Beta T2 Research Articles

Adipose tissue cadmium concentrations as a potential risk factor for insulin resistance and future type 2 diabetes mellitus in GraMo adult cohort.

Adipose tissue has been recently highlighted as a promising matrix for evaluation of cadmium's (Cd) long-term exposure although not frequently considered in epidemiological studies. The association between Cd exposure and type 2 Diabetes Mellitus (T2DM) remains unclear. This work aimed to explore the association between adipose tissue Cd levels and T2DM incidence over a 16-year follow-up in an adult cohort from Southern Spain considering smoking status. We also performed complementary cross-sectional analyses focused on subclinical markers of glucose homeostasis at recruitment. Clinical information was obtained from hospital databases. Socio-demographic characteristics, lifestyle and diet were collected by face-to-face interviews. Homeostatic model assessment (HOMA) values of insulin sensitivity/resistance and β-cell function were calculated using fasting serum glucose, insulin, and C-peptide levels at recruitment. Adipose tissue Cd concentrations were quantified by inductively coupled plasma mass spectrometry. Statistical analyses were performed by means of Cox-regression and multivariable linear regression models. Participants in the 4th quartile (Q4) of Cd concentrations showed a non statistically-significant increased T2DM risk (Hazard Ratio (HR) Q4 vs Q1: 1.97; 95% Confidence Interval (CI): 0.69, 5.66). This association was particularly strong and suggestive in current smokers (HR: 2.19; 95% CI: 0.98, 4.98). Interestingly, smokers in the 2nd tertile (T2) of adipose tissue Cd levels showed increased log-transformed insulin resistance (beta T2 vs T1: 0.52; 95% CI: 0.07, 0.97), as well as higher log-transformed insulin levels (beta T2 vs T1: 0.52; 95% CI: 0.08, 0.95). We found evidences supporting that Cd exposure, particularly from tobacco smoking, could be a risk factor for T2DM. In addition, our results support the potential relevance of adipose tissue as a matrix for Cd exposure assessment.

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone α-subunit (αGSU) gene

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.

GnRH-Mediated DAN Production Regulates the Transcription of the GnRH Receptor in Gonadotrope Cells

The primary function of gonadotropin-releasing hormone (GnRH) is the regulation of pituitary gonadotropin hormone gene transcription, biosynthesis and release. These effects are mediated through intracellular mobilization of Ca2+ and activation of PKC isoforms and MAP kinases. We show here that DAN (differential screening-selected gene aberrative in neuroblastoma) which is a secreted bone morphogenic protein (BMP) antagonist belonging to the TGFbeta protein superfamily, is controlled by GnRH in murine gonadotrope cells. Acute GnRH stimulation induced a rapid, 27-fold, elevation of DAN mRNA, accompanied by an approximate 3-fold increase in the amount of mature DAN glycoprotein in the cell cytoplasm and in DAN secretion into the culture medium. Incubation of L beta T2 cells in DAN-containing medium altered the levels of a number of cellular proteins. Two of these were identified as the steroidogenic acute regulatory protein (StAR) and the actin-related protein 2/3 complex subunits 2 (p34-ARC) which are primarily involved in steroidogenesis and cytoskeleton remodelling, respectively. DAN caused an approximate 2-fold specific elevation in the cytoplasmic levels of both these proteins in L beta T2 cells. We further tested the effects of DAN on classical GnRH effects viz. gonadotropin and GnRH receptor gene expression. Co-transfection of L beta T2 cells with DAN and gonadotropin subunit promoter luciferase reporter genes had no effect on GnRH stimulation of alpha GSU and LH beta or on the additive GnRH and activin induction of FSH beta subunit transcription. However, co-transfection of DAN markedly inhibited the synergistic activation of GnRH and activin on GnRH receptor gene expression thus implicating DAN as a novel autocrine/paracrine factor that modulates GnRH function in pituitary gonadotropes.

Smad3 Mediates Activin-Induced Transcription of Follicle-Stimulating Hormone β-Subunit Gene

Synthesis of FSH by the anterior pituitary is regulated by activin, a member of the FSH(beta) superfamily of ligands. Activin signals through a pathway that involves the activation of the transcriptional coregulators Smad2 and Smad3. Previous work from our laboratory demonstrated that Smad3, and not Smad2, is sufficient for stimulation of the rat FSH(beta) promoter in a pituitary-derived cell line L(beta)T2. Here, we used RNA interference technology to independently decrease the expression of Smad proteins in L(beta)T2 cells to further investigate Smad2 and Smad3 roles in activin-dependent regulation of the FSHbeta promoter. Down-regulation of Smad2 protein by small interfering RNA duplexes affects only basal transcription of FSH(beta), whereas decreased expression of Smad3 abrogates activin-mediated stimulation of FSH(beta) transcription. Although highly related, Smad2 and Smad3 differ in their Mad homolog (MH) 1 domains, where the Smad2 protein contains two additional stretches of amino acids that prevent this factor from binding to DNA. We investigated whether these structural features contribute to differential FSH(beta) transactivation by Smad2 and Smad3. A variety of Smad chimera constructs were generated and used in transient transfection studies to address this question. Only cotransfection of chimera constructs that contain the MH1 domain of Smad3 results in activin-mediated stimulation of the rat FSH(beta) promoter. Furthermore, the insertion of Smad2 loops into Smad3 protein renders it inactive, suggesting that DNA binding is necessary for Smad3-mediated stimulation of the rat FSH(beta) promoter. Taken together, these results indicate that the functional differences between Smad2 and Smad3 in their ability to transactivate the rat FSH(beta) promoter lie primarily within the MH1 domain and involve structural motifs that affect DNA binding.

Gonadotropin-releasing hormone regulates expression of the DNA damage repair gene, Fanconi anemia A, in pituitary gonadotroph cells.

Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta T2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature alpha T3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

The rat pituitary promoter of the neuronal nitric oxide synthase gene contains an Sp1-, LIM homeodomain-dependent enhancer and a distinct bipartite gonadotropin-releasing hormone-responsive region.

The neuronal nitric oxide synthase (NOS I) is expressed and hormonally regulated in rat anterior pituitary gonadotropes. In the present study, we investigated the mechanisms that underlie the constitutive and GnRH up-regulated activity of the pituitary exon 1p promoter of the NOS I gene in these cells. Through the use of 5'-deletions and transient transfections in L beta T2, a gonadotrope-derived cell line, we delineated a NOS I cell-specific (NCS) enhancer region (-73/-59) that is required for constitutive activity. Independently of the NCS enhancer, GnRH responsiveness is supported by a bipartite regulatory domain referred to as the GnRH response element I and II located between -33/-10 and -4/+4, the latter consisting of a cAMP-like response element. By combining transient transfections, gel shift, and supershift assays, we demonstrate that Sp1 and LIM-homeodomain-related protein bind the NCS enhancer, whereas cAMP response element binding protein and cAMP regulatory element modulator-like factors bind the GnRH response element II motif. We further show that factors involved in GnRH regulation are also implicated in constitutive activity, suggesting intimate links between constitutive and regulated promoter activity. We speculate that specific expression of the NOS I gene in gonadotropes together with its regulation by GnRH is suggestive of a critical participation of NOS I in gonadotrope function.

Nuclear factor Y and steroidogenic factor 1 physically and functionally interact to contribute to cell-specific expression of the mouse Follicle-stimulating hormone-beta gene.

FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH beta-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSH beta using the mouse L beta T2 and alpha T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of L beta T2 and alpha T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied L beta T2 cell-specific expression of FSH beta using 398 bp of the mouse FSH beta regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSH beta promoter can direct reporter gene expression specifically to L beta T2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including alpha T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in L beta T2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSH beta gene expression in an L beta T2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSH beta protein in L beta T2 cells and to identify specific elements within the FSH beta promoter that contribute to basal and cell-specific expression of the gene.

Differential regulation of gonadotropin subunit gene promoter activity by pulsatile gonadotropin-releasing hormone (GnRH) in perifused L beta T2 cells: role of GnRH receptor concentration.

The pulsatile release of GnRH by the hypothalamus is required to stimulate the pituitary-gonadal axis, and variations in GnRH pulse frequency are associated with differential synthesis and release of LH and FSH by pituitary gonadotropes. How gonadotropes differentiate between GnRH pulse frequencies and subsequently differentially regulate the expression of the LH beta and FSH beta genes remains to be determined. In the present study, using a perifusion system that allows us to replicate the GnRH pulsatility occurring in vivo, we have systematically characterized the effects of varying GnRH pulse frequencies on LH beta, FSH beta, alpha, and GnRH receptor (GnRHR) gene promoter stimulation in L beta T2 cells. We demonstrate that LH beta gene promoter activity is stimulated to the greatest extent at higher GnRH pulse frequencies, whereas the FSH beta gene promoter is preferentially stimulated at lower GnRH pulse frequencies, reflecting previous observations in primary rat pituitary cells in vivo and in vitro. By measuring GnRH binding, we demonstrate that cell-surface GnRHR number is increased at higher frequencies of pulsatile GnRH and that this increase precedes the differential regulation of LH beta and FSH beta gene promoter activity. To test the role of GnRHR number in mediating the differential effects of pulsatile GnRH, the rat GnRHR was overexpressed in L beta T2 cells, and the response to pulsatile GnRH was again assessed. Interestingly, although overexpression of GnRHR had no effect on the frequency-dependent regulation of LH beta, the induction of FSH beta gene promoter activity by pulsatile GnRH was reduced, and frequency dependence was abrogated. Our results demonstrate that L beta T2 cells represent a suitable model for the study of the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH. Furthermore, our studies indicate that cell-surface GnRHR density is a critical mediator of this differential regulation.

Embryonic Expression of the Luteinizing Hormone β Gene Appears to Be Coupled to the Transient Appearance of p8, a High Mobility Group-related Transcription Factor

A comparison between two pituitary-derived cell lines (alpha T3-1 and L beta T2) that represent gonadotropes at early and late stages of development, respectively, was performed to further elucidate the genomic repertoire required for gonadotrope specification and luteinizing hormone beta (LH beta) gene expression. One isolated clone that displayed higher expression levels in L beta T2 cells encodes p8, a high mobility group-like protein with mitogenic potential that is up-regulated in response to proapoptotic stimuli and in some developing tissues. To test the functional significance of this factor in developing gonadotropes, a knockdown of p8 in L beta T2 cells was generated. The loss of p8 mRNA correlated with loss of endogenous LH beta mRNA and the loss of activity of a transfected LH beta promoter-driven reporter, even upon treatment with gonadotropin-releasing hormone. In addition, expression of p8 mRNA in developing mouse pituitary glands mirrored its expression in the gonadotrope-derived cell lines and coincided with the first detectable appearance of LH beta mRNA. In contrast, p8 mRNA was undetectable in the pituitary glands of normal adults. Taken together, our data indicate that p8 is a stage-specific component of the gonadotrope transcriptome that may play a functional role in the initiation of LH beta gene expression during embryonic cellular differentiation.

Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in L beta T2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LH beta genes in these cells. Signaling via the G(i) subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LH beta induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters G beta gamma-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of G alpha(q) (Q209L) and was blocked by a cell-permeable peptide that uncouples G alpha(q) from GPCRs. Furthermore, chronic activation of G alpha(q) signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples G alpha(s) from receptors was also able to inhibit ERK, c-Fos, and LH beta, indicating that both G(q/11) and G(s) proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on G(s) and G(q/11) and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of G alpha(q) (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both G(q) and G(s) signaling to regulate gene expression in L beta T2 cells.

Isolation and characterization of a rat nitric oxide synthase type I gene promoter that confers expression and regulation in pituitary gonadotrope cells.

Nitric oxide synthase type I (NOS I) is expressed and up-regulated in rat pituitary gonadotrophs. Using rapid amplification of cDNA ends-PCR, 2 major transcripts with 5' ends corresponding to exon 1a but truncated of its first 369 or 384 nucleotides, indicative of two pituitary-specific transcription start sites, were identified. By chromosome walking, we isolated 5'-upstream of this truncated exon termed 1p, a novel -1653/+384-bp genomic region. Transient transfections, using the gonadotrope-derived alpha T3-1 and L beta T2 cell lines and the full-length or 5'-deleted sequences fused to a luciferase reporter gene, demonstrated that cell-specific positive and negative regions were present especially within the -246/-73 region, whereas the +12/+384 region was crucial for transcription. Moreover, in L beta T2 cells, the luciferase activity was increased by GnRH, with the full-length sequence being the most efficient and the -73/+60 region corresponding to the essential zone. The latter region was also crucial for cholera toxin-induced activation. Interestingly, GnRH and cAMP effects were not additive, implying a convergent step in the transduction cascade. These data provide evidence for the presence of several elements controlling NOS I expression in gonadotrophs and demonstrate that GnRH, the prime regulator of gonadotrope function, and cAMP may induce the transcription of NOS I in these cells.

CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene.

The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5' flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBP beta-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBP beta, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBP beta is the predominant C/EBP family member expressed in the L beta T2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBP beta protein in nuclear extracts from L beta T2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in L beta T2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBP beta transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

Calcitonin is expressed in gonadotropes of the anterior pituitary gland: its possible role in paracrine regulation of lactotrope function.

Previous studies from this laboratory have shown that salmon (S) calcitonin (CT)-like immunoreactive peptide (CTI) is synthesized and secreted by the anterior pituitary (AP) gland. These studies also co-localized CTI to gonadotropes, and demonstrated that SCT is a potent inhibitor of lactotrope function. However, the molecular structure of putative gonadotrope-derived CTI that inhibits lactotrope function has not been defined. The present studies cloned CT cDNA (pit-CT cDNA) from a mouse gonadotrope L beta T2 cell line using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Alignment of nucleotide sequences of pit-CT and mouse CT revealed greater than 99% homology between the sequences. The pit-CT cDNA was ligated into a mammalian expression vector, and the construct was transfected into L beta T2 cells. Two stable transfectant cell lines (CT.U6/A and B) were obtained by selection in G418. Subsequent S1-nuclease protection assay and immunocytochemistry results have shown that: (1) pit-CT peptide expressed by CT.U6 cell lines immunoreacted with GCT1-anti-SCT serum; (2) secretions of CT.U6 cells inhibited prolactin (PRL) release, PRL mRNA abundance and DNA synthesis of PRL-secreting GGH3 cells; and (3) CT.U6-induced inhibition was abolished by GCT1-anti-SCT serum. The studies also generated a riboprobe from the cloned pit-CT cDNA, and localized CT mRNA expression in gonadotropes of rat AP gland by in situ hybridization histochemistry. These results demonstrate that pit-CT mRNA is closely homologous to mouse CT mRNA; it is expressed by gonadotropes of the rat AP gland, and the peptide may significantly affect lactotrope function by inhibiting PRL release and cell proliferation.

Regulation and Actions of Smad7 in the Modulation of Activin, Inhibin, and Transforming Growth Factor-β Signaling in Anterior Pituitary Cells1

Activins and transforming growth factor-beta (TGF beta) are crucial autocrine, paracrine, and endocrine modulators of anterior pituitary function. Activins regulate most pituitary cells and lactotropes are targets of TGF beta. Smad2 and Smad3 are two cellular mediators of activin/TGF beta signaling, whereas Smad7 is as an inducible, negative modulator of the pathway. This study was undertaken to evaluate Smad7 regulation in the pituitary. Activin A rapidly and transiently increased Smad7 messenger RNA (mRNA) levels of rat anterior pituitary (RAP), clonal gonadotrope (alpha T 3-1 and L beta T2), and corticotrope (AtT20) cells with an EC(50) of 0.1-0.2 nM. In RAP cells, activin A or TGF beta 1 had equivalent effects that were additive. Follistatin, known to bind and inactivate activins, prevented Smad7 induction by activin. Inhibin A partially antagonized activin A, perhaps reflecting gonadotrope-selective actions. This antagonism was also evident with alpha T 3-1 and L beta T2 gonadotropes. Forskolin had no measurable effect in RAP cells, but increased Smad7 mRNA levels in alpha T3-1 cells and decreased them in L beta T2 cells. Transient transfection of Smad7 along with 3TPLux, an activin/TGF beta-responsive reporter, blocked activin-mediated promoter activation in alpha T3-1 and AtT20 cells. In alpha T3-1 cells, which express endogenous follistatin mRNA, a follistatin-luciferase reporter, rFS(rin3)-Luc, was transcriptionally activated by activin A, or when cotransfected with a constitutively active ActRIB [Alk4(T>D)], Smad2, or Smad3. Smad7 blocked rFS(rin3)-Luc activation by activin A or Alk4(T>D). Together, these results point to a role of Smad7 in modulating activin/TGF beta signaling in the pituitary.

The L beta T2 clonal gonadotrope: a model for single cell studies of endocrine cell secretion

We have used single gonadotropes from the newly derived line, LbetaT2, to investigate the modulation of Ca2+ signaling and exocytosis by the steroid hormone environment. This cell line, derived by targeted oncogenesis in transgenic mice, has recently been shown to secrete LH in response to GnRH. We have characterized the effects of both GnRH and membrane depolarization on exocytosis and intracellular [Ca2+] ([Ca2+]i) in individual LbetaT2 cells. GnRH (1-100 nM) evoked concentration-dependent increases in [Ca2+]i and secretion, as monitored by measurement of plasma membrane capacitance (Cm) using the whole-cell perforated-patch technique, and the extent of these changes were dependent upon steroid hormone background. GnRH treatment of cells cultured in medium containing charcoal-treated FBS (ct-FBS) showed smaller changes in [Ca2+]i than cells cultured in untreated FBS. However, when estradiol (E2) and dexamethasone (Dex) were added to the ct-FBS medium (E2/Dex-ct-FBS), the elevations in [Ca2+]i stimulated by GnRH increased almost 2-fold. Additionally, the rates of secretion in the E2/Dex-ct-FBS-cultured cells were greater than in either ct-FBS- or FBS-cultured cells. The increase in secretory response observed with E2/Dex-ct-FBS appeared to be due to both an increase in the peak [Ca2+]i stimulated by GnRH and a shift toward increased sensitivity of the Ca2+ dependency of exocytosis. In contrast to GnRH-evoked responses, the increases in [Ca2+]i elicited by depolarization were greater in cells cultured in ct-FBS than in E2/Dex-ct-FBS; however, the secretory rates were no different in the two groups. Likewise, there was no apparent effect of steroid treatment on the Ca2+ dependency of depolarization-evoked exocytosis. In summary, these results 1) clearly demonstrate the utility of this cell line for single-cell studies of both agonist- and depolarization-evoked secretion; 2) reveal that steroid hormone background has profound effects on LbetaT2 cells, both on stimulus-induced calcium mobilization and on the apparent Ca2+ -sensitivity of exocytosis; and 3) show that expression of the steroid hormone effect on Ca2+ -sensitivity is dependent upon receptor occupation by GnRH.

The L beta T2 clonal gonadotrope: a model for single cell studies of endocrine cell secretion.

We have used single gonadotropes from the newly derived line, LbetaT2, to investigate the modulation of Ca2+ signaling and exocytosis by the steroid hormone environment. This cell line, derived by targeted oncogenesis in transgenic mice, has recently been shown to secrete LH in response to GnRH. We have characterized the effects of both GnRH and membrane depolarization on exocytosis and intracellular [Ca2+] ([Ca2+]i) in individual LbetaT2 cells. GnRH (1-100 nM) evoked concentration-dependent increases in [Ca2+]i and secretion, as monitored by measurement of plasma membrane capacitance (Cm) using the whole-cell perforated-patch technique, and the extent of these changes were dependent upon steroid hormone background. GnRH treatment of cells cultured in medium containing charcoal-treated FBS (ct-FBS) showed smaller changes in [Ca2+]i than cells cultured in untreated FBS. However, when estradiol (E2) and dexamethasone (Dex) were added to the ct-FBS medium (E2/Dex-ct-FBS), the elevations in [Ca2+]i stimulated by GnRH increased almost 2-fold. Additionally, the rates of secretion in the E2/Dex-ct-FBS-cultured cells were greater than in either ct-FBS- or FBS-cultured cells. The increase in secretory response observed with E2/Dex-ct-FBS appeared to be due to both an increase in the peak [Ca2+]i stimulated by GnRH and a shift toward increased sensitivity of the Ca2+ dependency of exocytosis. In contrast to GnRH-evoked responses, the increases in [Ca2+]i elicited by depolarization were greater in cells cultured in ct-FBS than in E2/Dex-ct-FBS; however, the secretory rates were no different in the two groups. Likewise, there was no apparent effect of steroid treatment on the Ca2+ dependency of depolarization-evoked exocytosis. In summary, these results 1) clearly demonstrate the utility of this cell line for single-cell studies of both agonist- and depolarization-evoked secretion; 2) reveal that steroid hormone background has profound effects on LbetaT2 cells, both on stimulus-induced calcium mobilization and on the apparent Ca2+ -sensitivity of exocytosis; and 3) show that expression of the steroid hormone effect on Ca2+ -sensitivity is dependent upon receptor occupation by GnRH.

Steroid and pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing hormone and GnRH receptor in a novel gonadotrope cell line.

Properties of a pituitary gonadotrope include the capacity to regulate gonadotropin synthesis and secretion in response to a GnRH signal. Progress in identifying the steps involved in these processes has been impeded by the lack of a homogeneous in vitro model of gonadotropes. This paper presents functional characterization of a L beta T2 gonadotrope cell line generated by tumorigenesis in transgenic mice carrying the rat LH beta-subunit regulatory region linked to the SV40 T-antigen oncogene. This cell line expresses LH beta, alpha-subunit, and GnRH-receptor (GnRH-R) mRNAs (though not FSH beta), responds to glucocorticoid treatment with a reversible dampening of proliferation, and responds to pulsatile, concentration-dependent GnRH administration with LH secretion. L beta T2 cells presented with four GnRH pulses (10 nM, 90-min interpulse interval) on each of 4 days respond with incremental increases in LH secretion on successive days. This increase was greatest (15-fold) in the presence of estradiol and dexamethasone. Part of the enhanced responsiveness is apparently due to an increase in GnRH-R; pulsatile GnRH treatment alone as well as steroid treatment alone led to an increase in GnRH-R mRNA levels. When secretion was stimulated on day 4 with 54 mM [K+] pulses, bypassing the GnRH-R, the LH-secretory response indicated that the GnRH pulse history as well as estradiol and dexamethasone have actions on L beta T2-secretory capacity distinct from changes in the GnRH-R. This increase can be explained in part by the marked up-regulation of LH beta, but not alpha-subunit, mRNA observed in GnRH-pulsed cells. In summary, L beta T2 clonal gonadotropes exhibit functional characteristics consistent with those of normal pituitary gonadotropes such as LH secretion via a regulated pathway and changes in GnRH-R and LH beta gene expression in response to signaling by GnRH and steroid hormones and therefore should be a useful tool for dissecting the cellular and molecular events involved in these fundamental gonadotrope properties.

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO.

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.