Autocrine Regulation of Gonadotropin-Releasing Hormone Secretion in Cultured Hypothalamic Neurons

Abstract Episodic hormone secretion is a characteristic feature of the hypothalamo-pituitary-gonadal system, in which the profile of gonadotropin release from pituitary gonadotrophs reflects the pulsatile secretory activity of GnRH-producing neurons in the hypothalamus. Pulsatile release of GnRH is also evident in vitro during perifusion of immortalized GnRH neurons (GT1–7 cells) and cultured fetal hypothalamic cells, which continue to produce bioactive GnRH for up to 2 months. Such cultures, as well as hypothalamic tissue from adult rats, express GnRH receptors as evidenced by the presence of high-affinity GnRH binding sites and GnRH receptor transcripts. Furthermore, individual GnRH neurons coexpress GnRH and GnRH receptors as revealed by double immunostaining of hypothalamic cultures. In static cultures of hypothalamic neurons and GT1–7 cells, treatment with the GnRH receptor antagonist, [d-pGlu1, d-Phe2, d-Trp3,6]GnRH caused a prominent increase in GnRH release. In perifused hypothalamic cells and GT1–7 cells, treatment with the GnRH receptor agonist, des-Gly10-[d-Ala6]GnRH N-ethylamide, reduced the frequency and increased the amplitude of pulsatile GnRH release, as previously observed in GT1–7 cells. In contrast, exposure to the GnRH antagonist analogs abolished pulsatile secretion and caused a sustained and progressive increase in GnRH release. These findings have demonstrated that GnRH receptors are expressed in hypothalamic GnRH neurons, and that receptor activation is required for pulsatile GnRH release in vitro. The effects of GnRH agonist and antagonist analogs on neuropeptide release are consistent with the operation of an ultrashort-loop autocrine feedback mechanism that exerts both positive and negative actions that are necessary for the integrated control of GnRH secretion from the hypothalamus.

Download Full-text

The relationship between pulsatile GnRH secretion and cAMP production in immortalized GnRH neurons

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00081.2011 ◽

2011 ◽

Vol 300 (6) ◽

pp. E1022-E1030 ◽

Cited By ~ 3

Author(s):

John L. Frattarelli ◽

Lazar Z. Krsmanovic ◽

Kevin J. Catt

Keyword(s):

Initial Increase ◽

Rapid Decline ◽

Camp Production ◽

Gnrh Secretion ◽

Gnrh Receptors ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Gnrh Release ◽

High Concentrations ◽

The Relationship

In perifused immortalized GnRH neurons (GT1–7), simultaneous measurements of GnRH and cAMP revealed that the secretory profiles for both GnRH and cAMP are pulsatile. An analysis of GnRH and cAMP pulses in 16 independent experiments revealed that 25% of pulses coincide. Inversion of the peak and nadir levels was found in 33% and random relationship between GnRH and cAMP found in 42% of analyzed pulses. The random relation between GnRH and cAMP pulse resets to synchronous after an inverse relation between pulses occurred during the major GnRH release, indicating that GnRH acts as a switching mechanism to synchronize cAMP and GnRH release in perifused GT1–7 neurons. Activation of GnRH receptors with increasing agonist concentrations caused a biphasic change in cAMP levels. Low nanomolar concentrations increased cAMP production, but at high concentrations the initial increase was followed by a rapid decline to below the basal level. Blockade of the GnRH receptors by peptide and nonpeptide antagonists generated monotonic nonpulsatile increases in both GnRH and cAMP production. These findings indicate that cAMP positively regulates GnRH secretion but does not participate in the mechanism of pulsatile GnRH release.

Download Full-text

Transcript and protein profiling identifies signaling, growth arrest, apoptosis, and NF-κB survival signatures following GNRH receptor activation

Endocrine Related Cancer ◽

10.1530/erc-12-0192 ◽

2012 ◽

Vol 20 (1) ◽

pp. 123-136 ◽

Cited By ~ 7

Author(s):

Colette Meyer ◽

Andrew H Sims ◽

Kevin Morgan ◽

Beth Harrison ◽

Morwenna Muir ◽

...

Keyword(s):

Target Genes ◽

Cultured Cells ◽

Receptor Activation ◽

Gnrh Receptor ◽

Phase Changes ◽

Protein Profiling ◽

Proteomic Profiling ◽

Pathway Gene

GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60)in vitroandin vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene,CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenograftsin vivoduring Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60in vitro, and p-NF-κB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation.

Download Full-text

Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations

Endocrinology ◽

10.1210/en.2008-0002 ◽

2008 ◽

Vol 149 (6) ◽

pp. 3118-3129 ◽

Cited By ~ 13

Author(s):

Kevin D. G. Pfleger ◽

Adam J. Pawson ◽

Robert P. Millar

Keyword(s):

Ligand Binding ◽

Receptor Activation ◽

Gnrh Receptor ◽

Binding Modes ◽

Extracellular Loop ◽

Gnrh Analog ◽

Chimeric Receptors ◽

Gnrh Receptors ◽

Extracellular Loops ◽

Loop Conformation

GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations.

Download Full-text

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons

Biochemistry and Cell Biology ◽

10.1139/o00-058 ◽

2000 ◽

Vol 78 (3) ◽

pp. 359-370 ◽

Cited By ~ 19

Author(s):

Fredrick Van Goor ◽

Lazar Z Krsmanovic ◽

Kevin J Catt ◽

Stanko S Stojilkovic

Keyword(s):

Calcium Influx ◽

Hormone Secretion ◽

Gonadotropin Releasing Hormone ◽

Pacemaker Activity ◽

Sk Channels ◽

Hypothalamic Neurons ◽

Adult Rats ◽

Releasing Hormone ◽

Gnrh Receptors ◽

Store Depletion

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.

Download Full-text

α1-Adrenergic Receptors Mediate LH-Releasing Hormone Secretion through Phospholipases C and A2 in Immortalized Hypothalamic Neurons

Endocrinology ◽

10.1210/endo.142.11.8506 ◽

2001 ◽

Vol 142 (11) ◽

pp. 4839-4851 ◽

Cited By ~ 10

Author(s):

Silvia M. Kreda ◽

Martina Sumner ◽

Silvia Fillo ◽

Carla M. Ribeiro ◽

Guo X. Luo ◽

...

Keyword(s):

Adrenergic Receptors ◽

Critical Role ◽

Hormone Secretion ◽

Receptor Activation ◽

Dependent Manner ◽

Adrenergic Agents ◽

Releasing Hormone ◽

Lh Releasing Hormone

Abstract Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-releasing hormone (LHRH). In vivo and in vitro studies indicate that these effects are mediated primarily through α1-adrenergic receptors (α1-ARs). With the immortalized hypothalamic LHRH neurons, we have found that α1-adrenergic agents directly stimulate the secretion of LHRH in a dose-dependent manner. Ligand binding and RNA studies demonstrate that the GT1 cells contain both α1A- and α1B-ARs. Competition binding experiments show that approximately 75% of the binding is due toα 1B-ARs; the remainder is made up ofα 1A-ARs. Receptor activation leads to stimulation of PLC. PLCβ1 and PLCβ3 are expressed in GT1 neurons, and these PLCs are probably responsible for the release of diacylglycerol and IP as well as the increase in intracellular calcium. The mobilization of cytoplasmic calcium is sufficient to stimulate cytosolic PLA2 (cPLA2) and release arachidonic acid. A dissection of the contributions of the phospholipases to LHRH secretion suggests that cPLA2 acts downstream of PLC and that it significantly augments the PLC-stimulated LHRH secretory response. Inasmuch as the α1-ARs are known to play a critical role in LHRH physiology, we propose that both PLC and cPLA2 are critical in regulating and amplifying LHRH release.

Download Full-text

Pituitary gonadotrophin-releasing hormone receptor up-regulation in vitro: dependence on calcium and microtubule function

Journal of Endocrinology ◽

10.1677/joe.0.1070049 ◽

1985 ◽

Vol 107 (1) ◽

pp. 49-56 ◽

Cited By ~ 9

Author(s):

L. S. Young ◽

S. I. Naik ◽

R. N. Clayton

Keyword(s):

Luteinizing Hormone ◽

Gnrh Receptor ◽

Ionophore A23187 ◽

Pituitary Cells ◽

Hormone Release ◽

Releasing Hormone ◽

Luteinizing Hormone Release ◽

Gnrh Receptors ◽

Gonadotrophin Releasing Hormone

ABSTRACT Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7–10 h routinely resulted in a 50–100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40–60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 μmol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 μmol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH. Vinblastine (1 μmol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release. These results indicate that receptor stimulation by GnRH, K+ and dbcAMP is a calcium-dependent process requiring the integrity of the microtubule system and there is a different calcium requirement for the processes of GnRH receptor up-regulation and LH secretion. J. Endocr. (1985) 107, 49–56

Download Full-text

Expression, Structure, Function, and Evolution of Gonadotropin-Releasing Hormone (GnRH) Receptors GnRH-R1SHS and GnRH-R2PEY in the Teleost, Astatotilapia burtoni

Endocrinology ◽

10.1210/en.2006-1400 ◽

2007 ◽

Vol 148 (10) ◽

pp. 5060-5071 ◽

Cited By ~ 41

Author(s):

Colleen A. Flanagan ◽

Chun-Chun Chen ◽

Marla Coetsee ◽

Sipho Mamputha ◽

Kathleen E. Whitlock ◽

...

Keyword(s):

Gnrh Receptor ◽

Amino Acid Sequences ◽

Mrna Levels ◽

Ligand Selectivity ◽

Gnrh Receptors ◽

Reproductive Control ◽

Astatotilapia Burtoni ◽

Brain Mrna ◽

Highly Correlated

Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which receptor type regulates reproduction via the hypothalamic-pituitary-gonadal axis. The teleost fish, Astatotilapia burtoni, is useful for identifying the GnRH receptor responsible for reproduction, because only territorial males reproduce. We have cloned a second GnRH receptor in A. burtoni, GnRH-R1SHS (SHS is a peptide motif in extracellular loop 3), which is up-regulated in pituitaries of territorial males. We have shown that GnRH-R1SHS is expressed in many tissues and specifically colocalizes with LH in the pituitary. In A. burtoni brain, mRNA levels of both GnRH-R1SHS and a previously identified receptor, GnRH-R2PEY, are highly correlated with mRNA levels of all three GnRH ligands. Despite its likely role in reproduction, we found that GnRH-R1SHS has the highest affinity for GnRH2 in vitro and low responsivity to GnRH1. Our phylogenetic analysis shows that GnRH-R1SHS is less closely related to mammalian reproductive GnRH receptors than GnRH-R2PEY. We correlated vertebrate GnRH receptor amino acid sequences with receptor function and tissue distribution in many species and found that GnRH receptor sequences predict ligand responsiveness but not colocalization with pituitary gonadotropes. Based on sequence analysis, tissue localization, and physiological response we propose that the GnRH-R1SHS receptor controls reproduction in teleosts, including A. burtoni. We propose a GnRH receptor classification based on gene sequence that correlates with ligand selectivity but not with reproductive control. Our results suggest that different duplicated GnRH receptor genes have been selected to regulate reproduction in different vertebrate lineages.

Download Full-text

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model

Endocrinology ◽

10.1210/endocr/bqaa186 ◽

2020 ◽

Vol 162 (1) ◽

Author(s):

Siew Hoong Yip ◽

Pauline Campos ◽

Xinhuai Liu ◽

Robert Porteous ◽

Allan E Herbison

Keyword(s):

Median Eminence ◽

Pulse Generator ◽

Ectopic Expression ◽

Brain Slices ◽

Gnrh Neuron ◽

External Zone ◽

Pulsatile Gnrh ◽

Gnrh Neurons ◽

Gnrh Release ◽

Presynaptic Nerve Terminals

Abstract The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed “dendrons,” and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

Download Full-text

Differential Actions of Prolactin on Electrical Activity and Intracellular Signal Transduction in Hypothalamic Neurons

Endocrinology ◽

10.1210/en.2011-2005 ◽

2012 ◽

Vol 153 (5) ◽

pp. 2375-2384 ◽

Cited By ~ 46

Author(s):

R. S. E. Brown ◽

R. Piet ◽

A. E. Herbison ◽

D. R. Grattan

Keyword(s):

Signal Transduction ◽

Electrical Activity ◽

Fluorescent Protein ◽

Janus Kinase ◽

Anatomical Location ◽

Transcriptional Responses ◽

Hypothalamic Neurons ◽

Periventricular Nucleus ◽

Gnrh Neurons

In many tissues, including brain, prolactin action is predominantly mediated by the Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway, leading to changes in gene transcription. However, prolactin can also exert rapid actions on electrical activity of hypothalamic neurons. Here, we investigate whether both responses occur in a single cell type, focusing on three specific populations known to be influenced by prolactin: GnRH neurons, tuberoinfundibular dopamine (TIDA) neurons, and neurons in the anteroventral-periventricular nucleus in female mice. We performed phosphorylated STAT5 (pSTAT5) immunohistochemistry to identify prolactin-responsive neurons after in vivo prolactin treatment. In addition, we carried out in vitro electrophysiology in slices from transgenic mice expressing green fluorescent protein driven by the GnRH or tyrosine hydroxylase promoters as well as from C57BL/6J mice to assess acute electrical responses to prolactin. Approximately 88% of TIDA neurons expressed pSTAT5 in diestrous mice, rising to 97% after prolactin treatment. All TIDA neurons also showed a rapid increase in firing rate after prolactin treatment. In contrast, very few GnRH neurons (11%) showed pSTAT5 in response to prolactin, and none showed a change in electrical activity. Finally, in the anteroventral-periventricular nucleus, most neurons (69%) responded to prolactin treatment with an increase in pSTAT5, but only 2/38 (∼5%) showed changes in electrical activity in response to prolactin. These observations show that prolactin recruits different combinations of electrical and transcriptional responses in neurons depending upon their anatomical location and phenotype. This may be critical in establishing appropriate responses to prolactin under different physiological conditions.

Download Full-text

Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02005 ◽

2006 ◽

Vol 36 (3) ◽

pp. 601-610 ◽

Cited By ~ 13

Author(s):

M R Silver ◽

S A Sower

Keyword(s):

Intact Cell ◽

Functional Characterization ◽

Kinetic Studies ◽

Receptor Activation ◽

Gnrh Receptor ◽

Type I ◽

Type Ii ◽

Binding Assays ◽

Gnrh Receptors ◽

Series Of Experiments

The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependant manner when treated with either lamprey GnRH-I (LogEC50 −6.57±0.15) or lamprey GnRH-III (LogEC50 −8.29±0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708±0.245 nM)=chicken GnRH-II (Ki 0.765±0.160 nM) > mammalian GnRH (Ki 12.9±1.96 nM) > dAla6Pro9NEt mammalian GnRH (Ki 21.6±9.68 nM) > lamprey GnRH-I (Ki 118.0±23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependant internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.

Download Full-text