Concordance of 3 alternative teratogenicity assays with results from corresponding in vivo embryo-fetal development studies: Final report from the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) DruSafe working group 2.

An IQ DruSafe working group evaluated the concordance of 3 alternative teratogenicity assays (rat whole embryo culture, rWEC; zebrafish embryo culture, ZEC; and murine embryonic stem cells, mESC) with findings from rat or rabbit embryo-fetal development (EFD) studies. Data for 90 individual compounds from 9 companies were entered into a database. In vivo findings were deemed positive if malformations or embryo-fetal lethality were reported in either species. Each company used their own criteria for deciding whether the alternative assay predicted the in vivo findings. Standard concordance parameters were calculated, positive and negative predictive values (PPV and NPV) were adjusted for the aggregate portfolio prevalence of positive compounds (established by a survey of participating companies), and positive and negative likelihood ratios (LR+ and iLR-) were calculated. Of the 3 assays, only rWEC data were robustly predictive, particularly for negative predictions (NPVadj = 92%). However, both LR+ (4.92) and iLR- (4.72) were statistically significant for the rWEC assay. When analyzed separately for rats, the NPVadj and iLR-values for the rWEC assay increased to 96% and 9.75, respectively. These data suggest that a negative rWEC outcome could defer or replace a rat EFD study in certain regulatory settings.

GSK2245035, a TLR7 agonist, Does Not Increase Pregnancy Loss in Cynomolgus Monkeys.

GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.

Comparative Aspects of Pre- and Postnatal Development of the Male Reproductive System.

This review describes pre- and postnatal development of the male reproductive system in humans and laboratory animals, and highlights species differences in the timing and control of hormonal and morphologic events. Major differences are that the fetal testis is dependent on gonadotropins in humans, but is independent of such in rats; humans have an extended postnatal quiescent period, whereas rats exhibit no quiescence; and events such as secretion by the prostate and seminal vesicles, testicular descent, and the appearance of spermatogonia are all prenatal events in humans, but are postnatal events in rats. Major differences in the timing of the developmental sequence between rats and humans include: gonocyte transformation period (rat: postnatal day 0-9; human: includes gestational week 22 to 9 months of age); masculinization programming window (rat: gestational day 15.5-17.5; human: gestational week 9-14); and mini-puberty (rat: 0-6 hr after birth; human: 3-6 months of age). Endocrine disruptors can cause unique lesions in the prenatal and early postnatal testis; therefore, it is important to consider the differences in the timing of the developmental sequence when designing preclinical studies as identification of windows of sensitivity for endocrine disruption or toxicants will aid in interpretation of results and provide clues to a mode of action. Birth Defects Research 110:190-227, 2018. © 2017 Wiley Periodicals, Inc.

Osteoblast apoptosis and bone turnover.

With the discoveries of different death mechanisms, an emerging definition of apoptosis is the process of cell death associated with caspase activation or caspase-mediated cell death. This definition accepts that caspases represent the final common mechanistic pathway in apoptosis. Apoptosis may be triggered either by activation events that target mitochondria or endoplasmic reticulum or by activation of cell surface "death receptors," for example, those in the tumor necrosis factor (TNF) superfamily. In the postnatal and adult skeleton, apoptosis is integral to physiological bone turnover, repair, and regeneration. The balance of osteoblast proliferation, differentiation, and apoptosis determines the size of the osteoblast population at any given time. Although apoptosis has been recorded in many studies of bone, the selective mechanisms invoked in the different models studied rarely have been identified. This review offers a broad overview of the current general concepts and controversies in apoptosis research and then considers specific examples of osteoblast apoptosis pertinent to skeletal development and to the regulation of bone turnover. In reviewing selected work on interdigital apoptosis in the developing skeleton, we discuss the putative roles of the bone morphogenetic proteins (BMPs), Msx2, RAR-gamma, and death inducer obliterator 1 (DIO-1). In reviewing factors regulating apoptosis in the postnatal skeleton, we discuss roles of cytokines, growth factors, members of the TNF pathway, and the extracellular matrix (ECM). Finally, the paradoxical effects of parathyroid hormone (PTH) on osteoblast apoptosis in vivo are considered in the perspective of a recent hypothesis speculating that this may be a key mechanism to explain the anabolic effects of the hormone. An improved understanding of the apoptotic pathways and their functional outcomes in bone turnover and fracture healing may facilitate development of more targeted therapeutics to control bone balance in patients with osteoporosis and other skeletal diseases.

In vivo comparison of activated protein-1 gene activation in response to human parathyroid hormone (hPTH)(1-34) and hPTH(1-84) in the distal femur metaphyses of young mice.

Intermittent parathyroid hormone (PTH) treatment increases bone mass in humans and animals. Although intact human PTH has 84 amino acids, the N-terminal 31 to 38 amino acids are sufficient for bone anabolic activity in vivo. Prior studies have evaluated hPTH(1-34) and hPTH(1-84) with respect to bone mass increase and quality, but there have been no in vivo comparisons of dose-dependent molecular responses. After confirming that young male BALB/c mice respond to daily PTH with increased bone mass, we profiled the steady-state mRNA levels of activating protein-1 (AP-1) genes regulated by hPTH(1-34) and hPTH(1-84) at doses ranging from 0 to 19.4 nmol/kg in the distal femur metaphyses. We selected AP-1 genes, which include jun and fos, as they play a fundamental role mediating signals for proliferation, differentiation, and apoptosis in cells of different origins, including bone, and are known to be regulated by PTH. Human PTH(1-34) and hPTH(1-84) increased steady-state mRNA expression of c-jun, junB, c-fos, and fra-2 in an equivalent dose- and time-dependent manner. Expression of fosB or fra-1 was not detected with either peptide. When averaged across dose and time, responses to hPTH(1-34) and hPTH(1-84) were not significantly different from each other. Expression of c-jun, junB, and c-fos peaked 30 minutes after the injection while fra-2 expression peaked 30 minutes later. All AP-1 genes stimulated by PTH returned to the levels of vehicle treated controls by 3 h after injection. The expression level of junD, which was abundant in the distal metaphysis, was not altered by either peptide. No change in magnitude was observed after 1, 3, or 7 days of once-daily subcutaneous treatment of either peptide. When individual comparisons for each dose between peptides were made, the minimum effective dose necessary to stimulate a significant increase in c-fos and junB expression was equivalent for both peptides. The minimum effective dose for hPTH(1-34) was at least tenfold lower than hPTH(1-84) in stimulating c-jun and fra-2 expression. Area under the curve for the highest dose (19.4 nmol/kg) of either peptide showed no significant differences in the expression of any of the genes. In conclusion, in young mice given once-daily subcutaneous injections up to 7 days, hPTH(1-34) and hPTH(1-84) induced equivalent responses by time and dose in the selected AP-1 genes. These data on molecular regulation in mouse bone confirm and extend prior data from rat studies showing equivalence on bone mass at equimolar doses.

In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment.

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.

Structure-activity relationships of G protein-coupled receptors.

The primary function of cell-surface receptors is to discriminate the specific signaling molecule or ligand from a large array of chemically diverse extracellular substances and to activate an effector signaling cascade that triggers an intracellular response and eventually a biological effect. G protein-coupled cell-surface receptors (GPCRs) mediate their intracellular actions through the activation of guanine nucleotide-binding signal-transducing proteins (G proteins), which form a diverse family of regulatory GTPases that, in the GTP-bound state, bind and activate downstream membrane-localized effectors. Hundreds of GPCRs signal through one or more of these G proteins in response to a large variety of stimuli including photons, neurotransmitters, and hormones of variable molecular structure. The mechanisms by which these ligands provoke activation of the receptor/G-protein system are highly complex and multifactorial. Knowledge and mapping of the structural determinants and requirements for optimal GPCR function are of paramount importance, not only for a better and more detailed understanding of the molecular basis of ligand action and receptor function in normal and abnormal conditions, but also for a rational design of early diagnostic and therapeutic tools that may allow exogenous regulation of receptor and G protein function in disease processes.

Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone.

A central question in endocrinology is how a single ligand interacting with a single receptor can mediate multiple responses. GnRH interaction with receptor offers a prime example, leading to the regulation of synthesis and release of at least three molecules, regulation of target cell responsiveness and receptor number. The present study suggests a molecular model consistent with extant data that provides a mechanism by which this may occur and, further, which allows for coordinate regulation.

Gonadotropin-releasing hormone receptor couples to multiple G proteins in rat gonadotrophs and in GGH3 cells: evidence from palmitoylation and overexpression of G proteins.

There is evidence in several cell systems suggesting that the GnRH receptor couples to multiple G proteins. Presently there are no published studies showing GnRH receptor coupling to Gialpha, Gsalpha, and Gq/11alpha in a single cell type. To examine this possibility we measured palmitoylation of G proteins in response to GnRH receptor occupancy, since this event is a measure of G-protein activation by cognate receptors. GnRH stimulated time (0-120 min)- and dose (10(-12)-10(-6) g/ml)-dependent palmitoylation of both Gialpha and Gsalpha. Palmitoylation is G-protein activation dependent; accordingly, pertussis toxin (100 ng/ml; PTX), phorbol myristic acid (100 ng/ml), and Antide (50 nM; a GnRH antagonist) did not stimulate palmitoylation of Gialpha or Gsalpha above basal levels. However, cholera toxin (5 microgram/ml), an activator of Gsalpha, stimulated palmitoylation of Gsalpha but not Gialpha. We used a lactotrope-derived cell line expressing the GnRH receptor (GGH3) to examine whether the ability of the receptor to couple multiple G proteins is gonadotroph specific. GGH3 cells were transfected with specific cDNA coding for different G proteins, and agonist-stimulated second messenger production was assessed. Buserelin (a GnRH agonist) stimulated increased cAMP release in Gsalpha cDNA-transfected GGH3 cells, whereas in Gialpha cDNA-transfected cells, both inositol phosphate (IP) production and cAMP release were decreased in response to buserelin. Transfection of Gqalpha, G11alpha, G14alpha, and G15alpha cDNA into GGH3 cells resulted in an increased IP production in response to buserelin, indicating that GnRH receptor couples to this PTX-insensitive G-protein family. The observations presented in this study provide evidence for GnRH receptor coupling to multiple G proteins in a single cell type.

Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gqalpha and G11alpha knockout mice.

In this study, we used mice lacking the G11alpha [G11 knockout (KO)] or Gqalpha gene (Gq KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G11alpha and Gqalpha appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 microg/100 microl, sc) resulted in a rapid increase of serum LH levels (reaching 328 +/- 58 pg/25 microl for G11 KO; 739 +/- 95 pg/25 microl for Gq KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G11 KO or the heterozygous mice for Gq KO, resulted in an increase in serum LH levels (428 +/- 57 pg/25 microl for G11 KO; 884 +/- 31 pg/25 microl for Gq KO) at 75 min. Both G11 KO and Gq KO male mice released LH in response to Buserelin (2 microg/100 microl of vehicle; 363 +/- 53 pg/25 microl and 749 +/- 50 pg/25 microl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 microl within the first hour, then subsided to a steady level (approximately 100 pg/25 microl) for 109 h. In male G11 KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G11 KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the Gq KO male mice released less LH (531 +/- 95 pg/25 microl) after 13 h from the start of treatment than the heterozygous male mice (865 +/- 57 pg/25 microl), but the female KO mice released more LH (634 +/- 56 pg/25 microl) after 1 h from the start of treatment than the heterozygous female mice (346 +/- 63 pg/25 microl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G11 KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G11 KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 microg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between Gq KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female Gq KO mice compared with control groups of mice. However, female G11 KO mice produced less estradiol in response to Buserelin (2 microg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other.

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells.

The GnRH receptor (GnRH-R) belongs to the rhodopsin/beta-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane-proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH(3)1' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both Gs and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other Gs-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH(3)1' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH(3)1' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the alpha1B-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach-muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the Gs protein). Transfection of loop 3i from the D1A -dopamine receptor (coupled to the Gs protein) produced a selective attenuation (40%) in Gs-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and alpha2A-adrenergic receptors). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH(3)1' cell line, this loop is also involved in signal transduction mediated through the Gs protein pathway.

Redistribution of G(q/11)alpha in the pituitary gonadotrope in response to a gonadotropin-releasing hormone agonist.

In the present study, we took advantage of high-resolution multilaser confocal microscopy to examine the distribution of the alpha-subunit of the guanyl nucleotide binding protein subfamily G(q/11) (G(q/11)alpha). Dispersed cultures of pituitary cells were prepared from female weanling rats, fixed, permeabilized, and then stained with monoclonal antiserum (mouse) to the gonadotrope-specific form of secretogranin (SIIp), which was then tagged with Texas Red. Accordingly, the subpopulation of gonadotropes (approximately 15% of total cells) could be identified against a background of other pituitary cell types. G(q/11)alpha was localized with antiserum made in rabbit, then tagged with fluorescein. Hoechst 33258 nuclear stain was also used in some experiments for topological reference. The data indicate localization of the G(q/11)alpha in a cellular region near the plasma membrane and external to the border of the layer occupied by secretory granules. In the absence of activation, there were an average of six clusters of G(q/11)alpha in a section 1 microm thick and through the center of the cell. This corresponds to an average of 60 clusters per cell, assuming a mean gonadotrope diameter of 10 microm. Following continuous treatment with 0.1 microg/ml Buserelin, a metabolically stable GnRH agonist, the average number of clusters increased to 200/cell after 40 min and remained approximately constant for 120 min. This increase was blocked by the protein synthesis inhibitor, cycloheximide. In response to Buserelin, there was an additional increase in the number of clusters inside the cell in the area occupied by the secretory granules and in the perinuclear area. Prolonged (24 h) treatment with Buserelin, sufficient to provoke the onset of desensitization, did not significantly change total numbers of G(q/11)alpha clusters, although more were located in the peripheral compartment, an increase that occurred at the expense of the cytoplasmic compartment. Redistribution of the G(q/11)alpha family may be functionally significant, because this moiety may be rate limiting at the site of regulation of signal transduction.

Characterization of revertants of unc-93(e1500) in Caenorhabditis elegans induced by N-ethyl-N-nitrosourea.

Phenotypic reversion of the rubber-band, muscle-defective phenotype conferred by unc-93(e1500) was used to determine the utility of N-ethyl-N-nitrosourea (ENU) as a mutagen for genetic research in Caenorhabditis elegans. In this system, ENU produces revertants at a frequency of 3 x 10(-4), equivalent to that of the commonly used mutagen, EMS. The gene identity of 154 ENU-induced revertants shows that the distribution of alleles between three possible suppressor genes differs from induced by EMS. A higher percentage of revertants are alleles of unc-93 and many fewer are alleles of sup-9 and sup-10. Three revertants complement the three known suppressor genes; they may therefore identify a new gene product(s) involved in this system of excitation-contraction coupling in C. elegans. Molecular characterization of putative unc-93 null alleles reveals that the base changes induced by ENU are quite different from those induced by EMS; specifically we see an increased frequency of A/T-->G/C transitions. The frequency of ENU-induced intragenic deletions is found to be 13%. We suggest that ENU, at concentrations below 5 mM, will be a superior mutagen for studies of protein function in C. elegans.

Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.

Biphasic action of cyclic adenosine 3',5'- monophosphate in gonadotropin-releasing hormone (GnRH) analog-stimulated hormone release from GH3 cells stably transfected with GnRH receptor complementary deoxyribonucleic acid.

GH3 cells are a PRL-secreting adenoma cell line derived from pituitary lactotropes. These cells have been stably transfected with rat GnRH receptor complementary DNA to produce four cell lines: GGH(3)1', GGH(3)2', GGH(3)6', and GGH(3)12'. In response to either GnRH or Buserelin (a metabolically stable GnRH agonist), these cell lines synthesize PRL in a cAMP-dependent manner. Only GGH(3)6' cells desensitize in response to persistent treatment with 10(-7) g/ml Buserelin. GGH(3)1', GGH(3)2', and GGH(3)12' cells, however, can be made refractory to Buserelin stimulation by raising cAMP levels either by the addition of (Bu)2cAMP to the medium or by treatment with cholera toxin. In GGH(3) cells, low levels of cAMP fulfill the requirements for a second messenger, whereas higher levels appear to mediate the development of desensitization. The observation that in GGH(3)6' cells, cAMP production persists after the onset of desensitization is consistent with the view that the mechanism responsible for desensitization is distal to the production of cAMP. Moreover, the absence of any significant difference in the amount of cAMP produced per cell in GGH(3)2', GGH(3)6', or GGH(3)12' cells suggests that elevated cAMP production per cell does not explain the development of desensitization in GGH(3)6' cells. We suggest that Buserelin-stimulated PRL synthesis in GGH(3)6' cells is mediated by a different cAMP-dependent protein kinase pool(s) than that in nondesensitizing GGH(3) cells. Such a protein kinase A pool(s) may be more susceptible to degradation via cAMP-mediated mechanisms than the protein kinase pools mediating the Buserelin response in nondesensitizing GGH(3) cells. A similar mechanism has been reported in other systems.

Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid.

Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.