Effect of pulsatile infusion of progesterone on the in vivo activity of the luteinizing hormone-releasing hormone neural apparatus of awake unrestrained female and male rabbits

Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aimed at the tuberal region of the hypothalamus and were used in 19 perfusion experiments. Animals were treated under 2 conditions: a control condition in which female (n = 4) and male (n = 4) rabbits were perfused only with artificial medium for 5-9 h, and an experimental condition in which female (n = 4) and male (n = 5) rabbits were subjected to perfusion with artificial medium, followed by 6 pulses of progesterone (P4) (10 min on, 30 min off; 10 ng/ml) during a 240-min period. Two female rabbits were also subjected to perfusion with artificial medium, followed by 6 pulses of cholesterol (10 min on, 30 min off; 10 ng/ml) during a 240-min period. The LHRH concentration in perfusates collected every 10 min was measured by RIA. In the 4 females undergoing 6- to 7-h control push-pull perfusions, spontaneous pulses were observed, with about 1 pulse every 60 min, and a variable amplitude of the LHRH signal, with a mean release of about 0.91 pg/10 min. In the 4 does treated with pulsatile P4 at 10 ng/ml, the mean LHRH release rate increased significantly from 0.91 +/- 0.13 to 1.66 +/- 0.20 pg/10 min (P less than 0.035), primarily due to an increase in the amplitude of the LHRH pulses, which were significantly greater than controls. This response occurred with an apparent mean latency of about 50 min. Cholesterol pulses did not affect the spontaneous activity of the LHRH neural apparatus. In the 4 males undergoing 5- to 9-h control push-pull perfusions, spontaneous LHRH pulses were observed with about 1 pulse every 60 min, and the mean release increased from 1.25 +/- 0.56 to 1.54 +/- 0.55 (P less than 0.035, by Wilcoxon's matched pairs signed rank test) in the late afternoon primarily due to an increase in the amplitude of the pulses. P4 pulses did not affect the spontaneous activity of the LHRH neural apparatus compared to that in the control animals. Overall, these results clearly demonstrate that although spontaneous pulsatile LHRH release from the hypothalamus of awake unrestrained female and male rabbits is similar, pulses of P4 can activate only the female LHRH neural apparatus, with an apparent latency to peak LHRH release of approximately 50 min. In addition, there appears to be an increase in LHRH mean release levels in the late afternoon to early evening in male rabbits.     

Functional characteristics of the luteinizing hormone-releasing hormone pulse generator in conscious unrestrained female rabbits: activation by norepinephrine

We probed the activity of the neural LHRH apparatus by means of push-pull cannulae implanted in the hypothalamus of conscious intact anestrous unrestrained New Zealand White female rabbits raised under natural light. The data revealed that the neuropeptide LHRH is released in a pulsatile manner (one pulse per 46.4 +/- 3.9 min; amplitude, 1.63 +/- 0.39 pg; n = 7) from a rather extensive area within the basal hypothalamus of the rabbit covering the rostral, medial, and posterior regions in a rostro-caudal direction and about 1 mm lateral. A remarkable change in mean LHRH output was observed during the year, and two distinct types of LHRH output were detected: low (0.72 +/- 0.07 pg/10 min; n = 7) when these reflex ovulators were perfused during late spring to early summer and high (2.92 +/- 0.29 pg/10 min; n = 5) when perfused during late summer, early fall to spring months. This 4-fold annual change in LHRH output was mainly due to changes in the amplitude of the LHRH signal and was not correlated with the position of the push-pull cannula within a restricted area in the hypothalamus. These changes may be related to the state of the ovaries, since rabbits with high LHRH output showed larger ovaries with well developed follicles than rabbits with low LHRH output. Norepinephrine, a hypothalamic neurotransmitter known to be involved in the mounting-evoked ovulation reflex in this induced ovulator, elicited a 2-fold greater mean LHRH release (P less than 0.05) when infused in doses of 10(-7) - 10(-6) M directly into the hypothalamic perfusion area. The data indicate that the activity of the LHRH pulse generator in this species presents remarkable annual variations in its mean LHRH output mainly due to a greater amplitude of the LHRH signal and that norepinephrine is a potent stimulator of LHRH release in conscious unrestrained female rabbits.     

Infusion of progestins into the hypothalamus of female New Zealand white rabbits: effect on in vivo luteinizing hormone-releasing hormone release as determined with push-pull perfusion

Previously, we have reported that intermittent infusion of progesterone (P4; 10 ng/ml) into the hypothalamus of conscious unrestrained female New Zealand White rabbits stimulates LHRH release in vivo. To further investigate this phenomenon, in the present studies, the effect of pulsatile (six pulses; 10 min on, 30 min off) and continuous infusion of P4 (Exp I) and 4-pregnen-20 alpha-ol-3-one or 20 alpha-hydroxyprogesterone (20 alpha-OH-P; Exp II) on LHRH release were studied in vivo using push-pull cannulae. Furthermore, the effect of pulsatile infusion (six pulses; 10 min on, 30 min off) of low doses of the following three progestins [5 beta-pregnan-3 beta-ol-20-one (pregnanolone), 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-5 alpha-P), and 20 alpha-OH-P] into the hypothalamus of does using push-pull cannulae were examined in Exp III. In Exp I, continuous infusion of P4 at 10 ng/ml was unable to stimulate mean LHRH release. However, pulses of P4 at 0.01 ng/ml (n = 4) were found to significantly increase the amplitude of the largest LHRH pulse (control period, 1.18 +/- 0.41; versus treatment period, 3.15 +/- 0.75 pg; P less than 0.035) as well as the frequency of LHRH pulses (control period, 0.72 +/- 0.26; treatment period, 1.37 +/- 0.12 pulses/h; P less than 0.035). On the other hand, there was no effect of pulses of P4 at 0.001 ng/ml (n = 4) on the activity of the LHRH neural apparatus. In Exp II, pulses of 20 alpha-OH-P at 10 ng/ml (n = 4) were found to significantly increase the mean LHRH release (control period, 1.24 +/- 0.10; treatment period, 2.07 +/- 0.52 pg/10 min) as well as the amplitude of the largest LHRH pulse (control period, 0.99 +/- 0.36; treatment period, 8.15 +/- 3.75 pg). Interestingly, continuous infusion of 20 alpha-OH-P (10 ng/ml) also significantly increased the mean amplitude (control period, 0.58 +/- 0.34; treatment period, 1.90 +/- 0.29 pg) as well as the amplitude of the largest LHRH pulse (control period, 0.64 +/- 0.37; treatment period, 4.30 +/- 1.97 pg).(ABSTRACT TRUNCATED AT 400 WORDS)     

Roles of somatostatin and growth hormone-releasing factor in ether stress inhibition of growth hormone release.

In order to evaluate the release of somatostatin (SRIF) and growth hormone-releasing factor (GRF) into the pituitary gland in response to ether stress, a push-pull perifusion (PPP) technique has been used in freely moving rats. Push-pull cannulae (PPC) were implanted into the anterior pituitary (AP) glands of male rats. After a 7-day recovery period the rats were fitted with indwelling jugular catheters. The next day the animals were subjected to PPP of the AP during 1 h followed by ether stress (2 min) and another hour of perifusion. The perifusion flow was 20 microliters/min and 10-min fractions were collected and assayed for SRIF and GRF by RIA. Plasma growth hormone (GH) levels were assayed every 10 min. At the end of the experiments, the accuracy of PPC tip placements was ascertained with a dissecting microscope. Under basal conditions there were 2.9 pulses/h of SRIF with an amplitude of 12.76 +/- 0.46 pg. The output of SRIF and GRF in the 10-min period beginning with application of ether was increased 2-fold (p less than 0.005 and p less than 0.01, respectively). Interestingly, the increased release of SRIF continued for an additional 10 min, whereas GRF output decreased and was almost undetectable. The release of both GRF and SRIF had returned to basal values 20-30 min after stress. Mean plasma GH levels were significantly lowered 10 min after stress. Each of the 9 animals showed a restoration of pulsatile GH release to basal levels within 20-30 min after stress. Our findings provide compelling evidence that SRIF plays a prominent role in stress-induced inhibition of GH release in the rat by blocking the response to the transient elevation of GRF and continuing to suppress GH release for 20 min.     

Copper stimulation of LHRH release from median eminence explants. III. A process dependent on extracellular sodium

Copper, complexed to histidine (CuHis), stimulates LHRH release from explants of the median eminence area (MEA). To gain further understanding of the mechanism of copper action, in this study, we assessed the Na+ and energy requirements for CuHis stimulation of LHRH release. MEA explants, obtained from adult male rats, were incubated at 37 degrees C for 15 min with 100 microM CuHis and then for 45 min in CuHis-free medium (Krebs-Ringer-phosphate buffer, pH 7.4). LHRH released into the medium was evaluated by RIA. When the incubation buffer contained 143 mM Na+, CuHis stimulated the release of LHRH from a basal level of 17.2 +/- 1.26 (mean +/- SEM, n = 7) to 74.5 +/- 6.2 pg/60 min per MEA. When [Na+] was reduced to 16 mM Na+ (by substituting with Li+), CuHis-stimulated LHRH release was inhibited by 80% (p less than 0.001); indicating a requirement for Na+. In addition, we found that CuHis-stimulated LHRH release was a saturable function of Na+ concentration; saturation achieved with about 100 mM Na+. To assess the requirement for Na+ transport, we evaluated the effect of 1 mM ouabain, 10 microM tetrodotoxin (TTX), or 100 microM amiloride on CuHis stimulation of LHRH release. Ouabain inhibited CuHis stimulation of LHRH release by 80%, whereas TTX and amiloride were ineffective. In addition, we observed that CuHis did not stimulate LHRH release when incubation was carried out at 4 degrees C or at 37 degrees C in the presence of 5 mM KCN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep

The push-pull perfusion technique was used in combination with a sequential bleeding schedule to estimate simultaneously the release patterns of LHRH and LH in unanesthetized ovariectomized sheep and to determine the temporal relationship between the release of these two hormones. Ovariectomized (greater than 30 days) ewes received unilateral push-pull cannula (PPC) implants (od, 0.85 mm) into the median eminence. After at least 6 days of recovery, each ewe was fitted with an indwelling jugular catheter. For push-pull perfusion, a stylette was removed from the outer PPC, and an inner cannula assembly (od, 0.40 mm) was inserted. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 20 microliters/min. Continuous 10-min perfusate fractions were collected, acidified, and stored at -20 C for LHRH RIA. Blood samples were obtained every 10 min via the jugular catheter, each being drawn 5 min after the start of a perfusate collection interval. Plasma LH levels were determined by RIA. The duration of the sampling was 3-7 h. LHRH output was distinctly pulsatile, occurring at a frequency of approximately one pulse every 30-40 min (n = 5 sheep). LHRH pulse amplitude and frequency remained relatively constant throughout each perfusion. Plasma LH values also were pulsatile, and all LH peaks occurred either during the same interval or during the interval after a LHRH pulse. LH pulses always were accompanied or directly preceded by LHRH pulses, but LHRH pulses were not always followed by LH pulses. The amplitudes of LH pulses and corresponding LHRH pulses were highly correlated (r = 0.81; P less than 0.01). Histological examination revealed that detection of LHRH in perfusates depended upon the placement of the PPC tip into either the zona externa of the central median eminence or adjacent areas. These experiments demonstrate that 1) hypothalamic LHRH release in the Ovx ewe occurs in discrete pulses, with a mean interpulse interval of 38.7 +/- 1.5 min, 2) LH pulses invariably are preceded or accompanied by LHRH pulses, and 3) LH pulse amplitude is highly correlated with LHRH pulse amplitude.     

In vitro LHRH release from superfused hypothalamus as a function of the rat estrous cycle: effect of progesterone

The present study examines the effect of progesterone (P) on in vitro LHRH release from hypothalamic fragments from intact adult rats throughout the estrous cycle. Estrous cyclicity was monitored by daily vaginal smears, and animals which exhibited at least two consecutive 4-day estrous cycles were used. Animals were sacrificed between 10.00 and 13.00 h and the mediobasal hypothalamic-preoptic area-suprachiasmatic nucleus units were removed and transferred into superfusion chambers (one unit/chamber). Following a 2-hour control period, in which the spontaneous LHRH release was established, P (10 ng/ml) was infused in an intermittent mode (10 min-on, 20 min-off). Effluents were collected at 10-min intervals and LHRH concentrations were determined by RIA. The spontaneous LHRH release from control preparations was episodic throughout all stages of the estrous cycle with a significant low release rate and low LHRH amplitude only during estrus. Interestingly, intermittent infusion of P significantly stimulated LHRH release solely in hypothalamic fragments derived from proestrous rats. The P-stimulated LHRH release during the 1st-hour period after P infusion was significantly higher (p less than 0.05) than that observed in the control preparations during the same time period as well as from its own basal pre-P values (1-hour post-P: 4.26 +/- 0.96 vs. 1-hour control and pre-P: 2.34 +/- 0.38 and 2.32 +/- 0.57 pg/10 min, respectively). P administration did not stimulate in vitro LHRH release during the other stages of the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo release of luteinizing hormone releasing hormone increases with puberty in the female rhesus monkey

To determine the regulatory mechanism of the LHRH release associated with puberty, episodic release of LHRH from the stalk-median eminence was measured using a push-pull perfusion technique in conscious prepubertal and peripubertal female monkeys. After insertion of a push-pull cannula into the stalk-median eminence, a modified Krebs-Ringer phosphate buffer solution was infused through the push-cannula, and perfusates were collected through the pull-cannula at 200 microliters/10 min. LHRH in perfusates was determined by RIA. Two 6-h sampling sessions, in the morning (0600-1200 h; lights on 0600 h) and in the evening (1800-2400 h; lights off 1800 h) were performed in each animal. LHRH release patterns were analyzed in prepubertal (15.7 +/- 0.7 months of age; mean +/- SEM, n = 6) early pubertal (premenarcheal; 26.1 +/- 1.0 months, n = 7), and midpubertal (40.0 +/- 1.4 months, n = 6) monkeys. Results were as follows: 1) LHRH release was pulsatile in all age groups. While LHRH release in five of six prepubertal animals consisted of small (amplitude less than 2.5 pg/ml) pulses, in all peripubertal animals LHRH release was a mixture of small and large (amplitude greater than 2.5 pg/ml) pulses. 2) There was a significant developmental increase in mean LHRH release (P less than 0.02), and this was particularly apparent in the evening. Mean LHRH release in the early and midpubertal groups was higher than that in the prepubertal group (P less than 0.05 for morning and P less than 0.01 for evening). The mean release in the evening of the midpubertal group further increased over that of the early pubertal group (P less than 0.05). 3) Similarly, LHRH pulse amplitude increased developmentally (P less than 0.01). Pulse amplitudes in early and midpubertal groups were higher than those in the prepubertal group (P less than 0.05 for morning and P less than 0.02 for evening). Again the amplitude in the evening further increased from the early pubertal to the midpubertal period (P less than 0.05). 4) There was also a developmental increase in basal LHRH release (P less than 0.01). The evening values in the early pubertal and midpubertal groups were higher than those in the prepubertal group (P less than 0.05). 5) The interpulse interval decreased developmentally (P less than 0.001). Interpulse intervals in early and midpubertal groups were shorter than those in the prepubertal group (P less than 0.01 for morning and P less than 0.025 for evening).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of estrogen on episodic secretory patterns of luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) in hypergonadotropic hypogonadism

The secretory dynamics of plasma luteinizing hormone-releasing hormone (LHRH) and serum luteinizing hormone (LH) were studied in three hypogonadal women before and after chronic administration of mestranol. Blood samples were obtained through an indwelling iv line every 15 min over 3 hours, and plasma levels of LHRH and LH were measured by radioimmunoassay. LHRH and LH pulses were defined as rising from nadir to peak that exceed 2 times the intraassay coefficient of variation. All patients showed pulsatile LHRH and LH release before mestranol administration. The mean LH levels (89 +/- 20 mIU/ml) and pulse amplitude (33 +/- 14 mIU/ml) were significantly reduced after mestranol administration. On the other hand, the mean LHRH levels (1.87 +/- 0.49 pg/ml) and pulse amplitude (0.92 +/- 0.41 pg/ml) did not change significantly after mestranol administration. Pulse frequency (2 approximately 3 times/3 hrs) of LHRH and LH did not change after mestranol administration. These data show that the chronic administration of estrogen to such patients cause a decrease in mean LH levels and amplitude of LH pulse without a decrease of pulsatile LHRH secretions. These results suggest that the chronic negative feedback action of estrogen on episodic LH release in women may be at the level of the pituitary gland and estrogen may change the pituitary sensitivity to LHRH.     

Copper stimulation of LHRH release from median eminence explants. II. A process dependent on chloride transport

We have previously shown that chelated copper stimulates LHRH release from explants of the median eminence area (MEA). Characteristics of this release process are: ligand and metal specificity, the involvement of a limited number of copper interactive sites, and a lack of dependence on extracellular calcium. Since chloride transport is essential for exocytosis of peptides and biogenic amines, we wished to ascertain if chloride transport is essential for the process of CuHis-stimulated release of LHRH. MEA explants were incubated for 15 min with 100 microM CuHis (phase I) and then for 15 min in copper-free medium (phase II) and LHRH released into the medium was evaluated by RIA. In the presence of 136 mM Cl, CuHis stimulated the release of LHRH from a basal level of 5 +/- 0.4 pg/15 min per MEA to 17 +/- 0.9 pg during phase I and to 30 +/- 1.2 pg during phase II. In the absence of Cl-, the CuHis-stimulated release of LHRH during phases I and II was inhibited by 80 and 90%, respectively. In the presence of 136 mM Cl- and the anion transport inhibitor SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) the stimulated release was completely inhibited in both phases. When the selectivity of this release process for monovalent anions was tested, the effectiveness of the anions in supporting CuHis-stimulated LHRH release was in this decreasing order: Cl- greater than Br- greater than SCN- = acetate greater than I- = isethionate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of LH release and ovulation in the rabbit with inhibitory analogues of luteinizing hormone releasing hormone.

Plasma LH levels and ovulation were studied in female rabbits following administration of several inhibitory analogues of luteinizing hormone-releasing hormone (LHRH) before and after mating with experienced males. Administration of (D-Phe2, D-Leu6)-LHRH (1.5 mg/kg sc) to does 30 min before mating did not prevent either LH release or ovulation. However, a single sc injection of (D-Phe2, L-Phe3, D-Phe6y-LHRH (6 mg/kg) given 30 min before mating in 4 rabbits resulted in a 30-60 min delay in the coitus-induced release of LH when compared with post-coital changes in the same animals injected with vehicle; however, all of the does ovulated. When multiple dosages of 4 mg/kg (D-Phe2, L-Phe3, D-Phe6)-LHRH were administered 3-5 times at half-hourly intervals beginning 30 min prior to mating there was a considerable reduction in plasma LH elevations at 0.5, 1.0, 2.0 and 4.0 h after mating and 3/5 treated rabbits showed partial or complete blockade of ovulation. Quite similar results were obtained with the same dosage of (D-Phe2, D-Trp3, D-Phe6)-LHRH. An early sharp peak in LH release and full ovulation were stimulated in 6 out of 6 does by a single iv injection of synthetic LHRH (500 ng/kg). However, in another experiment, three half-hourly sc injections (4 mg/kg) of (D-Phe2, L-Phe3, D-Phe6)-LHRH beginning 30 min before administering LHRH markedly reduced the rise in plasma LH (P less than 0.01) and completely blocked ovulation in all of the same 6 animals. An unsuccessful attempt was made to provide a test animal for LHRH analogue investigations by implanting 4 cm of silastic tubing filled with crystalline estradiol (E2) sc in ovariectomized (OVX) AND INTACT DOES. In OVX does the silastic E2 implants resulted in a progressive decline in the ability to release LH in response to mating at 6 and at 20 days after implantation. With ovaries present, the E2 implant permitted post-coital LH release and ovulation at 4 d but not at 30 d post-implantation. At 30 d after removal of the E2 implant three out of four does had fully recovered the ability to release LH and ovulate post-coitally. Collectively, these results indicate that inhibitory analogues of LHRH can effectively block the ovulatory response to exogenous LHRH in rabbits, but that coitally-induced LH release and ovulation are less susceptible to blockade by the analogues. Silastic E2 implants, however, progressively and reversibly reduce the capacity of does to release LH after mating.     

Variations in hypothalamic luteinizing hormone releasing hormone content and release in vitro and plasma concentrations of luteinizing hormone and testosterone in developing cockerels

An in-vitro superfusion system was used to study age-dependent changes in the functional activity of LH releasing hormone (LHRH) neurones terminating in the mediobasal hypothalamus (MBH) of the cockerel. Fragments of MBH tissue were obtained from cockerels killed at 8, 12, 16 and 20 weeks of age and both the rate of release of LHRH in vitro and the residual content of LHRH were determined by radioimmunoassay. Blood was collected from a similar group of cockerels of the same age for determination of plasma LH and testosterone concentrations. Superfused cockerel MBH showed both basal and depolarization-induced release of LHRH and the calcium-dependency of the release process was demonstrated. The viability of MBH in vitro was indicated by the observation that the rate of CO2 production by the tissue remained constant during a 3-h period of superfusion. Both the basal rate of release of LHRH (7.24 +/- 0.63 pg/2MBH per h; mean +/- S.E.M., n = 4) and the residual content of LHRH (1.10 +/- 0.24 ng/2MBH) were lowest in the 8-week-old cockerel and increased progressively to reach levels three (P less than 0.001) and 13 (P less than 0.001) times greater, respectively, by 20 weeks of age. Concentrations of LH and testosterone in plasma did not increase significantly until 16 weeks of age when the respective values were 5.86 +/- 0.37 micrograms/l and 1.88 +/- 0.31 nmol/l (n = 11). Whereas plasma testosterone increased further to 5.76 +/- 0.42 nmol/l (n equal 11) at 20 weeks of age, plasma LH fell significantly (P less than 0.02) to a concentration of 4.08 +/- 0.41 micrograms/l (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)     

Puberty-related increase in episodic LHRH release from rat hypothalamus in vitro

The retrochiasmatic hypothalamus (RCH) was removed from brains of male rats between 12 and 50 days of age, and immediately studied in vitro. The release of LHRH from the RCH was evaluated by periodic (7.5-min) collections of culture medium and subsequent RIA. With synthetic LHRH in the experimental system, the mean (+/- 1 SD) recovery was 94 +/- 7% with a variation coefficient of 14 +/- 3%. An increase in LHRH release was considered to be significant when it exceeded 6 pg/7.5 min. Biological viability of RCH in vitro was assessed by an increased release of LHRH in response to the depolarizing effect of veratridine. As age increased, from 12 to 50 days, the hypothalamic LHRH content steadily increased. However, a significant increase in veratridine - induced release of LHRH occurred only at 23 days and thereafter. At various ages, single hypothalami were studied during a mean 112-min period to evaluate the spontaneous release of LHRH. In all age groups, the in vitro LHRH release occurred in pulses. However, mean pulse frequency increased significantly with age: in 12- and 17-day-old rats, 0.3 pulse/112 min was observed; at 23, 25 and 27 days, this frequency varied between 1.8 and 3.0 pulses/112 min. At 50 days of age, the observed frequency was within the same range. We conclude that the RCH obtained from rats of various ages may retain in vitro its capacity to release LHRH episodically and that the frequency of these episodic pulses markedly increases with age to the time of the onset of puberty in male rats.     

Infusion of neuropeptide Y into the stalk-median eminence stimulates in vivo release of luteinizing hormone-release hormone in gonadectomized rhesus monkeys

Studies in the rat and rabbit indicate that facilitatory effects of neuropeptide Y (NPY) as well as norepinephrine (NE) on LH and LHRH release are dependent on the presence of the ovarian steroid estrogen. However, we have previously found the NE and an alpha-1-adrenergic agonist are both stimulatory to pulsatile LHRH release in ovariectomized rhesus monkeys. In the present experiment the effects of NPY on LHRH release were examined in conscious monkeys using a push-pull perfusion method. Twelve gonadectomized monkeys (8 females and 4 males) were used. Perfusate samples from the stalk-median eminence (S-ME) were obtained through a push-pull cannula at 10-min intervals for 12 h, and the amount of LHRH in samples were determined with RIA. NPY dissolved in a modified Krebs-Ringer phosphate buffer solution at concentrations of 10(-8), 10(-7), 10(-6), and 10(-5) M was directly infused into the S-ME through the push cannula for 10 min at 90-min intervals. Vehicle was infused as a control. Since sex differences in LHRH response to NPY were not present, data from males and females were combined for analysis. NPY infusion into the S-ME stimulated LHRH release in a dose-dependent manner (P less than 0.001). The peak LHRH responses (mean +/- SEM) to NPY at different concentrations were: 10(-8) M = 2.1 +/- 0.4 pg/ml; 10(-7) M = 2.6 +/- 0.5 pg/ml; 10(-6) M = 6.5 +/- 1.1 pg/ml; 10(-5) M = 15.1 +/- 2.9 pg/ml, whereas to vehicle 0.37 +/- 0.17 pg/ml. All NPY doses tested were significantly effective as compared to vehicle (P less than 0.01). The LHRH response to 10(-6) M was greater (P less than 0.01) than that of 10(-8) M or 10(-7) M, and the response to 10(-5) M was greater (P less than 0.01) than that of all lower doses. The results indicate that NPY infusion into the S-ME elicits the release of LHRH in vivo in a dose-dependent manner in the monkey. The data further suggest that LHRH neurons and/or neuroterminals in the monkey are responsive to NPY stimulation in the absence of gonadal steroids. It is concluded that in addition to NE, NPY is an important regulator of pulsatile LHRH release in the nonhuman primate.     

Evidence for alpha 1-adrenergic involvement in neuropeptide Y-stimulated GnRH release in female rabbits.

Both neuropeptide Y (NPY) and norepinephrine stimulate gonadotropin-releasing hormone (GnRH) secretion in intact or ovariectomized (OVx) estradiol-treated rabbits. The mechanism by which NPY stimulates GnRH is currently unknown. We have tested the hypothesis that NPY increases GnRH release via an alpha-adrenergic pathway. Adult female rabbits were OVx and had Silastic capsules containing 17 beta-estradiol inserted subcutaneously that maintained plasma estradiol levels similar to those in ovarian intact rabbits. One week later, push-pull (PP) perfusion cannulae, with tips positioned in the mediobasal hypothalamus (MBH), and jugular vein catheters were placed in all does. Blood and PP perfusate samples were obtained every 20 min during 7 h perfusion of the MBH with Krebs-Ringer phosphate buffer (KRP). Intrahypothalamic treatment with NPY (n = 5), prazosin (alpha 1-adrenergic antagonist; n = 7), yohimbine (alpha 2-adrenergic antagonist; n = 7), NPY plus prazosin (n = 7) or NPY plus yohimbine (n = 6) dissolved in KRP occurred during hours 4 through 6. GnRH in hypothalamic perfusate and luteinizing hormone (LH) and prolactin (PRL) in peripheral plasma were measured by specific radioimmunoassays. As anticipated, NPY alone significantly increased MBH-GnRH secretion (0.93 +/- 0.24 vs. 2.46 +/- 0.37 pg/ml; p less than 0.05). In contrast, NPY infused concomitantly with prazosin did not increase MBH-GnRH release (1.26 +/- 0.50 vs. 0.78 +/- 0.19 pg/ml; p greater than 0.05) whereas NPY plus yohimbine did stimulate GnRH secretion (1.15 +/- 0.13 vs. 2.65 +/- 0.89 pg/ml; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

A model for combined morphological and functional investigations on the isolated mediobasal rat hypothalamus

We have developed a model for combined morphological and functional in vitro studies of the isolated mediobasal hypothalamus (MBH) by considering two prerequisites: (1) the tissue must be well preserved, free of morphological artefacts and functionally unimpaired until the end of the in vitro incubation, and (2) the tissue must be processed for morphology in optimal conditions. To test our model we have studied some aspects of the luteinizing hormone-releasing hormone (LHRH) system in 4-month-old male Sprague-Dawley rats. After decapitation the MBH was isolated and put in a flask containing 0.5 ml Hepes-buffered Locke's medium gassed by 5 ml/min of O2/CO2 (95%/5%) and shaken in a water bath at 37 degrees C. After a 10-min washing, the medium was changed twice at an interval of 20 min. After the in vitro incubation the tissue was satisfactorily preserved as judged by light- and electron-microscopic analysis. LHRH, somatostatin and thyrotropin-releasing hormone could be demonstrated by alkaline phosphatase or peroxidase-antiperoxidase immunohistochemistry on semithin sections and by immunogold technique on thin sections. The LHRH secretion was close to basal values after 30 min of incubation (22.1 +/- 4.8 pg/MBH) and then remained constant for another period of 20 min (17.6 +/- 2.6 pg/MBH). During the second 20 min of incubation LHRH secretion increased in presence of 61.6 mM K+ (110.7 +/- 8.7 pg/MBH). Thus the isolated hypothalamus was excitable until the end of the in vitro incubation. We conclude that this model can be successfully used for combined morphological and functional studies.     

Inhibition of sympathoadrenal activity by atrial natriuretic factor in dogs

In six conscious, trained dogs, maintained on a normal sodium intake of 2 to 4 mEq/kg/day, sympathetic activity was assessed as the release rate of norepinephrine and epinephrine during 15-minute i.v. infusions of human alpha-atrial natriuretic factor. Mean arterial pressure (as a percentage of control +/- SEM) during randomized infusions of 0.03, 0.1, 0.3, or 1.0 microgram/kg/min was 99 +/- 1, 95 +/- 1 (p less than 0.05), 93 +/- 1 (p less than 0.01), or 79 +/- 6% (p less than 0.001), respectively, but no tachycardia and no augmentation of the norepinephrine release rate (up to 0.3 microgram/kg/min) were observed, which is in contrast to comparable hypotension induced by hydralazine or nitroglycerin. The release rate of epinephrine (control, 6.7 +/- 0.6 ng/kg/min) declined immediately during infusions of atrial natriuretic factor to a minimum of 49 +/- 5% of control (p less than 0.001) during 0.1 microgram/kg/min and to 63 +/- 5% (0.1 greater than p greater than 0.05) or 95 +/- 13% (not significant) during 0.3 or 1.0 microgram/kg/min. Steady state arterial plasma concentrations of atrial natriuretic factor were 39 +/- 10 pg/ml (n = 6) during infusions of saline and 284 +/- 24 pg/ml (n = 6) and 1520 +/- 300 pg/ml (n = 9) during 0.03 and 0.1 microgram/kg/min infusions of the factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y is a neuromodulator of pulsatile luteinizing hormone-releasing hormone release in the gonadectomized rhesus monkey.

In a previous study, we have demonstrated that infusion of neuropeptide Y (NPY) into the stalk-median eminence (S-ME) of gonadectomized rhesus monkeys stimulated LHRH in a dose-dependent manner. This finding led us to address the following questions: 1) What are the characteristics of NPY release in vivo? 2) How does NPY release relate to LHRH release? 3) Is endogenous NPY essential to pulsatile LHRH release? To answer these questions, three experiments using push-pull perfusion were performed in adult gonadectomized rhesus monkeys. Perfusate samples from the S-ME were collected at 10-min intervals for 6 to 12-h periods, and the concentrations of LHRH and NPY in perfusates were determined by RIA. In Exp I, the release pattern of NPY and LHRH in the S-ME was independently determined in a group of 11 conscious monkeys: NPY release in the S-ME was pulsatile with an interpulse interval of 44.9 +/- 3.3 min (n = 11). This interpulse interval was similar to that seen for LHRH release (43.8 +/- 1.1 min, n = 7). Exp II was designed to determine whether NPY pulses and LHRH pulses occur synchronously and to examine whether NPY release in the S-ME is correlated with circulating LH pulses. NPY and LHRH concentrations in aliquots of the same perfusate sample from the S-ME and circulating LH levels were concurrently measured in 8 monkeys sedated with Saffan. It was found that NPY pulses were temporally correlated (P less than 0.001) with LHRH pulses, which were also temporally correlated (P less than 0.001) with LH pulses. Moreover, NPY pulses were correlated (P less than 0.05) with LH pulses. NPY peaks preceded LHRH peaks by 4.5 +/- 0.6 min, LHRH peaks preceded LH peaks by 5.5 +/- 0.6 min, and NPY peaks preceded LH peaks by 9.7 +/- 0.8 min. In Exp III, the role of endogenous NPY in LHRH release was evaluated by infusing a specific antiserum to NPY into the S-ME during push-pull perfusion in 8 conscious monkeys. Infusion of a specific antiserum to NPY into the S-ME at 1:100 and 1:1000 dilutions suppressed pulsatile LHRH release significantly (P less than 0.05). Infusion of nonimmune serum as a control was without effect. These results are summarized as follows: 1) NPY release in the S-ME is pulsatile, 2) NPY pulses occur synchronously with LHRH and LH pulses, and 3) immunoneutralization of endogenous NPY in the S-ME suppresses pulsatile LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship between luteinizing hormone releasing hormone concentration in hypophysial portal blood and luteinizing hormone release in intact, castrated, and electrochemically-stimulated rats.

The concentration of luteinizing hormone releasing hormone (LHRH) in hypophysial portal plasma was determined in pentobarbital anesthetized,intact and castrated rats of both sexes, including proestrous rats following electrochemical stimulation of the medial preoptic area (MPOA). Mean LHRH levels in portal plasma obtained between 1400--1700 h from estrous and diestrous rats and from rats ovariectomized for 8 weeks were similar and ranged from 50--55 pg/ml, but the LHRH levels in proestrous rats were less than 12 pg/ml. In addition, hypophysial portal plasma collected during 1100 to 1400 h from animals orchidectomized for 8 weeks and from intact male rats contained mean LHRH concentrations that ranged from 50--65 pg/ml and 30--35 pg/ml, respectively. Electrochemical stimulation of the MPOA in the female rat on the afternoon of proestrus resulted in a marked increase in the concentration of LHRH in portal plasma. LHRH levels in portal plasma during the 0 to 30, 30 to 60, 60 to 90, 90 to 120, and 120 to 150-min periods after electrochemical stimulation of the MPOA were 105 +/- 24.2, 61 +/- 10.8, 51 +/- 8.2, 36 +/- 5.3, and 32 +/- 4.1 pg/ml, respectively. LHRH levels in portal plasma from the unstimulated rats were not detectable (less than 12 pg/ml) in most of the animals. In another group of proestrous rats, the effect of rabbit anti-LHRH serum or normal rabbit serum (NRS) on the release of LH after electrochemical stimulation of MPOA was examined. Pretreatment of proestrous rats with anti-LHRH serum blocked the release of LH due to MPOA stimulation, whereas pretreatment with NRS did not inhibit LH release. On the basis of these findings, it is concluded that electro-chemical stimulation of the MPOA in proestrous rats increases LHRH levels in portal blood and that the enhanced secretion of LHRH stimulates the release of LH from the pituitary gland.     

Release of luteinizing hormone-releasing hormone, beta-endorphin and noradrenaline by the nucleus infundibularis/median eminence during periovulatory period in the sheep.

The relationships between the release of LHRH, beta-endorphin (beta-END) and noradrenaline (NA) from the hypothalamic infundibular nuclei/median eminence (NI/ME) during the periovulatory period in the ewe was studied. Neurohormone release was assayed in perfusates collected from the NI/ME via push-pull cannulae. LHRH concentrations in perfusates ranged from below detectable values (5 pg) to 50 pg and from 15 to 240 pg/20 min perfusate on the days of proestrus and estrus, respectively. beta-END concentrations in perfusates ranged from 320 to 6,000 pg on the day of proestrus and fell to a range between 100 and 380 pg/20 min perfusate on the day of estrus. The NA content of perfusates ranged from an undetectable level to 0.9 x 10(4) pg/perfusate during proestrus, and rose from 1.0 x 10(4) to 6.6 x 10(4) pg/perfusate shortly before the preovulatory release of LHRH and LH. On the basis of the present observations, the following sequence of events leading to the massive LH ovulatory surge in the sheep is suggested: (1) increased secretion of beta-END in the NI/ME on the day of proestrus generates an increase in the releasable pool of LHRH through inhibition of LHRH release; (2) on the day of estrus a decreased release of beta-END allows the expression of NA activity in the NI/ME and the augmentation of NA tone facilitates the release of newly accumulated LHRH; (3) the resultant intensified LHRH output with its significantly changing pattern of release triggers the preovulatory surge of LH.