neurotransmitters with increased activity of the enzyme phospholipase C and a subsequent rise in the concentration of intracellular free calcium ([Ca2+]i). The increase in [Ca2+]i occurs as a result of the release of Ca2+ from intracellular stores and an influx of Ca2+ through the plasma membrane; this influx of Ca2+ may or may not be store-dependent. Drosophila transient receptor potential (TRP) proteins and some mammalian homologues (TRPC proteins) are thought to mediate capacitative Ca2+ entry. Here we describe the molecular mechanism of store-depletion-independent activation of a subfamily of mammalian TRPC channels. We find that hTRPC6 is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited fashion, independently of protein kinases C activated by diacylglycerol. Although hTRPC3, the closest structural relative of hTRPC6, is activated in the same way, TRPCs 1, 4 and 5 and the vanilloid receptor subtype 1 are unresponsive to the lipid mediator. Thus, hTRPC3 and hTRPC6 represent the first members of a new functional family of second-messenger-operated cation channels, which are activated by diacylglycerol.

Animals↗

PubMed1999

A case of referred pain evoked by remote light touch after partial nerve injury.

An unusual case of referred pain is presented in which a 63-year-old man, who suffered a severe injury to his right hand and arm during young adulthood, describes the later development of dysesthesia and shooting pain in his arm subsequent to stimulation of the ipsilateral scalp, the temporal and infrazygomatic region of the face, and the back. Referred sensations of this type are usually reported following amputation of an arm. Clinical examination of the sensory and motor function of the arm and hand revealed partial damage to the radial, ulnar and median nerves as well as possible brachial plexus involvement. Interestingly, pain could be evoked by repeated light touches applied to the remote trigger areas suggesting the involvement of a 'wind-up'-like process.

Arm↗

PubMed1999

Levels of transforming growth factor alpha (TGF-alpha) in human cerebrospinal fluid.

In this study, we investigated cerebrospinal fluid of patients with various neurological symptoms for the presence of transforming growth factor alpha (TGF-alpha). 41 samples of cerebrospinal fluid were collected by lumbar puncture performed routinely due to the clinical suspicion of neurological disease from 22 females (age 15-80 years, median 42 years) and from 19 males (age 18-82 years, median 48 years). A highly sensitive and specific radioimmunoassay was used to determine the concentration of TGF-alpha in the samples. The detection limit of the assay was about 200 pg TGF-alpha. There was no cross-reactivity to human EGF. We showed CSF indeed does contain TGFalpha. As TGF-alpha was detected in all 41 samples investigated, this growth factor appears to be a constant component of CSF. The mean concentration was 5.5 ng TGF-alpha (S.D. +/- 2.7 pg/ml, range 1.1 to 13.9 pg/ml). There was no significant correlation between TGF-alpha concentration in CSF and age (r = -0.006) and there was no significant difference between females (mean 5.8+/-3.10 pg/ml) and males (mean 5.2+/-1.96 pg/ml). No diagnosis was over represented in patients with TGF-alpha concentrations above or below 1 S.D. off the mean. However, highest concentrations of TGF-alpha were found in the group of patients with peripheral neurological sensory dysfunctions and polyneuropathy. We conclude that TGF-alpha is not only a constant component of human cerebrospinal fluid in adults but could also be significantly involved in the pathophysiology of various neurological diseases. The earlier hypothesis that TGF-alpha could mainly have a role in brain development needs hence to be re-evaluated.

Adolescent↗