Receptor number determines latency and amplitude of the thyrotropin-releasing hormone response in Xenopus oocytes injected with pituitary RNA

TRH evokes depolarizing membrane electrical responses in Xenopus laevis oocytes injected with RNA from pituitary cells. We have shown previously that the amplitude of this response is directly proportional to the dose of TRH and the amount of RNA injected. Herein we show that the number of TRH receptors expressed on oocytes after injection of rat pituitary (GH3) cell RNA or mouse thyrotropic (TtT) tumor RNA determines the latency as well as the amplitude of the response. In oocytes injected with a maximally effective amount of GH3 cell RNA, the latency of the response decreased from a maximal duration of 103 +/- 16 to 10 +/- 1 sec when the TRH concentration was increased from 5 to 3000 nM. When oocytes injected with different amounts of GH3 cell RNA were stimulated with 3000 nM TRH, the latency decreased from 31 +/- 4 to 11 +/- 0.5 sec when the amount of RNA injected was increased from 30 to 400 ng. Specific binding of [3H]methylhistidine-TRH increased when increasing amounts of TtT poly(A)+ RNA was injected, and binding correlated with increased response amplitude. To show that these effects were caused by mRNA for the TRH receptor and did not depend on other mRNAs, TtT poly(A)+ RNA was fractionated on a sucrose gradient. Using RNA from each fraction, there was an inverse correlation between response amplitude and latency. For size-fractionated RNA, as for unfractionated RNA, there was a direct correlation between specific [3H]methylhistidine-TRH binding and response amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of membrane electrical response to thyrotropin-releasing hormone in Xenopus oocytes injected with GH3 pituitary cell messenger ribonucleic acid

TRH evoked a complex electrical membrane response in Xenopus laevis oocytes injected with either total cytosolic or poly(A)(+)-enriched RNA from GH3 pituitary cells but not in uninjected oocytes. A typical response consisted of a transient, rapid depolarizing current followed by a prolonged depolarizing current with superimposed current fluctuations. The reversal potentials of the rapid and the slow components of the response were -23.0 and -22.6 mV, respectively, and were markedly affected by CI- concentration indicating that the TRH response was mainly an increase in Cl- conductance. The response to TRH was dose dependent and was inhibited by the TRH antagonist, chlordiazepoxide. TRH caused rapid hydrolysis of labeled phosphatidylinositol 4,5-bisphosphate and a marked, prolonged increase in 45Ca2+ efflux from injected oocytes. The depolarizing response to TRH was not diminished in oocytes incubated in a Ca2(+)-free medium, but was inhibited by microinjection of EGTA. These data suggest that TRH evokes an electrophysiological response in oocytes injected with RNA from GH3 cells via activation of the same biochemical pathway that mediates its actions in GH3 cells. This pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate, forming inositol trisphosphate that causes mobilization of cellular Ca2+. We suggest that oocytes injected with GH3 cell RNA, because of their large size and easy access to their intracellular milieu, will be a useful intact cell model in which to define the molecular details of signal transduction by TRH.     

G alpha 11 and G alpha q guanine nucleotide regulatory proteins differentially modulate the response to thyrotropin-releasing hormone in Xenopus oocytes.

Xenopus oocytes that express mouse thyrotropin-releasing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R cDNA respond to THR by a depolarizing current. This response is transduced by activation of phosphoinositide-specific phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotide-binding regulatory (G) protein(s). The alpha subunit of G11 and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differences between these proteins, we have co-expressed the TRH-R with either alpha 11 or alpha q. alpha 11 potentiated the response to TRH (by 61 +/- 16%), while alpha q inhibited the response (by 37 +/- 9%). The changes in amplitudes were accompanied by inverse changes in response latencies. These data show that alpha 11 and alpha q differentially modulate signal transduction in Xenopus oocytes.     

Gonadotropin-releasing hormone receptor expression in Xenopus oocytes

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.     

Age-related differences in the pituitary prolactin response to thyrotropin-releasing hormone

We have previously reported that human subjects undergoing surgery for inguinal hernias exhibit an age-related attenuation in the plasma prolactin response, with no differences during resting conditions. We suggested that these differences were due to age-related neuroendocrine changes, but that peripheral factors may play a role as well. In the present study, we have assessed the pituitary response to 500 micrograms of thyrotropin-releasing hormone (TRH) in the very same subjects previously studied during surgery. Blood samples were drawn immediately prior to, as well as 10, 20, 40 and 60 minutes following the intravenous administration of TRH. There was a clear-cut age-related attenuation in the pituitary prolactin response with no difference in the thyrotropin (TSH) response. Maximum prolactin response in the young subjects was 31.7 micrograms/l and 19.2 micrograms/l in old subjects (F(4) = 3.5, p less than .01, two-way ANOVA). These results indicate that the age-related differences in the prolactin response to stress are mainly due to pituitary changes. However, prolactin-secreting cells are under the control of the hypothalamus. Therefore, the possibility must be considered that aging or other concurrent factors could be exerting their influence via the hypothalamus and not necessarily directly at the pituitary level.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Ribosomal frameshifting in response to hypomodified tRNAs in Xenopus oocytes

We used Xenopus oocytes as an intracellular system to study ribosomal frameshifting. Microinjection of oocytes with a construct encoding the naturally occurring UUU or AAC codon at the frameshift site demonstrated that the level of frameshifting was similar or lower than found normally in retroviral frameshifting in mammalian cells, suggesting that oocytes are a reliable system to study this event. Phenylalanine (Phe) or asparagine (Asn) tRNAs with and without the highly modified wyebutoxine (Y) or queuosine (Q) base, respectively, were microinjected to assess their ability to promote frameshifting. inhibited the level of frameshifting, while promoted frameshifting providing evidence that the hypomodified form does not act only to enhance frameshifting, but is an essential requirement. Both and were used indiscriminately in frameshifting, whether the frameshift site contained the wild-type AAC, or the mutant AAU codon, suggesting that Q base modification status does not influence this process.     

Growth hormone response to thyrotropin-releasing hormone in acromegalic patients: reproducibility and dose-response study.

The aim of the study was to analyze 14 consecutive patients with active acromegaly who had not undergone any therapy, the dose response of growth hormone (GH) to thyrotropin-releasing hormone (TRH), the existence of reproducibility of such response as well as to rule out the possibility of spontaneous fluctuations of GH which would mimic this response. On several nonconsecutive days, we investigated the GH response to saline serum, 100, 200 (twice) and 400 micrograms of TRH administration. We also studied both basal serum prolactin, serum prolactin after TRH administration and thyrotropin values. Our results show an absence of GH response after saline serum infusion, whereas after TRH doses, 36.3 42.8 and 45.4% positive responses were obtained, respectively. All GH responders were concordant to the different doses administered. The mean of GH concentrations of the different doses at different times did not reach significant differences. The response to the administration of the same dose brought about a significative increase, although it was not identical. It demonstrated a progressive increase of the area under the response curve, as did the means of increments after each TRH administration, albeit without reaching statistical significance. Between the GH-responding and GH-nonresponding groups there were no differences in either basal serum prolactin or serum prolactin and thyroid-stimulating hormone levels after TRH stimulation. The present study clearly shows that TRH elicits serum GH release from GH-secreting pituitary tumors. The response was reproducible in qualitative terms rather than quantitative, and no dose-response relationship was found between the TRH concentrations and the amounts of GH secreted.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.     

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.     

Growth hormone response to thyrotropin-releasing hormone in liver cirrhosis: unique alteration in anterior pituitary responsiveness to hypothalamic hormones

Patients with chronic liver diseases were evaluated for: 1) the ability of somatostatin to affect the thyrotropin-releasing hormone (TRH) induced growth hormone (GH) rise; 2) the competence of luteinizing-hormone releasing hormone (LH-RH) to release GH; 3) the non-specific releasing effect of TRH and LH-RH on other anterior pituitary (AP) hormones. In 6 patients, infusion of somatostatin (100 micrograms iv bolus + 375 micrograms i.v. infusion) completely abolished the TRH (400 micrograms i.v.)-induced GH rise; in none of 12 patients, of whom 7 were GH-responders to TRH, did LH-RH (100 micrograms i.v.) cause release of GH; 4) finally, LH-RH (12 patients) did not increase plasma prolactin (PRL) and TRH (7 patients) did not evoke a non-specific release of gonadotropins. It is concluded that: 1) abnormal GH-responsiveness to TRH is the unique alteration in AP responsiveness to hypothalamic hormones present in liver cirrhosis; 2) the mechanism(s) subserving the altered GH response to TRH is different from that underlying the TRH-induced GH rise present in another pathologic state i.e. acromegaly, a condition in which the effect of TRH escapes somatostatin suppression and LH-RH evokes GH and PRL release.     

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.     

Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes

Shallow injection of inositol 1,4,5-trisphosphate (IP3) 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 CaCl2 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, beta-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP3 injections at either hemisphere but did not affect the amplitudes of the responses to CaCl2. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl2. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.     

The role of vagal afferents and carbon dioxide in the respiratory response to thyrotropin-releasing hormone

Rats treated neonatally with pargyline and 5,7-dihydroxytryptamine to decrease central serotonin-containing neurons have an accentuated respiratory response to i.c.v. thyrotropin-releasing hormone (TRH). Since these treated rats also evidence an elevated PaCO2, we sought to evaluate the importance of CO2 in determining the magnitude of the respiratory response to TRH. Neonatal treatment with capsaicin or acute vagotomy also produced adult animals whose basal PaCO2 was elevated and whose respiratory response to TRH was greater than that seen in control rats with lower PaCO2 values. In normal rats, however, administration of CO2 immediately before and after TRH administration does not alter the subsequent response to TRH. Thus, it appears that TRH facilitates the processing of CO2-dependent afferent impulses, and that CO2 does not alter disposition or pharmacokinetics of TRH.