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G Gimpl

Publications and source records attributed to G Gimpl.

26 records · Page 2Linked to original sources
PubMed1994

Molecular structure analysis of the pituitary adenylate cyclase activating polypeptide type I receptor from pig brain.

In pig brain a type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP) has been identified and structurally characterized. Scatchard analysis of the equilibrium binding data indicates a single class of binding sites with Kd of 0.50 nM and Bmax of 2.52 pmol/mg. The receptors could be efficiently solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonate (Chapso) without altering their ligand binding parameters (Kd = 0.60 nM; Bmax = 2.46 pmol/mg). In both preparations the ligand-receptor complex was identified as a 68 kDa polypeptide by affinity crosslinking with 125I-PACAP-27 and disuccinimidyl suberate. Employment of nonreducing conditions decreased the electrophoretic mobility of the complex to a 60 kDa species suggesting that the native PACAP receptor exists in a compact conformation stabilized by intramolecular disulfide bridges. Disulfide bonds are also important for the ligand binding activity of the PACAP receptor since pretreatment of the membranes with dithiothreitol led to complete inhibition of PACAP binding and to 61% dissociation of the ligand-receptor complex. Among different sulfhydryl selective reagents tested, p-chloromercuriphenylsulfonic acid was found to inhibit PACAP binding to its receptor in a dose-dependent fashion (IC50 = 0.5 mM) suggesting that one or more sulfhydryl groups are located close to the ligand binding domain of the receptor. Desialylation of the affinity-labeled PACAP receptor with neuraminidase revealed a 61 kDa protein, whereas deglycosylation with N-glycosidase F decreased the M(r) to 52,000. Chromatography on a series of lectin agaroses showed the highest affinity for wheat germ agglutinin (WGA). WGA was also effective in dose-dependent inhibition of PACAP binding activity. Our results characterize the pig PACAP receptor as a tri- or tetraantennary non- or low fucosylated complex type glycoprotein with a protein core of about 49 kDa containing several terminal sialyl residues.

Animals↗
PubMed1994

Extracellular ATP-induced currents in astrocytes: involvement of a cation channel.

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P2-purinoceptor-mediated depolarization. ATP (100 microM) induced an inward current with a mean amplitude of 130 pA and an EC50 of 17 microM. The response desensitized during a 1 min application. Replacement of extracellular Na+ with NMDG or K+ abolished the ATP-evoked inward current. Replacement of Na+ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na+ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K(+)-sensitive microelectrodes showed that 100 microM ATP lowered the intracellular K+ concentration. Replacement of extracellular Ca2+ or Cl- did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca2+ concentration during ATP application. Removal of extracellular Ca2+ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P2x receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca2+ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K+ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells.

Adenosine Triphosphate↗
PubMed1993

Direct identification of an extracellular agonist binding site in the renal V2 vasopressin receptor.

To purify the renal V2 receptor and identify domains involved in hormone binding, photoaffinity labeling of the membrane-bound bovine V2 receptor with a tritium-labeled photoreactive vasopressin agonist was performed. The labeled 30,000 M(r) protein was purified to homogeneity by anion-exchange chromatography, isoelectric focusing, gel filtration, gel electrophoresis, and reversed-phase HPLC. N-terminal sequencing showed that the isolated protein which contains the covalently bound hormonal ligand, represents an N-terminal truncated bovine V2 receptor. The purified labeled V2 vasopressin receptor protein was digested with V8 protease, and peptide fragments were isolated. Protein microsequencing and comparison with the cDNA sequence of a cloned PCR product identified two extra- and two intracellular peptides of the bovine V2 receptor. Radioactivity was incorporated into two amino acid residues localized in the second extracellular domain. Our results indicate that this extracellular domain is involved in peptide agonist binding of the V2 receptor.

Affinity Labels↗
PubMed1993

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain.

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 x 10(5) cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: 1) electrophysiological responses and 2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20-70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin.

Adenosine Diphosphate Ribose↗
PubMed1992

Bradykinin receptors in cultured astrocytes from neonatal rat brain are linked to physiological responses.

Specific binding sites for bradykinin (BK) have recently been demonstrated on astrocytes of primary cultures from neonatal rat brain. In this study we demonstrate that BK induces membrane currents in concert with an elevation of [Ca2+]i. In 67% of astrocytes, BK induced an inward current as determined with the perforated patch-clamp technique in the whole-cell recording configuration. In a small population of astrocytes (20%), a BK-activated outward current was observed, while in the remainder of the cells (13%) no apparent current responses were detected. As recorded by fura-2 microfluorimetry, the peptide induced a transient rise of [Ca2+/bdi even when the extracellular calcium was removed. In the majority of astrocytes, the selective B1-agonist des-Arg9-BK elicited physiological responses with a much lower potency, indicating that the BK receptors are predominantly of the B2 subtype. A minor population of astrocytes was present which only responded to des-Arg9-BK.

Animals↗
PubMed1991

Identification of a receptor protein for neuropeptide Y in rabbit kidney. G-protein association and inhibition of adenylate cyclase.

We previously identified the receptor for neuropeptide Y (NPY) on rabbit kidney membranes as a 100 kDa protein [(1990) J. Biol. Chem. 265, 18142-18143]. It is demonstrated in this study that the stable guanine nucleotide analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), is capable of decreasing the labeling of the 100 kDa protein in a concentration-dependent manner, with an IC50 value of 50 microM. This suggests that the 100 kDa protein represents a G-protein coupled receptor type. In isolated proximal tubules from rabbit kidney NPY decreases the parathyroid hormone stimulated cAMP production in a dose-dependent fashion. This indicates that the NPY receptor in rabbit kidney is negatively coupled to adenylate cyclase by a G1-like protein.

Adenylyl Cyclases↗
PubMed1990

Solubilization and characterization of active neuropeptide Y receptors from rabbit kidney.

Active neuropeptide Y receptors were solubilized from rabbit kidney membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). In membrane fragments and soluble extracts neuropeptide Y binding was time dependent, saturable, reversible, and of high affinity. Scatchard analysis of equilibrium binding data indicated a single class of binding sites with respective KD and Bmax values of 0.09 nM and 530 fmol/mg of protein for the membrane-bound receptors and 0.10 nM and 1585 fmol/mg of protein for the soluble receptors. Neuropeptide Y binding was specifically inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) in a concentration-dependent manner, with IC50 values of 28 and 0.14 microM for membrane-bound and soluble receptors, respectively, suggesting that neuropeptide Y receptors are functionally coupled to GTP-binding regulatory proteins. Cross-linking studies were performed with the heterobifunctional N-hydroxysuccinimidyl-4-azidobenzoate and the monofunctional neuropeptide Y derivative, azidobenzoyl and led to the identification of a 100 kDa peptide that should represent the covalently labeled neuropeptide Y receptor.

Affinity Labels↗