A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

Ellinwood, William E.; Rønnekleiv, Oline K.; Kelly, Martin J.; Resko, John A.

doi:10.1016/0196-9781(85)90075-0

Cited by 97 publications

(44 citation statements)

References 33 publications

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“…Radioimmunoassay of GnRH was performed as described previously (Chappell and Levine, 2000) using the EL-14 GnRH antiserum (Ellinwood et al, 1985). The intra-assay and interassay coefficients of variance for the assays were 14.2 and 8.2%, respectively.…”

Section: Methodsmentioning

confidence: 99%

Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Zhang¹,

Bosch²,

Levine³

et al. 2007

J. Neurosci.

113

139

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Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner that is dependent on circulating 17␤-estradiol (E2) and glucose concentrations. However, the intrinsic conductances responsible for the episodic firing pattern underlying pulsatile release and the effects of E2 and glucose on these conductances are primarily unknown. Whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons revealed that the K ATP channel opener diazoxide induced an outward current that was antagonized by the sulfonylurea receptor 1 (SUR1) channel blocker tolbutamide. Single-cell reverse transcription (RT)-PCR revealed that the majority of GnRH neurons expressed Kir6.2 and SUR1 subunits, which correlated with the diazoxide/tolbutamide sensitivity. Also, a subpopulation of GnRH neurons expressed glucokinase mRNA, a marker for glucose sensitivity. Indeed, GnRH neurons decreased their firing in response to low glucose concentrations and metabolic inhibition. The maximum diazoxide-induced current was approximately twofold greater in E2-treated compared with oil-treated ovariectomized females. In current clamp, estrogen enhanced the diazoxide-induced hyperpolarization to a similar degree. However, based on quantitative RT-PCR, estrogen did not increase the expression of Kir6.2 or SUR1 transcripts in GnRH neurons. In the presence of ionotropic glutamate and GABA A receptor antagonists, tolbutamide depolarized and significantly increased the firing rate of GnRH neurons to a greater extent in E2-treated females. Finally, tolbutamide significantly increased GnRH secretion from the preoptic-mediobasal hypothalamus. Therefore, it appears that K ATP channels and glucokinase are expressed in GnRH neurons, which renders them directly responsive to glucose. In addition, K ATP channels are involved in modulating the excitability of GnRH neurons in an estrogen-sensitive manner that ultimately regulates peptide release.

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Section: Methodsmentioning

confidence: 99%

Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Zhang¹,

Bosch²,

Levine³

et al. 2007

J. Neurosci.

113

139

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“…These sections were washed with 0.1 M sodium phosphate buffer (pH 7.4), and then processed with streptavidin-FITC as described previously [11]. After localizing the biocytin-filled neuron, the slides containing the appropriate sections were processed with either a monoclonal tyrosine hydroxylase (TH) antibody (Ink-Star, Stillwater, Minn., USA) at a 1:3,000 dilution, a GnRH antiserum [12] at a 1:2,500 dilution or a β-endorphin antiserum [13] at a 1:1,000 dilution, using fluorescence immunohistochemistry [11]. …”

Section: Methodsmentioning

confidence: 99%

The Peptide Orphanin FQ Inhibits β-Endorphin Neurons and Neurosecretory Cells in the Hypothalamic Arcuate Nucleus by Activating an Inwardly-Rectifying K<sup>+</sup> Conductance

Wagner¹,

et al. 1998

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Orphanin FQ (OFQ) is a novel heptadecapeptide whose structure resembles that of dynorphin A_1–17. Its receptor shares appreciable homology with µ-, δ- and ĸ-opioid receptors, and is highly expressed in the hypothalamus. The present study examined the effects of OFQ on neurons within the arcuate nucleus (ARC) of the mediobasal hypothalamus, using intracellular recordings from coronal slices. In current clamp, OFQ produced a hyperpolarization of ARC neurons, including those immunopositive for β-endorphin, tyrosine hydroxylase and gonadotropin-releasing hormone. This hyperpolarization was dose-dependent, insensitive to antagonism by naloxone and was associated with a decrease in input resistance. In voltage clamp, OFQ produced an outward current associated with an increase in conductance. Varying the extracellular K⁺ concentration shifted the reversal potential for the OFQ response to the degree predicted by the Nernst equation. Furthermore, barium chloride markedly attenuated both the OFQ-induced hyperpolarization and decrease in input resistance. Administration of maximally effective concentrations of OFQ, followed by coadministration of maximal concentrations of either OFQ and the µ-opioid receptor agonist DAMGO or OFQ and the GABA_B receptor agonist baclofen produced additive hyperpolarizations and outward currents. If DAMGO was applied first, followed by the coadministration of DAMGO and OFQ, then the responses were occluded. Taken together, these results indicate that OFQ inhibits β-endorphin neurons, as well as A₁₂ dopamine and GnRH neurosecretory cells, within the ARC by activating a subset of inwardly-rectifying K⁺ channels. This suggests that OFQ is not only an antiopioid peptide, but that it also modulates the hypothalamo-pituitary axis and, ultimately, reproductive behavior.

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“…hpg mice, obtained from Harry Charlton (Oxford University, England), were maintained in our animal facilities in temperature-and light-controlled (14 hr light/10 hr dark) rooms, with food and water available ad libitum. Female mice were 2-5 months old and males 4-5 (T and W nos., see (8,9). RM 8/5 (1:1000) is thought to identify a secondary cleavage product of the precursor molecule (10), whereas ARC II (1:2000) identifies the dibasic amino acid cleavage site (11).…”

Section: Methodsmentioning

confidence: 99%

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

Silverman

Roberts

Dong

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the i jection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.The mutant hypogonadal (hpg) mouse lacks a functional gene for gonadotropin-releasing hormone (GnRH) (1). The hypogonadism can be reversed by the implantation of normal fetal preoptic-area tissue into the third ventricle of the affected mouse (2, 3). An immortalized cell line expressing the GnRH gene and producing releasable peptide was made by targeted tumorigenesis (4). In cell culture conditions, some of these cells assume a neuronal morphology and produce the entire GnRH prohormone (5) and then metabolize this protein to the GnRH decapeptide (6). We have injected these cells into the preoptic/anterior hypothalamic regions of adult hpg male and female mice to determine whether they send axons to appropriate targets and support gonadal development. METHODSCell Culture and Collection. GT1 cells from GT1-3 or GT1-7 subclones (4) were cultured in our laboratory on 100-mm plates in Dulbecco's modified Eagle's medium with 10o fetal bovine serum. Cells were plated at 2 x 103 per cm2. Under these conditions, confluence is reached 4-6 days after seeding, and 2-to 4-day preconfluent cells were used in the present study. The cells were harvested by trypsinization of a monolayer, washed with phosphate-buffered saline, and collected by centrifugation at 4TC. Cells (_J107) were then resuspended in 1 ml of cold phosphate-buffered saline and were counted with a hemocytometer. Appropriate dilutions were made with phosphate-buffered saline to yield the concentrations of cells required in each experiment (se...

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A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

Cited by 97 publications

References 33 publications

Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

The Peptide Orphanin FQ Inhibits β-Endorphin Neurons and Neurosecretory Cells in the Hypothalamic Arcuate Nucleus by Activating an Inwardly-Rectifying K<sup>+</sup> Conductance

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

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A new antiserum with conformational specificity for LHRH: Usefulness for radioimmunoassay and immunocytochemistry

Cited by 97 publications

References 33 publications

Gonadotropin-Releasing Hormone Neurons Express KATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Gonadotropin-Releasing Hormone Neurons Express KATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

The Peptide Orphanin FQ Inhibits β-Endorphin Neurons and Neurosecretory Cells in the Hypothalamic Arcuate Nucleus by Activating an Inwardly-Rectifying K<sup>+</sup> Conductance

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

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Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition

Gonadotropin-Releasing Hormone Neurons Express K_ATPChannels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition