Luteinizing Hormone-releasing Hormone Content Research Articles

Manganese protects against the effects of alcohol on hypothalamic puberty-related hormones

AimsSince manganese (Mn) is capable of stimulating the hypothalamic-pituitary unit and advancing female puberty, we assessed the possibility that this element might overcome some of the detrimental effects of prepubertal alcohol (ALC) exposure on the hypothalamic control of pituitary function. Main methodsRats received either saline or Mn (10mg/kg) daily by gastric gavage from day 12 to day 31. After weaning, all rats were provided Lab Chow diet ad libitum until day 27 when they began receiving either the Bio Serv control or ALC diet regime. On day 31, the medial basal hypothalamus (MBH) was collected to assess luteinizing hormone-releasing hormone (LHRH) and cyclooxygenase 2 (COX2) protein levels. Release of prostaglandin-E2 (PGE2), LHRH and serum luteinizing hormone (LH) were also assessed. Other animals were not terminated on day 31, but remained in study to assess timing of puberty. Key findingsShort-term ALC exposure caused elevated hypothalamic LHRH content, suggesting an inhibition in peptide release, resulting in a decrease in LH. Both actions of ALC were reversed by Mn supplementation. COX2 synthesis, as well as PGE2 and LHRH release were suppressed by ALC exposure, but Mn supplementation caused an increase in COX2 synthesis and subsequent PGE2 and LHRH release in the presence of ALC. Mn supplementation also ameliorated the action of ALC to delay puberty. SignificanceThese results suggest that low level Mn supplementation acts to protect the hypothalamus from some of the detrimental effects of ALC on puberty-related hormones.

Luteinizing-hormone-releasing-hormone-containing biodegradable polymer micelles for enhanced intracellular drug delivery

A biodegradable triblock copolymer, poly(ethylene oxide)-block-poly(allyl glycidyl ether)-block-poly(dl-lactide) (mPEG-b-PAGE-b-PLA), with allyl groups on its middle block was designed and synthesized through anionic polymerization of allyl glycidyl ether (AGE) with PEG monomethyl ether sodium salt as the macroinitiator and subsequent ring-opening polymerization of dl-lactide. Luteinizing hormone-releasing hormone (LHRH) was conjugated to the PAGE block through a linkage of -SCH2CH2C([double bond, length as m-dash]O)NH- between the allyl and LHRH residues. The LHRH content in the conjugate was ca. 25 wt%. Owing to the amphiphilic nature of the conjugate, it was self-assembled into micelles of 15-40 nm in diameter and with the LHRH moieties in the hydrophilic corona of the micelles. Cellular uptake experiments were carried out using flow cytometry and confocal laser scanning microscopy (CLSM) with HeLa cells as LHRH-receptor overexpressing cells and HepG-2 cells as normal cells. HeLa cells displayed more cellular uptake of the LHRH-micelles than the normal cells. In vivo biodistribution of the LHRH-containing micelles and LHRH-free micelles was studied using a CRI Maestro™ imaging system. Preferred accumulation in tumor sites of LHRH-containing micelles was observed at 24 hours post injection. Therefore, LHRH-conjugated amphiphilic copolymers might be used as a potential drug delivery system for the treatment of LHRH receptor overexpressing carcinoma.

Stabilization of luteinizing hormone-releasing hormone in a dry powder formulation and its bioactivity

Abstract Background: Luteinizing hormone-releasing hormone (LHRH) is a naturally occurring hormone that controls sex hormones in both men and women. In general, LHRH is poorly absorbed through the gastrointestinal tract due to its large molecular size, high polarity, and loss from enzymatic degradation. Objective: Prepare and develop LHRH in a dry power formulation with stability and biological activity. Methods: Mannitol (M) and glycine (G) were chosen as ingredients to stabilize and protect LHRH during the freeze drying processes and during storage. The physicochemical properties of LHRH dry powders were examined by capillary electrophoresis, fluorescence spectrophotometry, scanning electron microscopy, and photon correlation spectroscopy. The release of LHRH from the dry powder was carried out in dissolution apparatus. In addition, a rat model was employed to study the bioactivity of LHRH in the dry powder form. Results: The LHRH dry powder formulations using M and G in the ratios of 6:4 and 7:3 were more stable than other formulations. LHRH colloids containing M:G showed no aggregation after storage at 4°C for one month. The concentration of LHRH in the dry powder form was more stable than that of LHRH in solution form. All the LHRH dry powder formulations were instantly dissolved within 10 seconds in an aqueous medium. After the LHRH dry powder (13 mg) was reconstituted and administered intraperitoneally to male rats during a one-month period, the testosterone level in the plasma was significantly decreased compared with an untreated group (15.0±1.0 ng/mL, 15.0±1.0 ng/mL and 20.0±2.0 ng/mL for LHRH containing M:G; 6:4, 7:3, and 8:2, respectively, compared to the control of 35±2 ng/mL, p<0.05). Conclusion: The LHRH dry powder formulations had good physicochemical properties and bioactivity.

Immunoreactive Substance P and LH-RH Content in Median Eminence and Pituitary Gland during Proestrus, Oestrus, Lactation and after Anterior Hypothalamic Deafferentation

Substance P (SP) and luteinizing hormone releasing hormone (LH-RH) content were measured by radioimmunoassay in median eminence of female rats, SP only in rats pituitary after decapitation on the day of oestrus (E), on the first and second day of dioestrus (D1 and D2), on the day of proestrus (P) and in "constant oestrus" (AD) caused by anterior hypothalamic deafferentation and in lactation (L). During four days of oestrus cycle the highest SP and LH-RH content of median eminence was found in dioestrus. Both during cyclic oestrus and "constant oestrus", SP and LH-RH were significantly lower than on the second day of dioestrus. These results may suggest the reciprocal functional relation of both hypothalamic peptides in the mechanism of the control of gonadotropin release. The highest LH-RH concentration of the median eminence was found in the animals on the 11th to 15th day of lactation, while the SP content in the median eminence of these animals is not significantly different from those of the remaining groups except the value for dioestrus (D2) rats. The SP content in pituitary of lactating rats was significantly higher than those of rats during cyclic or "constant oestrus".

The effect of prolonged stress on the hypothalamic luteinizing hormone-releasing hormone (LHRH) in the anoestrous ewe.

A study was performed to examine the effect of the intermittent long-term (20 min/h daily during 3 consecutive days) electric stress (footshocks) on the hypothalamic LHRH in anoestrous ewe. On the third day, immediately after the last stimulation, the animals were decapitated and the hypothalami and pituitaries were taken for analysis of LHRH and LH by immunocytochemistry; LHRH was also assayed by radioimmunoassay. In the stressed animals the concentration of immunoreactive LHRH (IR-LHRH) and LHRH analysed by radioimmunoassay (RI-LHRH) increased significantly in the median eminence (ME) and the medial preoptic area (MPOA) when compared with the controls. Additionally, IR-LHRH perikarya appeared in the MPOA of stressed animals, whereas they were not seen in the controls. RI-LHRH in the medial basal hypothalamus (MBH) of stressed ewes was markedly lower than in control ones. No significant differences in RI-LHRH concentration were found in the septum between stressed and control ewes. The present study clearly indicates that prolonged stressful stimuli modify LHRH concentration in the discrete areas of the hypothalamus of the anoestrous ewe. It is suggested that these changes are attributed mainly to an inhibition of LHRH release from the ME and a suppression of LHRH transport along neural fibres within the hypothalamus.

Participation of a pure neuronal mechanism in the control of gonadal functions.

The present review summarizes the data on the existence of a neural connection between the gonads and the central nervous system. As an experimental model the development of compensatory ovarian hypertrophy following unilateral ovariectomy was studied. Unilateral ovariectomy or orchidectomy resulted in a higher cellular activity in the hypothalamic arcuate neurons contralateral to hemigonadectomy and an increased luteinizing hormone-releasing hormone content in the medio-basal hypothalamus ipsilateral to the removed gonad. Local treatment of the remaining ovary with 6-hydroxy-dopamine prevented the development of compensatory ovarian hypertrophy after unilateral ovariectomy. Hemitransection of the spinal cord contralateral to hemigonadectomy, different unilateral hypothalamic deafferentations of the medio-basal hypothalamus as well as unilateral lesion of the medial preoptic area or of the arcuate nucleus blocked the compensatory ovarian growth when the brain surgery was on the same side as ovariectomy. Furthermore, in hypophysectomized + unilaterally ovariectomized rats a less severe ovarian atrophy occurred than in animals solely with hypophysectomy. All these data favour the view that - besides the hormonal control - a pure neuronal mechanism is also involved in the regulation of gonadal functions.

The effect of ketamine/xylazine and carbon dioxide on plasma luteinizing hormone releasing hormone and testosterone concentrations in the male Norway rat

The effect of a commonly used anaesthetic, ketamine/xylazine and/or carbon dioxide (CO(2)) on plasma luteinizing hormone releasing hormone (LHRH) and testosterone concentrations was determined in male Sprague-Dawley rats. These values were compared with values obtained from pre-anaesthetic control samples. Ketamine/xylazine treatment did not significantly affect testosterone concentrations. In contrast, LHRH started to decrease one hour after ketamine/xylazine administration and continued to significantly decrease after 24 h. In addition, in the CO(2) euthanasia-only group, LHRH concentrations were also significantly decreased. These results suggest that ketamine/xylazine anaesthesia followed by CO(2) euthanasia 24 h later is exerting a significant effect on LHRH concentrations 24 h after anaesthetizing, while only having a slight effect on testosterone, and that CO(2) is exerting an immediate significant effect on LHRH. In conclusion, LHRH analysis should be avoided after ketamine/xylazine anaesthesia and CO(2) euthanasia.

Hypothalamic Control of Mitogen-Induced Proliferative Responses and Luteinizing Hormone-Releasing Hormone Levels in Thymus and Peripheral Blood of Rat Fetuses

The role of endogenous luteinizing hormone-releasing hormone (LHRH) in the development of concanavalin A (ConA)-induced proliferative responses was studied in rat fetuses. Preliminary treatment of fetuses in utero with either the LHRH receptor antagonist or anti-LHRH antibodies resulted in the suppression of ConA-induced proliferative responses of thymocytes. LHRH and LHRH-immunopositive cells, morphologically similar to thymocytes, were detected in intact fetal thymus. A significant content of LHRH was also found in the peripheral blood of fetuses. The LHRH content in thymus and plasma was similar in males and females. Surgical ablation of the hypothalamus resulted in 2-fold decreases in thymus and plasma levels of LHRH in 21-day-old fetuses compared to sham-operated fetuses. It was concluded that LHRH regulates mitogen-induced proliferative responses of thymocytes during prenatal ontogenesis in the rat. The main source of plasma LHRH at that period is the hypothalamus. Moreover, LHRH is synthesized in the fetal thymus. Thus, LHRH is suggested to have not only a central effect but also to be involved in autocrine or paracrine regulation of proliferative immune responses.

Neonatal handling and reproductive function in female rats

Neonatal handling induces anovulatory estrous cycles and decreases sexual receptivity in female rats. The synchronous secretion of hormones from the gonads (estradiol (E2) and progesterone (P)), pituitary (luteinizing (LH) and follicle-stimulating (FSH) hormones) and hypothalamus (LH-releasing hormone (LHRH)) are essential for the reproductive functions in female rats. The present study aimed to describe the plasma levels of E2 and P throughout the estrous cycle and LH, FSH and prolactin (PRL) in the afternoon of the proestrus, and the LHRH content in the medial preoptic area (MPOA), median eminence (ME) and medial septal area (MSA) in the proestrus, in the neonatal handled rats. Wistar pup rats were handled for 1 min during the first 10 days after delivery (neonatal handled group) or left undisturbed (nonhandled group). When they reached adulthood, blood samples were collected through a jugular cannula and the MPOA, ME and MSA were microdissected. Plasma levels of the hormones and the content of LHRH were determined by RIA. The number of oocytes counted in the morning of the estrus day in the handled rats was significantly lower than in the nonhandled ones. Neonatal handling reduces E2 levels only on the proestrus day while P levels decreased in metestrus and estrus. Handled females also showed reduced plasma levels of LH, FSH and PRL in the afternoon of the proestrus. The LHRH content in the MPOA was significantly higher than in the nonhandled group. The reduced secretion of E2, LH, FSH and LHRH on the proestrus day may explain the anovulatory estrous cycle in neonatal handled rats. The reduced secretion of PRL in the proestrus may be related to the decreased sexual receptiveness in handled females. In conclusion, early-life environmental stimulation can induce long-lasting effects on the hypothalamus-pituitary-gonad axis.

Luteinizing hormone and luteinizing hormone-releasing hormone secretion is under locus coeruleus control in female rats

It has been suggested that norepinephrine (NE) from the locus coeruleus (LC) plays an important role in triggering the preovulatory surge of gonadotropins. This work intended to study the role of LC in luteinizing hormone (LH) secretion during the estrous cycle and in ovariectomized rats treated with estradiol and progesterone (OVXE 2P) and to correlate it with LH releasing hormone (LHRH) content in the medial preoptic area (MPOA) and median eminence (ME). Female rats on each day of the estrous cycle and OVXE 2P were submitted to jugular cannulation and LC electrolytic lesion or sham-operation, at 09:00 h. Blood samples were collected hourly from 11:00 to 18:00 h, when animals were decapitated and their brains removed to analyze LC lesion and punch out the MPOA and ME. Plasma LH levels and LHRH content of MPOA and ME were determined by radioimmunoassay. During metestrus, diestrus and estrus, LC lesion did not modify either LH plasma concentrations or LHRH content, but completely abolished the preovulatory LH surge during proestrus and the surge of OVXE 2P. These blockades were accompanied by an increased content of LHRH in the MPOA and ME. The results suggest that: (1) LC does not participate in the control of basal LH secretion but its activation is essential to trigger spontaneous or induced LH surges, and (2) the increased content of LHRH in the MPOA and ME may be due to a decreased NE input to these areas. Thus, LC activation may be required for depolarization of LHRH neurons and consequent LH surges.

Luteinizing hormone-releasing hormone (LHRH) biosynthesis and secretion in embryonic LHRH.

Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7-10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mM KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide.

Luteinizing Hormone-Releasing Hormone (LHRH) Biosynthesis and Secretion in Embryonic LHRH Neurons

Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7–10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mm KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide.

Ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone release by scavenging nitric oxide.

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormone-releasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K(+)] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), N(G)-monomethyl-l-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K(+)], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10(-5) to 10(-3) M) had no effect on basal LH-RH release but completely blocked high [K(+)]- and nitroprusside-induced LH-RH release. N-Methyl-d-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10(-5) to 10(-3) M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.

Effect of Nitric Oxide Synthase Inhibitors on Preventing Ethanol‐Induced Suppression of the Hypothalamic‐Pituitary‐Gonadal Axis in the Male Rat

Ethanol (EtOH) suppression of the hypothalamic-pituitary-gonadal (HPG) axis results in broad reproductive malfunction. In the HPG axis, the suppressive effects of EtOH are manifested by decreased serum testosterone, reduced testicular luteinizing hormone (LH) receptor numbers, lowered serum LH and pituitary beta-LH mRNA levels (in castrated animals), and impaired luteinizing hormone releasing hormone (LHRH) release from the hypothalamus. Increasing evidence has suggested that nitric oxide (NO) plays a role in regulation of the HPG axis. NO was shown to stimulate LHRH secretion from the hypothalamus and to have variable effects on LH release from the pituitary. At the gonadal level, NO is inhibitory to testosterone production. NO may directly inhibit some testicular steroidogenic enzymes. To investigate the effect of EtOH, NO, and their interaction on the male HPG axis, three NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester, N(G)-nitro-L-arginine, and 7-nitro indazole were used to study overall HPG function in the presence and absence of EtOH. Animals were given intraperitoneal injections of saline, EtOH, various NOS inhibitors, or EtOH, along with NOS inhibitors 2 hr before sacrifice. Serum testosterone and LH concentrations, pituitary beta-LH mRNA levels, hypothalamic LHRH mRNA levels, and LHRH content were determined. It was found that blocking NOS by these NOS inhibitors prevented EtOH-induced suppression of testosterone and, in some cases, serum LH. However, this was not accompanied by concurrent changes with NOS blockade on LHRH mRNA, hypothalamic pro-LHRH or LHRH content or pituitary LH beta mRNA levels. It appears that the protective effect of NOS blockade was largely, although not completely, due to a direct effect at the gonadal level.

Temporal effects of estradiol (E) on luteinizing hormone-releasing hormone (LHRH) and LH release in castrated male sheep

We tested the hypothesis that rapidly expressed inhibitory effects of estradiol (E) on luteinizing hormone (LH) release in the male are attributable, in part, to suppression of luteinizing hormone-releasing hormone (LHRH) release. Hypophyseal-portal cannulated, castrated male sheep were infused with E (15 ng/kg/hr) or vehicle. Portal and jugular blood samples were collected at 10-min intervals for 4 hr before, and for either 12 hr (E, n = 4; vehicle, n = 4) or 24 hr (E, n = 8; vehicle, n = 3) after the start of infusion. In animals sampled for 16 hr, temporal changes in both LHRH and LH were assessed. In animals sampled for 28 hr, only LH data were analyzed. Before either the 12-hr or 24-hr infusion, LHRH and/or LH mean concentrations, pulse amplitude and interpulse interval (IPI) did not differ between E- and vehicle-infused animals. In animals sampled for 16 hr, no effects of time or steroid × time interactions were detected for mean LHRH and LHRH pulse amplitude; however, both were greater (P < 0.01) in vehicle-infused than in E-infused males. LHRH IPI was unaffected by infusion. In contrast, both mean LH and LH pulse amplitude declined (P < 0.01) within 4–8 hr after the start of E infusion, whereas mean LH IPI was unaffected. In animals sampled for 28 hr, an effect of time (P < 0.01) and a steroid × time interaction (P < 0.01) was detected for mean LH, and there was an effect of time (P < 0.01) on LH pulse amplitude. Mean LH IPI was not affected. Our results show that in male sheep E rapidly reduces LH release in the absence of a detectable change in LHRH release.

The role of gonadectomy and testosterone replacement on thymic luteinizing hormone-releasing hormone production.

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.

Jurkat cell proliferative activity is increased by luteinizing hormone-releasing hormone

Jurkat cells were used to study the immunomodulatory role of luteinizing hormone-releasing hormone (LHRH) in immune cells. The Jurkat cell, a human mature leukemic cell line, phenotypically resembles resting human T lymphocytes and has been widely used to study T cell physiology. The data from this study demonstrate that the Jurkat cell concentration of immunoreactive LHRH was 210 +/- 36 pg/10(6) cells and that of proLHRH was 188 +/- 27 pg/10(6) cells (means +/- S.E.M.). The authenticity of this LHRH immunoreactivity is documented in two ways. First, both Jurkat LHRH and proLHRH immunoreactivity demonstrate dilutional parallelism with hypothalamic LHRH and proLHRH. Second, Jurkat lysates show LHRH bioactivity by releasing luteinizing hormone from rat anterior pituitary cells in culture. The presence of substantial amounts of LHRH in medium in which Jurkat cells were cultured for 72 h indicated that LHRH can be released from the cells. Using specific primers to exons 2 and 4 of the LHRH gene, we have found that Jurkat cells (like human T cells) express LHRH mRNA. The LHRH agonist, des-Gly10,D-Trp6-LHRH ethylamide, significantly increases the proliferative activity of Jurkat cells, as assessed by tritiated thymidine incorporation, from 15980 +/- 1491 c.p.m. in control to 28934 +/- 3395, 30457 +/- 3861 (P = 0.05 vs control) or 35299 +/- 5586 c.p.m. (P < 0.01 vs control) with 10(-11), 10(-9) or 10(-7) M agonist respectively. LHRH antagonist, [D-pGlu1,D-Phe2,D-Trp3,6]-LHRH, at a concentration of 10(-8) M decreases Jurkat cell proliferative activity form 17145 +/- 526 c.p.m. in control medium to 10653 +/- 1323 c.p.m. (P = 0.05). Co-incubation with the LHRH antagonist completely inhibits the proliferative stimulation induced by the LHRH agonist. Furthermore, applying monoclonal LHRH antibody to Jurkat cells inhibits the cell proliferative activity assessed by tritiated thymidine incorporation from 19900 +/- 2675 c.p.m. in control to 15680 +/- 2254, 15792 +/- 1854 and 9700 +/- 908 c.p.m. in media with 1:40, 1:20 and 1:10 dilution of purified antibody respectively (P < 0.01, 1:10 dilution compared with control). In addition, the cAMP level in LHRH-stimulated Jurkat cells is decreased to 74, 27 and 57% of control levels after 15, 30 and 45 min respectively of exposure to 10(-7) M LHRH agonist. In summary, Jurkat cells produce, process and release immunoreactive and bioactive LHRH, as do normal human T cells. Endogenous and exogenous LHRH increase Jurkat cell proliferative activity, and cAMP may be involved in LHRH-induced Jurkat cell proliferation. The Jurkat cell may be a useful model with which to study the role of LHRH in human T cell function.

The effect of castration on steady state levels of luteinizing hormone-releasing hormone (LHRH) mRNA and proLHRH processing: time course study utilizing semi-quantitative reverse transcription/polymerase chain reaction.

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH)--steady state levels of LHRH mRNA, post-translational processing and secretion--have, however, not been consistent. The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple postoperative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH. Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intra-pituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days. Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins. ProLHRH content in castrates was 1.8-times that seen in controls at 1 day post castration (P<0.05), concomitant with the rise in steady state levels of LHRH mRNA at that time point. However, proLHRH content in castrates was no different from that seen in controls at each of the later time points examined. LHRH content was unchanged through 7 days after castration, but then fell significantly to 57% of control levels in hypothalami from animals gonadectomized 14 to 21 days previously (P<0.001 vs control), and to 54% of sham-operated levels at 28 days postoperatively (P<0.001). We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal proLHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of proLHRH to LHRH is, likewise, small and transient. In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level.

Oramucosal delivery of LHRH: pharmacokinetic studies of controlled and enhanced transmucosal permeation.

Several transmucosal therapeutic systems (TmTs) were developed to study the enhanced/controlled delivery of luteinizing hormone-releasing hormone (LHRH) through oral mucosae for prolonged periods. TmTs is a track field-shaped bilayer mucoadhesive device consisting of fast-release and sustained-release layers. In vivo evaluations were performed in beagle dogs, and pharmacokinetic profiles were monitored to characterize the transmucosal permeation kinetics of LHRH delivered by the various TmTs formulations containing a stabilizer, cetylpyridinium chloride, and a permeation enhancer, such as bile salts, to enhance the stability and permeability of LHRH. The plasma LHRH concentrations were observed to reach the plateau level within 30 min and were maintained for 2 hr following application of TmTs, in contrast to a rapid elimination profile observed after IV administration. Addition of 5% bile salt into the fast-release layer was observed to produce an enhancement in the absorption rate, higher plateau plasma levels, and greater systemic bioavailability. Addition of pH modifiers was noted to affect the bile salt enhanced transmucosal delivery of LHRH. To prolong the plasma LHRH level, several loading doses of LHRH were incorporated into the sustained-release layer. The plasma levels were sustained and the area under the curve (AUC) values were found to be linearly dependent upon the combined loading doses of LHRH in the fast-release and sustained-release layers. Mucosal irritation was also measured, using buccal mucosa, and results were observed to be low and reversible for the single application. The results indicated that TmTs is relatively safe and capable of achieving enhanced and controlled transmucosal delivery of peptide drugs.

Calcium involvement in luteinizing hormone-releasing hormone release from the bovine infundibulum

Bovine infundibular (stalk median eminence) explants were incubated in vitro to test the hypothesis that calcium (Ca) is involved in the release of luteinizing hormone-releasing hormone (LHRH) from LHRH neuron terminals in cattle. Right and left infundibular halves from individual heifers and/or steers were randomly assigned to either control or treated (EGTA [a Ca chelator] or verapamil [an L-type Ca channel antagonist]) groups. Each half was incubated in 600 μl of KrebsRinger bicarbonate medium (KRB) in the presence or absence of a treatment agent for 180 min. At 30-min intervals, 500-μl samples were removed from each incubate and replaced with fresh media. Spontaneous (basal) and depolarization-induced (60 mM potassium) LHRH release was evaluated by radioimmunoassay of the LHRH content in the media incubated from 91 to 120 and 121 to 150 min of culture, respectively. The effect of treatment on depolarization-induced LHRH release was analyzed by comparing the differences between spontaneous and depolarization-induced LHRH release in control and treated groups. Spontaneous LHRH release was not different between control and 1.25 mM EGTA- or 100 μM verapamil-treated halves from steers. In contrast, steer infundibular halves incubated with EGTA (replacing Ca in KRB and chelating any Ca in the media) released less LHRH during depolarization than did control halves. In addition, verapamil-treated (to block Ca uptake by the terminal) infundibular halves from steers or heifers released less LHRH in response to depolarization than did control halves. In conclusion, these results: 1) support the hypothesis that Ca is involved in LHRH release from the bovine infundibulum, 2) suggest that the involvement of Ca may be independent of the reproductive state, and 3) demonstrate that bovine infundibular halves incubated in vitro are useful for studying selected mechanisms regulating bovine hypothalamic neurohormone release (exocytosis) from neuron terminals.