No abstract

Shallow injection of inositol 1,4,%trisphosphate (IP,) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCI, near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, &phorbol 1Zmyristate 13-acetate (PMA), significantly enhanced the rapid responses to IP, injections at either hemisphere but did not affect the amplitudes of the responses to CaCb. The PK-C inhibitor I-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaClr. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) K Ksubunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of K K-bungarotoxin (K K-BTX). We characterized the functional properties of a hybrid (K Kmongoose/L LQ QN N-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC 50 of K K-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC 50 1.3 vs. 23.3 W WM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.z 1998 Federation of European Biochemical Societies.

Native Xenopus oocytes exhibit dose-dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 microM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was <10% of the maximal. Subsequent addition of 0.5 microM LPA resulted in 50-70% desensitization, when compared to naïve controls. Injection of antisense oligodeoxyoligonucleotides (ASODNs) targeted at either of the two endogenous LPA receptors inhibited the LPA response by approximately 50%, but did not alter the degree of rapid desensitization. To study the involvement of G-proteins in rapid homologous desensitization of responses to LPA, we selectively depleted native G-proteins by injection of specific ASDONs. Injection of ASDONs targeted at Galphaq family mRNAs (mainly Galpha11) reduced the response to 0.5 microM LPA by 50%. ASDONs targeted at either Galphao or Galphao1 caused a large decrease in the amount of their cognate mRNAs and the Galphao family proteins, while the response to LPA was inhibited by up to 30%. Injection of ASDONs targeted at Galphao1 mRNA decreased rapid desensitization from 69 to 23%, while pertussis toxin (PTX) completely abolished it. Expression of two dominant negative mutants of the human Galphao family homologs either decreased or virtually abolished rapid desensitization. Microinjection of CaCl(2) demonstrated that 50% of rapid desensitization could be attributed to inhibition of Ca(2+) activation of chloride channels. We propose that the apparent degenerate coupling of different G-proteins to LPA receptors in Xenopus oocytes actually serves both the generation of the response (by Gq and Go G-protein families) and its desensitization (mostly by Go G-protein family).