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1.
S M Simasko 《Molecular and cellular endocrinology》1991,78(1-2):79-86
The effects of intracellular calcium buffering and increasing bath Ca2+ on spontaneous membrane depolarizations expressed by the clonal rat pituitary cell line GH3 were examined by use of the whole-cell patch-clamp technique. Increasing intracellular calcium buffering capacity caused the duration of spontaneous depolarizations to increase without altering other parameters of membrane potential activity. Increasing bath Ca2+ caused a decrease in duration. These results suggest that the duration of spontaneous membrane depolarization in GH3 cells is regulated by the accumulation of free intracellular Ca2+. The behavior of spontaneous depolarizations measured with the perforated-patch variation of whole-cell patch-clamp techniques closely resembled that obtained in standard whole-cell patch-clamp measurements with an intracellular calcium buffer of 200 microM EGTA with free Ca2+ adjusted to 100 nM. 相似文献
2.
The possible influence of growth hormone-releasing factor (GHRF) on epithelial cell proliferation in the digestive tract was investigated. Fasted young rats received five hourly subcutaneous injections of either GHRF or saline. They were killed 6, 12, or 18 h after the initial injection and 45 min after [3H]thymidine pulse labeling. At the time of death, blood was taken to determine circulating growth hormone and gastrin levels. After radioautography, DNA synthetic and mitotic activities were estimated in the fundic, antral, duodenal, jejunal, and colonic mucosae. Growth hormone-releasing factor significantly increased labeling indices 6, 12, and 18 h after the initial injection in fundic mucosa, and 6 and 18 h after injection in antral and duodenal mucosae. Furthermore, GHRF significantly increased mitotic indices at 12 h in fundic mucosa and at 12 and 18 h in jejunal mucosa. No effect was seen in the colon. At the three checkpoint times, circulating growth hormone showed no change, but plasma gastrin was increased in the rats treated with GHRF as compared with controls. However, whether the reported stimulatory effect of the GHRF on target cells is direct or indirect remains to be determined. 相似文献
3.
Growth hormone-releasing hormone stimulates cAMP release in superfused rat pituitary cells. 总被引:1,自引:0,他引:1
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J E Horváth K Groot A V Schally 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(6):1856-1860
The release of growth hormone (GH) and cAMP was studied in superfused rat pituitary cells by infusing growth hormone-releasing hormone (GHRH) at different doses or a combination of GHRH and somatostatin 14 (SS-14). Three-minute pulses of GHRH caused a dose-dependent GH and cAMP release (effective concentration of 50% of the maximal biological effect is 0.21 nM and 52.5 nM, respectively). The lowest effective doses of GHRH in the superfusion system were 0.03 nM for GH release and 0.3 nM for cAMP discharge when 3-min pulses were applied. The amount of cAMP liberated from the cells was not proportional to GH release: cAMP responses to low doses of GHRH were disproportionally small, and the gradual increase in the release of cAMP after high doses of GHRH was not followed by a parallel rise in GH release. The desensitization induced by repeated pulses or prolonged infusion of GHRH resulted in a greater reduction in GH release than in cAMP liberation. A simultaneous infusion of SS-14 completely blocked GH release stimulated by GHRH but did not inhibit the immediate release of cAMP caused by GHRH. An abrupt decrease in GHRH-stimulated GH release induced by SS-14 was followed by only a minimal reduction in cAMP liberation 9 min later. Our findings indicate that a discharge of cAMP is stimulated after a GHRH pulse, but this effect alone cannot maintain the release of GH. Other steps of the signal transduction mechanisms that are independent of the cAMP route may participate in the process of GH release. The nature of the mechanisms involved in the mediation of GH release may vary with the doses of GHRH used. 相似文献
4.
Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. 总被引:7,自引:2,他引:7
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N Billestrup L W Swanson W Vale 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(18):6854-6857
The mitogenic effect of the hypothalamic peptides growth hormone-releasing factor (GRF) and somatostatin on cultured growth hormone (GH)-producing cells (somatotrophs) was studied. Using autoradiographic detection of [3H]thymidine uptake and immunocytochemical identification of GH-producing cells, we show that 5 nM GRF causes a 20-fold increase in the percentage of somatotrophs labeled with [3H]thymidine. The total number of somatotrophs in GRF-treated cultures was increased by 60%. Somatostatin had no measurable effect on the labeling index by itself, but it partly inhibited the GRF-induced increase in both the labeling index and the total number of cells. Forskolin caused an increase in both the percentage of somatotrophs with a [3H]thymidine-labeled nucleus and the somatotroph number similar to that caused by GRF. GH secretion as well as cellular GH content in the GRF- or forskolin-treated cells increased with culture time over the entire period, whereas secretion and content of GH gradually decreased in control or somatostatin-treated cultures during the entire culture period. These data suggest that GRF and somatostatin regulate the mitotic activity of GH-producing cells and that the effect of GRF is possibly mediated by cyclic AMP. 相似文献
5.
Growth hormone-releasing factor desensitization in rat anterior pituitary cells in vitro 总被引:2,自引:0,他引:2
Preincubation of rat pituitary cells in primary culture with rat GH-releasing factor (rGRF) resulted in substantial desensitization to subsequent GRF stimulation. rGRF-directed GH release and intracellular cAMP accumulation decreased in the desensitized cells. Whereas prior treatment of rat pituitary cells caused partial depletion of intracellular GH levels, diminished cellular reserves could not entirely account for the decreased GH release. Cells that had been preexposed to 10 nM rGRF for 4 h demonstrated at 30-50% depletion of intracellular GH; subsequent stimulation of those cells with 10 nM rGRF elicited GH release which was only 5% of that seen in cells that were not desensitized [control, 112 +/- 3.2 ng/well (+/- SEM); GRF-stimulated, 435 +/- 32 ng/well; GRF-pretreated, control, 63 +/- 3 ng/well, GRF-pretreated, GRF-stimulated, 73 +/- 3.4 ng/well]. Despite the marked depletion of cellular GH stores and the greatly diminished rGRF-stimulated GH release in cells that had been preexposed to rGRF, both forskolin and (Bu)2cAMP were able to induce a 2-fold stimulation of GH release. Incubation of the rGRF-pretreated cells with fresh medium which lacked rGRF resulted in gradual recovery of the ability of rGRF to stimulate GH release without complete reconstitution of the intracellular GH stores. These results indicate that exposure of rat pituitary cells to rGRF results in 1) partial depletion of intracellular GH stores; 2) a diminished ability of a subsequent rGRF challenge to elicit GH secretion and intracellular cAMP accumulation, and 3) a sustained ability of forskolin and (Bu)2cAMP to stimulate GH release, indicating that rGRF desensitization occurs in vitro. 相似文献
6.
Besides its physiological role as a neurotransmitter, dopamine (DA) induces apoptosis in the central nervous system. This effect is mediated partly by the DA transporter (DAT) and involves reactive oxygen species (ROS) formation as well as oxidative stress. In the pituitary, the inhibitory control by DA of prolactin release and synthesis and lactotrope cell proliferation is well known, while the pro-apoptotic effect of DA remains unclear. Our aim was to study the pro-apoptotic effect of DA in the GH3 mammosomatotrope cell line and determine the DA mechanism that leads to apoptosis in these cells. Using flow cytometry, Western blot, and confocal microscopy, we showed for the first time that DA induced: (1) loss of mitochondrial potential; (2) relocation of Bax to the mitochondria; (3) cytochrome c release; (4) caspase-3 activation, and (5) nuclear fragmentation, resulting in apoptosis. We observed that DAT was expressed in GH3 cells and participated in the DA effect, as apoptosis was significantly reversed in the presence of DAT inhibitors. Direct measurement showed that DA rapidly increased the formation of intracellular ROS. The antioxidant N-acetyl-L-cysteine (NAC) effectively blocked DA-induced ROS formation and apoptosis. Neither JNK nor p38 were involved in this process, so we suggest that the mitochondrial pore of transition is the likely target of the ROS generated by DA. These data provide the first evidence that DA triggers apoptosis in pituitary cells via a mechanism involving DAT and oxidative stress. These findings may be particularly relevant in understanding lactotrope apoptosis during postnatal life. 相似文献
7.
Growth hormone-releasing peptide-2 stimulates GH secretion in GH-deficient patients with mutated GH-releasing hormone receptor. 总被引:6,自引:0,他引:6
R G Gondo M H Aguiar-Oliveira C Y Hayashida S P Toledo N Abelin M A Levine C Y Bowers A H Souza R M Pereira N L Santos R Salvatori 《The Journal of clinical endocrinology and metabolism》2001,86(7):3279-3283
GH-releasing peptides (GHRPs) are synthetic peptides that bind to specific receptors and thereby stimulate the secretion of pituitary GH. In vivo it is uncertain whether these peptides act directly on somatotroph cells or indirectly via release of GHRH from the hypothalamus. In this study we compared the pituitary hormone response to GHRP-2 in 11 individuals with isolated GH deficiency (GHD) due to a homozygous mutation of the GHRH receptor (GHRH-R) gene and in 8 normal unrelated controls. Basal serum GH levels were lower in the GHD group compared with controls [0.11 +/- 0.11 (range, <0.04 to 0.38) vs. 0.59 +/- 0.76 microg/L (range, 0.04-2.12 microg/L); P = 0.052]. After GHRP-2 administration there was a 4.5-fold increase in serum GH relative to baseline values in the GHD group (0.49 +/- 0.41 vs. 0.11 +/- 0.11 microg/L; P = 0.002), which was significantly less than the 79-fold increase in the control group (46.8 +/- 17.6 vs. 0.59 +/- 0.76 microg/L; P = 0.008). Basal and post-GHRP-2 serum levels of ACTH, cortisol, and PRL were similar in both groups. Basal levels of serum TSH were significantly higher in the GHD group than in the control group (3.23 +/- 2.21 vs. 1.37 +/- 0.34 microIU/mL; P = 0.003). TSH levels in both groups did not change after GHRP-2 administration. These results suggest that an intact GHRH signaling system is not an absolute requirement for GHRP-2 action on GH secretion and that GHRP-2 has a GHRH-independent effect on pituitary somatotroph cells. 相似文献
8.
Ionising radiation is used for the treatment of pituitary tumours as fractionated radiotherapy, where the total dose reaching the tumour area is in the range of 40-50 Gy, or during stereotactic radiosurgery, where the total dose reaching the tumour area during one session is in the range of 20-90 Gy. In this study, we investigated the effect of ionising radiation of (60)Co (dose rate of 3 Gy/min, similar to that used during gamma knife procedure) on the mode of cell death of the somatomammotroph pituitary cell line, GH3, an immortalized cell line derived from a rat pituitary adenoma. We found that the basic mechanism of cell death induced by irradiation of this GH3 cell line by gamma-rays was programmed cell death-apoptosis. Doses of 20-50 Gy were shown to inhibit proliferation in these cells. 24 hours after irradiation with a dose of 20 and 50 Gy, cells were shown to accumulate in the G(2)/M phase of cell cycle. This cell cycle arrest lasted for at least ten days. Apoptosis was detected 72 hours towards until the end of the study (10 days). However, a significant number of cells were still alive ten days following irradiation. We conclude that ionising radiation doses of 20 and 50 Gy induce pituitary GH3 cell apoptosis following cell cycle arrest in the G(2)/M phase. 相似文献
9.
GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, sequestration of the betagamma-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-gamma. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH. 相似文献
10.
Reevaluation of the electrophysiological actions of thyrotropin-releasing hormone in a rat pituitary cell line (GH3) 总被引:1,自引:0,他引:1
S M Simasko 《Endocrinology》1991,128(4):2015-2026
The electrophysiological actions of TRH were examined in the clonal pituitary cell line GH3 with the use of the perforated patch variation of the standard whole cell patch-clamp technique. The action of TRH on spontaneously spiking cells was to cause a brief hyperpolarization (first phase action), followed by a period during which action potential behavior was significantly modified (second phase action). The modifications during second phase action included a reduction in the slope of the up-stroke, a reduced peak potential, an increase in duration, and a depolarizing shift of the after-hyperpolarization. The modification of voltage- and calcium-dependent conductances that underlie these changes were investigated in voltage clamp experiments. During first phase action TRH was found to increase calcium-dependent potassium current. During second phase action TRH was found to significantly reduce the L-type calcium current (35%), with no alteration in the T-type calcium current. The second phase action of TRH on calcium-dependent potassium conductance was complex. First, a decrease was observed. This was followed by an increase that did not become fully manifest until after TRH was washed from the cell. TRH caused no change in voltage-dependent potassium current. These results indicate that the second phase action of TRH on action potential behavior in GH3 cells is mediated by a reduction in L-type calcium current and alterations in the behavior of calcium-dependent potassium currents, but not through changes in voltage-dependent potassium currents. 相似文献
11.
The effect of the anti-estrogen Tamoxifen (Tx) on membrane electrical properties and the underlying calcium (Ca2+) conductances was examined in the clonal pituitary cell line GH3/B6 which exhibits calcium action potentials at rest. Electrophysiological recordings (109 cells) were made using either high resistance intracellular microelectrodes or the whole-cell recording (WCR) patch-clamp technique. Electrical activities of 39 spontaneously active GH3/B6 cells were recorded with sharp microelectrodes filled with 3 M KCl. The spikes were Ca2+-dependent since they were blocked by Cobalt ions (Co2+, 5 mM). When applied directly to the recorded cell, Tx (10(-7) M) inhibited action potential firing. This blockade was accompanied by a discrete hyperpolarization of the membrane potential (-2.8 +/- 2 m V) from rest (-39.5 +/- 5 m V) and a 10% increase in the input membrane resistance. The effect stopped soon after Tx removal (mean 11.4 +/- 6 sec), and Tx solvent was unable to elicit the response. Current clamp WCR with pipettes containing potassium gluconate (KGlu, 140 mM) confirmed these results (30 cells), but the addition of cell extract to KGlu was necessary to prevent rundown of the response and to obtain reproducible action potential blockade. Short (1-5 min) or long term (48 h) pretreatment with estradiol (10(-7) to 10(-5) M) did not change the response to Tx, thus indicating that its effect was not mediated through estrogen receptors. Voltage clamp WCR study of the effect of Tx (10(-7) M) using pipettes filled with cesium chloride (140 mM) showed that both fast and slow inactivating calcium conductances were inhibited (38 cells). The fast inactivating Ca2+ current was reduced by about 60-80% whereas slow inactivating Ca2+ current was completely inhibited. This action may represent one way by which the antitumoral effect of this antiestrogen is mediated. 相似文献
12.
G Valenti A Banchini L Denti R Polotti G P Ceda 《Journal of endocrinological investigation》1986,9(2):165-169
Nineteen normal subjects (12 men and 7 women) were injected with 100 micrograms of ACTH 1-17. Additionally 6 male subjects were studied twice at 3-day intervals with random infusions of ACTH 1-17 and saline. A clear GH response to ACTH 1-17 infusions (GH peak higher than 5 ng/ml) was documented in 10 out of 12 males and in 6 out of 7 women. In the 6 male subjects studied twice, clear-cut GH increments were observed only after peptide administration. PRL levels decreased throughout the study period both in male and female subjects; however, when the PRL percentage decline was evaluated in the same group of subjects after saline and ACTH 1-17, the more obvious decrease of PRL levels after the peptide infusion was not statistically significant. No variation of LH, FSH and TSH levels was documented. With the exception of the specific increase of cortisol levels, no significant change in peripheral steroid pattern (Te, E2, DHEA-S) was observed. In this experiment the effect on GH secretion was quite evident in both sexes. This effect was obtained using the lowest dosage of ACTH preparation documented in the literature. 相似文献
13.
Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone that is essential for normal expansion of the somatotrope lineage during pituitary development. Decreased GHRH secretion and/or action leads to impairment of this process and somatotrope hypoplasia in both humans and experimental animals. Excessive GHRH secretion and/or action result in dysregulated somatotrope proliferation, leading to hyperplasia and neoplastic transformation. Our understanding of the molecular and morphologic bases for these effects from both animal and clinical studies has greatly increased during the past decade. However, many features of the cellular pathways remain to be defined, including the interaction of other genes in the multistep process of somatotrope tumorigenesis. 相似文献
14.
Epidermal growth factor induces the functional expression of dopamine receptors in the GH3 cell line. 总被引:3,自引:0,他引:3
GH3 cells are a clonal strain from a rat pituitary tumor that synthesizes and secretes both PRL and GH. The peculiarity of these cells is that they do not express receptors for dopamine; thus the hormone release is insensitive to the inhibitory effect of dopamine and D2 receptor agonists. Exposure of GH3 cells to epidermal growth factor for 4 consecutive days markedly altered the cell morphology, from a spherical appearance to an elongated flattened shape, and increased the cell size. These morphological changes were accompanied by the functional expression of D2 dopamine receptors as shown by the presence of a specific, saturable, and stereoselective high affinity binding for [3H]spiroperidol in epidermal growth factor-treated cells and by the fact that the selective D2 agonist quinpirole recovered the property to inhibit PRL secretion in the cell cultures exposed to the neurotrophic factor. The effect of EGF on the functional expression of D2 receptors was dose dependent (EC50 = 8 pM) and reversible. These data suggest that EGF elicits major effects on the expression of specific genes leading to the differentiation of GH3 cells into lactotroph-like cells endowed with dopamine D2 receptors. 相似文献
15.
Both vascular endothelial growth factor (VEGF) and its receptor Flk-1 are expressed in normal pituitary cells and in the prolactin-
and growth hormone-producing GH3 cell line of the rat, thus suggesting autocrine/paracrine function. Regulation of the Flk-1
receptor system in pituitary cells is poorly understood, but evidence suggests that up-regulated growth factors play a role
in its expression and activation. To study the role of growth factors in this process, we examined changes in VEGF and Flk-1
expression in GH3 cells following varied exposure to βFGF, EGF, and TGFβ1. Immunofluorescence labelling and laser scanning
cytometry were used to measure changes in VEGF and Flk-1 expression. Results showed that βFGF, EGF and TGFβ up-regulated the
VEGF/FLK-1 receptor system. Distinct patterns of activation were detected. At 2 hours, EGF and TGFβ caused no significant
changes in VEGF and Flk-1 expression; however, βFGF up-regulated VEGF expression in 99% of cells but only induced modest changes
in Flk-1 overexpression. A similar percentage of cells overexpressed VEGF after 24-hour incubation with βFGF, but more prominent
Flk-1 overexpression was detected. At 24 hours, EGF and TGFβ1 induced a significant increase in both VEGF and Flk-1 expression.
In summary, our findings show that VEGF/Flk-1 expression in pituitary cells may be altered by different growth factors. This
may affect angiogenesis and the progression of pituitary tumors. 相似文献
16.
17.
Growth hormone-releasing factor binding sites in rat anterior pituitary membrane homogenates: modulation by glucocorticoids 总被引:4,自引:0,他引:4
Specific high affinity binding sites for growth hormone releasing factor (GRF) were described in rat anterior pituitary homogenates with use of the analog [His1-mono-125I-Tyr10,Nle27]-hGRF(1-32)-NH2 as radioligand. Computerized analysis of competition experiments indicated one class of specific high affinity binding sites with a dissociation constant of 0.19 nM. The relative binding affinities of rGRF, hGRF(1-40) and various analogs correlated well with their in vitro biological potencies. Further, the number binding sites was drastically decreased after adrenalectomy; chronic dexamethasone treatment of these animals restored GRF binding capacity to control without changing binding affinity. These results may in part explain the enhanced responsiveness of the somatotroph after dexamethasone treatment. 相似文献
18.
Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells. 总被引:4,自引:0,他引:4
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G G Gick F N Zeytin P Brazeau N C Ling F S Esch C Bancroft 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(5):1553-1555
A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells. 相似文献
19.
20.
Several peptide hormones and neurotransmitters are produced by cleavage at the monobasic processing sites. An endoprotease capable of cleaving a dynorphin peptide at the monobasic processing site is secreted from the rat anterior pituitary lactotrophic cell line, GH4C1. When characterized by fast protein liquid chromatography using an ion exchange column, the majority of the endoprotease activity elutes as a single symmetrical peak around 0.3 M NaCl. The protease inhibitor profile suggests that the activity is due to putative thiol protease. These enzymatic properties are similar to a monobasic processing enzyme previously found in bovine pituitary and in the rat brain. The secretory pathway which contains the enzyme activity in GH4C1 cells was characterized by stimulation of secretion by thyrotropin releasing hormone, forskolin, phorbol ester, or potassium chloride. The secretion of the enzyme activity was substantially increased by these compounds suggesting that the GH4C1 cells secrete the activity via the regulated pathway. A hormonal treatment of the GH4C1 cells which has been previously shown to produce a substantial increase in the number of secretory granules and ir-prolactin has been found in this study to elevate this enzyme activity 2-fold. This increase is similar to that seen in the carboxypeptidase E activity, another putative peptide hormone processing enzyme activity. These data suggest that the peptide processing activity is regulated to a small but significant extent and is coordinately regulated with carboxypeptidase E activity. 相似文献