Amount Of Luteinizing Hormone-releasing Hormone Research Articles

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism.

Nicotine at very low doses (5-30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 microM). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (approximately 30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-D-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1-50 microM) plus Cd2+ (200 microM). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.

Luteinizing hormone-releasing hormone in undifferentiated and differentiated SK-N-SH human neuroblastoma cells

The presence of luteinizing hormone-releasing hormone (LHRH) in SK-N-SH human neuroblastoma cells was investigated by immunocytochemistry and enzyme-linked immunosorbent assays of whole cell extracts and culture medium. In addition, ribonuclease protection assays were utilized to quantitate LHRH messenger RNA. The expression of LHRH mRNA and LHRH protein level was correlated with neuronal differentiation induced by retinoic acid (RA). In differentiated SK-N-SH cells the LHRH mRNA level as well as the amount of LHRH in cell extracts and cell medium were significantly lower than in differentiated cells. These results suggest that RA affects the expression of LHRH mRNA and the level of LHRH protein in SK-N-SH cells. These data show that altering the growth state of the human neuroblastoma SK-N-SH cells toward more neuronal phenotype results in a significant decrease in expression of LHRH mRNA and the protein. The ability of RA to induce changes in LHRH at the mRNA and at the peptide levels will allow further study of RA regulation of LHRH at the neuronal level.

Effect of castration on eicosanoid and catecholamine-induced luteinizing hormone-releasing hormone release in vitro

The castraction of adult rats four weeks before experimentation did not alter either the basal production of prostaglandin (PG) E 2 (intract rats 1157 ± 160pg/mg protein, castrated rats 1093 ± 90 pg/mg protein) or the A 23187-induced production of PGE 2 (1591 ± 209 or 1701 ± 289 pg/mg protein respectively) or that of leucotriene (LT) C 4 (474 ± 33, 389 ± 39 pg/mg protein). Castration significantly reduced the absolute amounts of luteinizing hormone-releasing hormone (LHRH) relased under basal conditions (594 ± 56 pg/mg protein to 34% of intact controls) and after A 23187 stimulation (to 28% of controls). The LHRH released by the median eminence (ME) in castrated rats in the presence of PGE 2 (10 −6M), LTC 4 (10 −8M), norepinephrine (NE) (10 −5M) or dopamine (DA) (10 −5M and 10 −4M) was significnatly lower than the LHRH released by intact animals in the presence of these factors. However the relative values for LHRH relase (e.g. as % of controls) indicated that the degree of stimulation was identical in intact and castrated rats (2–3 times). These data suggest that the absence of gonadal steroid resulting from long-term castration does not block the stimulatory action of eicosanoids or catecholamines on the release of LHRH by the ME of adult male rats in vitro. Furthermore, castration does not intergere with the biosynthesis of PGE 2 and LTC 4.

Spontaneous changes in LHRH release during the rat estrous cycle, as measured with repetitive push-pull perfusions of the pituitary gland in the same female rats.

In the present study, we examined the in vivo release profiles of luteinizing hormone-releasing hormone (LHRH) during the rat estrous cycle by utilizing repetitive push-pull perfusions of the anterior pituitary in the same freely behaving animal. Throughout the estrous cycle, LHRH levels were higher than those reported from previous work using a push-pull cannula placed in the mediobasal hypothalamus. A significant increase in LHRH input to the anterior pituitary was obtained in proestrus (Pro), though the magnitude of increment was different among animals. This LHRH increment in Pro was attributable to a significant increase in the amplitude of LHRH pulses. Interestingly, no changes were observed in the frequency of LHRH pulses (about 1 pulse/50 min) throughout the estrous cycle. The data demonstrate that the anterior pituitary of female rats receives an increased amount of LHRH during Pro due to changes in the amplitude of the LHRH signal without changes in the frequency.

Immunocytochemical identification of luteinizing hormone-releasing hormone-positive fibres and terminals in the olfactory system of the rat

Luteinizing hormone-releasing hormone immunoreactivity was studied in the olfactory system of the rat in combination with acetylcholinesterase histochemistry. Neuronal perikarya containing luteinizing hormone-releasing hormone lie in the medial septal nucleus, the vertical limb of the diagonal band of Broca, the olfactory tubercule and the ganglionated plexus of the terminal nerve. Labelled fibres spread in the superficial layers of the main and accessory olfactory bulbs, some encompassing the strongly acetylcholinesterase-positive atypical glomeruli. Others are observed on the medial side of the bulb, running along the terminal nerve bundles and ganglia. These fibres join the vomeronasal nerve branches and proceed distally towards the nasal cavity. In the septal submucosa, immunoreactive fibres are partly associated with the terminal nerve network. Conspicuous endings filled with luteinizing hormone-releasing hormone are observed on blood vessels of the olfactory mucosa. Such well-differentiated terminals might be the neurosecretory afferents of a new neurohemal area. Immunoreactive terminals are also observed around the excretory ducts of the anterior medial glands. We have failed to observe any labelled fibres in the olfactory and vomeronasal epithelia. The results of the present study are discussed with respect to possible functional interpretations. It is suggested that significant amounts of luteinizing hormone-releasing hormone could be released in the submucosal capillaries in spite of the scarcity of immunoreactive fibres. Similar afferents could also modulate the secretory activity of some nasal glands. Synaptic events involving the neuropeptide might occur in the olfactory bulb, particularly in atypical glomerular areas previously characterized by their high acetylcholinesterase content. Finally, no anatomical support for a chemosensory function of fibres containing luteinizing hormone-releasing hormone has been brought out by our work.

Pulsatile infusion of luteinizing hormone-releasing hormone induces precocious puberty (vaginal opening and first ovulation) in the immature female guinea pig.

Previously we have hypothesized that an increase in luteinizing hormone-releasing hormone (LHRH) due to hypothalamic maturation is the key factor controlling the onset of puberty. This led to the working hypothesis that precocious puberty would be induced if LHRH is administered with an appropriate protocol. Thus, effects of pulsatile infusion of LHRH on the onset of first vaginal opening and first ovulation in immature female guinea pigs were studied. Luteinizing hormone-releasing hormone in hourly pulses of either 5 ng or 50 ng was infused through a chronically implanted jugular catheter for 9-29 days starting at 20 days of age. For the control experiment saline was infused in a similar manner. Infusion of 5 ng LHRH/h resulted in significantly earlier (P less than 0.001) ages at first vaginal opening (24.7 +/- 0.9 days) and at first ovulation (28.8 +/- 0.9 days) compared to saline controls (first vaginal opening 53.3 +/- 6.8 days; first ovulation 55.2 +/- 6.5 days). Infusion with a 10-fold higher LHRH dose (50 ng/h) also advanced the age at first vaginal opening (25.3 +/- 0.7 days), but precocious ovulation was no longer induced (53.7 +/- 5.3 days). Interestingly, LHRH infusion with the high dose resulted in a prolonged period of vaginal opening and cornification without ovulation. These results indicate that 1) pulsatile infusion of a small amount of LHRH with a constant frequency induces precocious puberty in a laboratory rodent, and 2) infusion of LHRH with a dose higher than the effective dose for the induction of early puberty results in a persistent estrous anovulatory syndrome. Therefore, the present study not only supports our hypothesis that an increase in endogenous LHRH release is responsible for the onset of puberty, but also further suggests that excessive release of LHRH or abnormal patterns of LHRH release may be involved in the etiology of the anovulatory persistent estrus syndrome.

Castration of male rats reduces the capacity of granules isolated from the median eminence to secrete luteinizing hormone-releasing hormone in response to copper.

The effect of castration of male rats on the secretory function of median eminence area (MEA) granules containing luteinizing hormone-releasing hormone (LHRH) was examined, using copper as a test substance. LHRH granules, isolated from MEA of sham or castrated rats 1, 2, or 12 weeks postoperatively, were incubated with various concentrations of copper complexed to histidine (CuHis) and the kinetic constants of LHRH release estimated. CuHis-stimulated release of LHRH was found to be a saturable function of the concentration of CuHis. Castration did not alter the apparent Km for CuHis-stimulated release of LHRH; the Km being 3 and 16.9 microM copper for granules obtained from 1-2 weeks and 12 weeks sham-operated rats, respectively, and 3.7-5.6 and 10.2 microM copper for the granules of castrated rats, respectively. Moreover, castration did not alter the fractional amount of LHRH released in response to CuHis. In contrast, castration markedly reduced the actual amount of LHRH released in response to CuHis; the Vmax being 1,933 and 2,942 pg LHRH released/6 min/MEA for 1-2 weeks and 12 weeks sham-operated rats, respectively, and 782 and 757 pg for the castrated rats, respectively. Similarly, the LHRH content of the MEA of castrated rats was lower than that of the shams. These results are suggestive that castration reduces the capacity of the MEA granules to release LHRH in response to a secretion stimulus such as copper and that this is due not to alterations in the biochemical parameters underlying the release process itself but to a reduced level of MEA LHRH available for release.

Effect of starvation on gonadotrophin secretion and on in vitro release of LRH from the isolated median eminence of the male rat.

Abstract. A perfusion system was developed in which isolated median eminences (ME) were stimulated in vitro by depolarizing agents such as potassium and veratridine. Potassium concentrations between 30 and 80 mm released increasing amounts of luteinizing hormone-releasing hormone (LRH) from the MEs of starved and control rats. Veratridine at a concentration of 50 μm caused a more prolonged LRH release in both starved and control animals. LRH secretion in vitro was slightly, though not under all conditions, significantly greater in rats starved for 5 days. The testosterone (T)-LH feedback was studied by castrating the animals and substituting various doses of T through implantation of T-releasing capsules of different sizes. The concentration in plasma, which can prevent the castration-induced much smaller in starved than in control rats. The in vitro release of LRH evoked by 80 mm potassium was not different for starved and fed rats under various feedback conditions. Both groups revealed decreased in vitro release of LRH when castrated animals were not substituted with T. The effect of castration was studied from 1 to 28 days. The plasma LH values rapidly increased in starved and control animals, indicating that the hypothalamic responsestration is not delayed by starvation. The release in vitro of LRH decreased from the first to the fifth day and remained constant thereafter. No significant difference between starved and fed rats was observed. The experiments indicate that the 'releasable pool' of LRH in vitro is greater under conditions of reduced LH release in vivo. The basic mechanism of depolarization-induced exocytosis of LRH from the ME is intact in starved animals.

Differential effect of althesin versus chloralose-urethane anesthesia on in vivo LHRH and LH release in the proestrous rat.

Anesthetic doses of Althesin and chloralose-urethane were compared as to their effects on neuroendocrine reproductive processes in diestrous and proestrous rats. Proestrous rats treated with Althesin experienced a 5-fold increase in immuno-reactive luteinizing hormone releasing hormone (LHRH) concentration in methanolic extracts of pituitary stalk plasma. This LHRH increase was 67% greater than that of proestrous rats administered chloralose-urethane. Moreover, Althesin given before the onset of the critical period did not suppress the proestrous surge of LHRH in stalk plasma. The Duncan's multiple range test indicated stalk plasma LHRH activity was significantly elevated in proestrous rats anesthetized with Althesin (p less than 0.05) but not with chloralose-urethane. However, the luteinizing hormone (LH) concentration in peripheral plasma increased 14 to 18-fold in all proestrous rats, including those anesthetized with chloralose-urethane. Evidently, in chloralose-urethane-treated rats, the smaller LHRH surge during proestrus is sufficient to elicit maximum release of LH from the anterior pituitary gland. This suggest that under normal conditions the amount of LHRH secreted into portal blood during proestrus is approximately 2-fold greater than that necessary to elicit maximum secretion of LH. The results also show that picogram concentrations of LH in the stalk plasma will stimulate the release of nanogram quantities of LH into the peripheral plasma of the same rats. Moreover, the sensitivity of the pituitary gland to LHRH stimulation via the portal circulation increases nearly 3-fold between diestrus and proestrus.

Effect of pre-incubation with different concentrations of LH-RH on subsequent LH release caused by supramaximally active amounts of LH-RH; Role of LH-RH-induced protein synthesis

Anterior pituitary glands from intact diestrous female rats were incubated for two consecutive periods of 3 hours. During the first period various submaximally active amounts of luteinizing hormone-releasing hormone (LH-RH) were added to the media, whereas during the second period a supramaximally active concentration of LH-RH was present. When during the second incubation period protein synthesis was inhibited by cycloheximide, the amount of luteinizing hormone (LH) released during that period was positively correlated to the concentration of LH present during the first incubation period. This relationship was not seen when cycloheximide was absent, or when cycloheximide was present throughout both periods. Total LH was not affected by LH-RH; thus no effect of LH-RH on LH synthesis was observed. It is concluded that the amounts of protein synthesized by the pituitary glands in response to the different amounts of LH-RH during the first incubation period can constitute a limiting factor for the response to the supramaximally active amount of LH-RH added during the second incubation period.

Distribution of luteinizing hormone-releasing hormone (LHRH) in the guinea pig brain.

With the use of the unlabeled antibody enzyme technique and antiserum to luteinizing hormone-releasing hormone (LHRH), the distribution of LHRH in the adult male guinea pig was studied. Immunoreactive deposits were found in the cell bodies of hypothalamic neurons in the medial preoptic, anterior hypothalamic, suprachiasmatic, and arcuate nuclei. Ten to thirty LHRH-containing perikarya were seen per brain. Four fiber tracts positive for LHRH were described: Tract I runs on the pre-commissural side of the anterior commissure from the bed nucleus of the stria terminalis and medial preoptic area to the suprachismatic nucleus: Tract II runs on the post-commissural side to the retrochiasmatic portion of the suprachiasmatic nucleus; Tract III originates in the arcuate nucleus and the ventral portion of the ventromedial nucleus and courses medially and caudally to enter the median eminence; Tract IV travels from the mammillary bodies to the mid-brain. Three weeks after surgical isolation of the medial basal hypothalamus there was a slight decrease in the amount of LHRH in the median eminence. These data suggest that LHRH is synthesized in more than one hypothalamic nuclear group but that the majority of axons containing the hormone in the median eminence originate in the medial basal hypothalamus.