Failure of neurotransmitter blockers to alter PGE2-induced LH release.

The purpose of this study is to ascertain whether or not prostaglandin (PG) E2 induces LH release by modifying or modulating the release or action of neural transmitters. PGE2 injected iv into spayed rats primed two days earlier with 10 microgram estradiol benzoate increased the plasma levels of LH 10 min later as measured by radioimmunoassay. The peak of plasma LH was not changed by prior treatment with beta- or alpha-adrenergic receptor blockers, propranolol or phenoxybenzamine. The peak level of plasma LH did not alter in rats treated with DL-alpha-methyl-p-tyrosine methyl ester HCl (alpha-MPT) or sodium diethyldithiocarbamate (DDC). Similarly, the peak of plasma LH was not changed by prior treatment with imipramine. Administration of PGE2 produced an increase in anterior pituitary and plasma, but not hypothalamic cyclic AMP concomitantly with the elevation in plasma LH. Although it is possible that the effect of PGE2 could be mediated by another transmitter system, as yet unknown, or that the effect of PGE2 on LH release could be mediated via the adenylate cyclase-cyclic AMP system, the results indicate that PGE2 does not act trans-synaptically, but probably acts directly on LH-RH neurons.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration.

Five min following a single iv injection of PGE2 into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE2 injection. Plasma LH le-el increased at 2 min after PGE2 and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland.

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration

Five min following a single iv injection of PGE₂ into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE₂ injection. Plasma LH level increased at 2 min after PGE₂ and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Effects of microinjection of various prostaglandins into the 3rd ventricle, median eminence and pituitary on plasma LH in rats.

The effects of microinjection of PG'S (PGE1, E2, F2a) into the 3rd ventricle, median eminence (ME) and anterior pituitary on plasma LH in rats were investigated. Blood samples were obtained by jugular puncture before, and 10 and 45 min after the injection of PGS (50 or 100 mug), plasma LH was measured by radioimmunoassay. In the 3rd ventricle microinjection, PGE2 prodiced a significant rise in plasma LH. PGE1 and F2a did not significantly after plasma LH levels. In the median eminence, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. In the pituitary, PGE2 and E1 produced a significant rise in plasma LH. PGF2a did not alter plasma LH levels. These observations indicate that PGs act directly on the hypothalamic-pituitary axis, and that particular PG may be involved in the release of particular horomones from the hypothalamus and pituitary.     

Site of the in vivo stimulatory effect of prostaglandins on LH release

A possible direct effect of prostaglandins E₁ and E₂ (PGE₁ and PGE₂) on luteinizing hormone (LH) release at the pituitary level was studied using anterior pituitary cells in primary culture, a system approximately 10-fold more sensitive to stimulation of LH release than previously used hemipituitaries. No effect of PGE₁ or PGE₂ could be detected on the time course of basal or LH-RH-stimulated LH release or on the LH responsiveness to LH-RH. This absence of a direct effect of PGEs at the pituitary level on LH release was confirmed by experiments using female rats under Surital anesthesia in the afternoon of proestrus. After intravenous injection, under these conditions, 15(S)-15-methyl PGE₂ was 3–5 times more potent than PGE₂ to increase plasma LH levels while PGE₁ had about 50% the potency of PGE₂. Injection of sheep anti-LH-RH serum one hour before PGE₁ or PGE₂ injection not only lowered basal plasma LH levels but prevented the rise induced by PGEs. These data indicate clearly that the increased plasma LH levels observed after PGE injection are secondary to a stimulation of LH-RH release while PGEs do not appear to have a significant effect on LH release at the pituitary level.     

The influence of prostaglandin E2 and indomethacin on progesterone production and ovulation in the rabbit ovary perfused in vitro

The effects of prostaglandin E2 (PGE2) on the ovulation process were studied in a recirculating perfusion model using ovaries from virgin rabbits. Ovulation frequency, time of ovulation, and progesterone release from the ovaries were examined after the addition of PGE2, either alone or with luteinizing hormone (LH) in the presence or absence of indomethacin. The stimulatory effect of LH on ovulation was totally blocked if the ovaries were exposed to indomethacin at the same time. Ovaries treated with PGE2 alone did not ovulate, and PGE2 was unable to restore indomethacin-blocked ovulation. Conversely, the frequency of ovulation was reduced in ovaries treated with PGE2 and LH compared with controls receiving only LH. There was no measurable difference in the pattern of progesterone release between ovaries simultaneously treated with LH and indomethacin and LH-treated controls. Ovaries exposed to PGE2 alone showed only a slight increase of progesterone release in the medium, while those treated with PGE2 in combination with LH in the perfusate showed a smaller progesterone release than those treated with LH alone. The results confirm the blocking effect on ovulation by indomethacin. PGE2 could not reverse this effect, but instead reduced the number of LH-induced follicular ruptures. Indomethacin had no effect on progesterone levels, while PGE2 (which alone showed a slight stimulating effect on the steroid concentration) together with LH counteracted the effect of LH on progesterone release.     

Stimulation by cyclic nucleotides of prostaglandin E production in isolated graafian follicles.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5' AMP and 8-Br-5' GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-l-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors. Administration of hCG in vivo or incubation with LH in vitro did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG. Collectively, the results suggest that stimulation of ovarian PGE production by cyclic nucleotides and LH is dependent on de novo synthesis of one or more components of the PG synthetase system rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production.     

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.     

Role of arginine vasopressin in control of ACTH and LH release during stress

To determine the role of arginine vasopressin (AVP) in stress-induced release of anterior pituitary hormones, AVP antiserum or normal rabbit serum (NRS) was micro-injected into the 3rd ventricle of freely-moving, ovariectomized (OVX) female rats. A single 3 microliter injection was given, and 24 hours later, the injection was repeated 30 min prior to application of ether stress for 1 min. Although AVP antiserum had no effect on basal plasma ACTH concentrations, the elevation of plasma ACTH induced by ether stress was lowered significantly. Plasma LH tended to increase following ether stress but not significantly so; however, plasma LH following stress was significantly lower in the AVP antiserum-treated group than in the group pre-treated with NRS. Ether stress lowered plasma growth hormone (GH) levels and this lowering was slightly but significantly antagonized by AVP antiserum. Ether stress also elevated plasma prolactin (Prl) levels but these changes were not significantly modified by the antiserum. To evaluate any direct action of AVP on pituitary hormone secretion, the peptide was incubated with dispersed anterior pituitary cells for 2 hours. A dose-related release of ACTH occurred in doses ranging from 10 ng (10 p mole)-10 micrograms/tube, but there was no effect of AVP on release of LH. The release of other anterior pituitary hormones was also not affected except for a significant stimulation of TSH release at a high dose of AVP. The results indicate that AVP is involved in induction of ACTH and LH release during stress. The inhibitory action of the AVP antiserum on ACTH release may be mediated intrahypothalamically by blocking the stimulatory action of AVP on corticotropin-releasing factor (CRF) neurons and/or also in part by direct blockade of the stimulatory action of vasopressin on the pituitary. The effects of vasopressin on LH release are presumably brought about by blockade of a stimulatory action of AVP on the LHRH neuronal terminals.     

Preovulatory changes in ovarian cyclic AMP and prostaglandins in immature rats injected with PMSG.

In 28-day-old rats injected with PMSG, ovarian cyclic AMP and PGE and PGF increased 2 and 3-4 h respectively after the LH peak.     

Prostacyclin (PGI2) effects on anterior pituitary hormones in the rat in vivo.

Intravenous injection of 600 microgram PGE2 or PGI2 significantly increased serum LH and prolactin levels in estradiol treated ovariectomized rats. There was no effect on serum FSH concentration. PGE2 and PGI2 stimulated LH release in a non-dose dependent manner, while prolactin levels were positively correlated with the dose administered following PGI2 treatment. 6-keto-PGF1 alpha at a comparable dose had no effect on pituitary hormone levels. Subcutaneous administration of 1 mg/kg or 60 mg/kg PGI2 for seven days significantly depressed serum LH level both in male and female rats. These doses had no effect on serum FSH or prolactin levels.     

Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen, progesterone-treated rats

We studied the effects on plasma LH levels of intracerebroventricular (ICV) administration of neuropeptide Y (NPY), NPY analog (NPY-A), galanin (GAL) and neuropeptide K (NPK) in ovariectomized (ovx) and in ovx rats pretreated with estradiol benzoate (EB) and progesterone (P). Plasma LH levels were estimated in blood drawn from an intrajugular cannula before (0 min) and at 10, 20, 30 and 60 min after the ICV injection of either saline (3 microliter) or one of the neuropeptides in saline. The three classes of peptides elicited different LH responses in the two experimental paradigms. NPY and NPY-A (0.5 or 2 micrograms) decreased LH release in ovx rats and stimulated LH release in EBP ovx rats. However, GAL (0.5, 2 or 10 micrograms) failed to suppress LH release in ovx rats, but it readily increased plasma LH levels in a dose-related fashion in EBP ovx rats. In contrast, NPK readily decreased LH release in ovx rats in a time-related fashion for up to 60 min, but was mildly effective in EBP ovx rats as only a high dose of 10 micrograms produced a small significant increase. Collectively, our results show that (1) NPY can differentially effect LH release in ovx and EBP ovx rats but this property is not equally shared by the neuropeptides that have a similar anatomical disposition in the hypothalamus and (2) the excitatory effects of GAL are demonstrable in the steroid-primed rats and the inhibitory effects of NPK are apparent in the steroid-unprimed ovx rats. Since NPK induced a long-lasting marked suppression with little evidence of LH excitation at low doses, we speculate that either NPK alone or in conjunction with other peptides may mediate the suppression of LH release induced by gonadal steroids.     

Studies on factors affecting release of gonadotrophins during the superfusion of isolated rat pituitaries.

Isolated pituitary glands from adult male rats were maintained in a continuous flow system. Gn-RH (1000 pmol/ml) caused a characteristic release of cyclic AMP, LH, and FSH. Cyclic AMP (1000 nmol/ml) liberated a similar amount of both gonadotrophins. Theophylline (1 mmol/ml) enhanced the effect of cyclic AMP by 21% for LH and 41% for FSH. The infusion of oestradiol (184 pmol/ml) alone or before Gn-RH infusion did not produce a significant effect on the secretion of either gonadotrophin or cyclic AMP. In contrast, there was a significant reduction in the amount of LH (P less than 0-025) and FSH (P less than 0-05) released by pituitaries infused with oestradiol and cyclic AMP.     

Cyclic response of hypophysectomized rats to ovulation induced by LHRH agonist: mediation by prostaglandins

Further confirmation that the LHRH/LHRH agonist-induced ovulation in the hypophysectomized (hypx) rat is due to a direct ovarian effect and not mediated by LH release from residual pituitary tissue or other CNS sites is provided by the persistence of this effect despite concomitant median eminence lesion or passive immunization to LH. Adrenalectomy did not affect the ovulatory activity of the LHRH agonist, D-Trp6-N alpha MeLeu7-DesGly10-Pro9-NHEt-LHRH (Wy-40,972), in the hypx rat. Prior administration of a potent LHRH antagonist blocked ovulation induced in hypx proestrous rats by Wy-40,972 but not by LH-S19. Ovulation can be induced by Wy-40,972 one day earlier (e.g. metestrus) in the intact rat than it can in the hypx rat. Results in the hypx metestrous rat indicate that the ovulatory responsiveness of the intact rat at this stage of the cycle may occur by complementary action of Wy-40,972-stimulated endogenous LH release and a direct ovarian effect of the agonist. Prostaglandins (PG) are involved in the ovulatory mechanism of Wy-40,972 in the hypx proestrous rat as evidenced by the dose-dependent inhibition of this effect by PG synthetase inhibitors, indomethacin and Fentiazac. Moreover, there were significant increases in ovarian concentrations of PGF2 alpha and PGE2-PGE1 in response to Wy-40,972 that could be prevented by indomethacin. However, exogenous administration of either of these PG's was not effective in inducing ovulation in the hypx rat.     

Role of ovarian secretion in the development of dysfunction in the regulation of luteinizing hormone secretion in female rats exposed to constant illumination.

Female rats in constant illumination (LL) fail to show the facilitation of LH release following steroid administration that is characteristic of animals in normal lighting. To determine whether this effect is mediated through changes in ovarian function, rats were spayed either at the time of placement into different lighting schedules (LL or a 14:10 light-dark (LD) schedule) or 10 weeks later, and their plasma LH responses to steroids were compared after an additional 3-week exposure to the experimental lighting conditions. To test the LH response, estradiol benzoate (EB) was injected at 12.00 h and followed 72 h later by injection of progesterone (P) or a second injection of EB. Neither steroid regime revealed differences in LH release between animals ovariectomized at the time of placement into LL and those spayed 10 weeks later. The duration of castration in animals in LD affected the LH response to a priming dose of EB, but not to a second dose of EB or to P. It is concluded that altered ovarian activity is not the factor which mediates the loss of a facilitatory response of LH release following administration of gonadal steroids to rats under constant illumination.     

Gonadotropin release as a function of mating and steroid feedback in the female rat

The aim of this investigation was to study possible relationships between mating-induced and steroid-induced luteinizing hormone (LH) release in spayed Long-Evans rats. Large amounts of LH were released approximately 7 hr following progesterone injection in rats primed with estradiol benzoate (EB). The amount of LH release varied widely depending on (1) the interval between the time of the progesterone injection and the EB priming; (2) the progesterone dose; and (3) the time of day when blood samples were collected. These findings provided confirmation of those of Caligaris, Astrada and Taleisnik (1971a). Females, prepared with estrogen-progesterone treatment in a variety of schedules in which the three above-mentioned variables were altered systematically, were allowed to mate with vigorous males. Mating under these various conditions did not significantly increase plasma LH levels even when the females showed high degrees of sexual receptivity. Sodium pentobarbital prevented the afternoon LH rise resulting from progesterone treatment 3 days after EB priming. Pituitary sensitivity to LRF was not enhanced in the afternoon and the mating did not significantly increase plasma LH in these barbiturate-blocked rats. Following administration of 5 large daily doses of EB without progesterone, however, significant increases in LH were produced by mating on the sixth day. Postcopulatory LH release in these circumstances was dependent on a diurnal factor since the effect of mating was greater in the afternoon than in the morning. Thus, although major LH release can be readily induced by mating in estrogen-treated spayed rats, this effect could not be obtained under conditions of progesterone administration to estrogen primed rats.     

Arachidonic acid metabolism in isolated pancreatic islets. VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release.

Pancreatic islets stimulated with D-glucose are known to liberate arachidonic acid from membrane phospholipids and release prostaglandin E2 (PGE2). A component of the eicosanoid release induced by D-glucose has been demonstrated to occur without calcium influx and must be triggered by other coupling mechanisms. In this study, we have attempted to identify mechanisms other than calcium influx which might couple D-glucose stimulation to hydrolysis of arachidonate from membrane phospholipids in islet cells. We have found that occupancy of the beta cell plasma membrane D-glucose transporter is insufficient and that D-glucose metabolism is required to induce islet PGE2 release because 3-O-methylglucose fails to induce and mannoheptulose prevents PGE2 release otherwise induced by 17 mM D-glucose. The carbohydrate insulin secretagogues mannose and D-glyceraldehyde have also been found to induce islet PGE2 release, but the non-secretagogue carbohydrates L-glucose and lactate do not. Carbohydrate secretagogues are known to be metabolized to yield ATP and induce depolarization of the beta cell plasma membrane. We have found that depolarization by 40 mM KCl induces PGE2 release only in the presence and not in the absence of extracellular calcium, but exogenous ATP induces islet PGE2 release with or without extracellular calcium. Carbachol is demonstrated here to interact synergistically with increasing concentrations of glucose to amplify PGE2 release and insulin secretion. Pertussis toxin treatment is shown here not to prevent PGE2 release induced by glucose or carbachol but to increase the basal rate of PGE2 release and the islet cyclic AMP content. Theophylline (10 mM) exerts similar effects. Eicosanoid release in pancreatic islets can thus be activated by multiple pathways including muscarinic receptor occupancy, calcium influx, increasing cAMP content, and a metabolic signal derived from nutrient secretagogues, such as ATP.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.