No changes in rat benzodiazepine receptors after withdrawal from continuous treatment with lorazepam and diazepam.

Five and 11 days after withdrawal from 8 weeks of treatment with 90 mg/kg/day of diazepam p.o. or 60 mg/kg/day of lorazepam p.o. there were no consistent changes in the number of benzodiazepine receptors or apparent affinity $\text{in vitro}$ for ³H-diazepam at 0°C in rat forebrain membranes. Daily exposure of rats from 10 days before birth until 7 days after birth was also without gross effects on the benzodiazepine receptor. Abstinence and tolerance to benzodiazepines were thus not attributable to changes in brain benzodiazepine receptors.     

Luteinizing hormone (LH) release after single injections of a synthetic LH-releasing hormone (LH-RH) in the ewe at three different reproductive stages and comparison with natural LH release at oestrus

The possibility was investigated of using single i.v. injections of a synthetic luteinizing hormone-releasing hormone (LH-RH) to manipulate the reproductive pattern of the ewe.Single i.v. injections of 150 μg synthetic LH-RH were given on Day 12 of the oestrous cycle, during seasonal anoestrus and on Day 16 $\text{post}$-$\text{partum}$ in ewes which lambed during the breeding season. Blood samples were obtained at 5-, 10- or 15-minute intervals for 1 hour before and for 3 hours after treatment. Plasma LH concentrations were measured using a specific double antibody radioimmunoassay, the development of which is described. Laparotomy was performed on each animal 2–3 days after treatment.The treatment induced LH peaks in all animals and ovulation in the majority. There was no significant difference between the groups in the LH response. The LH release was, however, much less than that found in untreated ewes sampled every 15 minutes for 18 hours during oestrus.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Release of LH after administration of an analog of synthetic LH-RH in cattle

The $\text{in vivo}$ effect of an analog of LH-RH, (Des-Gly-NH₂¹⁰, Proethylamide⁹)-LH-RH, on LH release, was studied in one bull with azooapermia, and one cow with cystic follicle. The single intravenous injection of 100 μg LH-RH analog caused abrupt and marked increase in serum LH and the high levels of LH were maintained for 3 hours. The highest levels of serum LH were 48 and 114 times as high as the pre-treatment levels in a bull and a cow, respectively. Further studies are being undertaken to evaluate this compound for improving reproductive function in cattle.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

Antagonism of benzodiazepine receptors by beta carbolines

A number of beta carbolines were found to interact at brain benzodiazepine sites $\text{in vitro}$. The compounds we evaluated had affinities in the low nanomolar range and had Hill slopes less than unity. In addition to being potential antagonists at benzodiazepine receptors, they may specifically label the BZ₁ site. After intravenous administration, these compounds antagonized benzodiazepine actions in the cat spinal cord and in a mouse free behavioral model. Aspects of this antagonism and their proposed role as endogenous benzodiazepine ligands in brain are discussed.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

The nature of the pituitary gonadotropins and their role in ovulation in a urodele amphibian (Ambystoma tigrinum).

Gonadotropic hormones were fractionated from pituitaries of the urodele amphibian $\text{Ambystoma}$$\text{tigrinum}$ (tiger salamander) by methods previously employed to separate these hormones in other species of tetrapod vertebrates. These procedures yielded two distinct fractions that resembled the FSH and LH from other species in regard to their biological profiles in several nonmammalian bioassays and by their chromatographic behavior. The $\text{Ambystoma}$-FSH was free of LH activity (< 0.1%). The major $\text{Ambystoma}$-LH was highly potent in ovulation assays for LH; it also had a high activity in the $\text{Anolis}$ lizard assay, but it is not clear whether this reflects high intrinsic activity or incomplete separation of FSH.$\text{In vitro}$ studies with these and other (frog, turtle, mammalian) gonadotropins indicate that the induction of ovulation in $\text{Ambystoma}$, as in anurans, is highly specific for LH, independent of the source of the gonadotropins. These data support the view that two separate gonadotropins existed early in tetrapod evolution.     

Regulation of steroidogenesis in the ovine corpus luteum

To examine the factor affecting LH-induced progesterone production $\text{in}$$\text{vitro}$ in ovine luteal slices, an experimental procedure was employed wherein each slice served as its own control. The role of microfilaments in steroidogenesis was studied in luteal slices treated with cytochalasin B (an inhibitor of microfilament function). Cytochalasin B treatment resulted in significant reduction of progesterone production by luteal slices in response to LH and the addition of serum to the medium did not alter this effect. The ability of luteal slices to respond to LH with increased progesterone secretion was restored when cytochalasin B was removed from the medium. Further studies indicated that inhibition of LH-induced progesterone production by treatment with cytochalasin B was not a result of a change in: 1) cyclic adenosine 3'-5'-monophosphate production in response to LH; 2) mitochondrial membrane permeability to cholesterol; or 3) activity of 3β-hydroxysteroid dehydrogenase, Δ⁵,Δ⁴-isomerase enzyme complex.The possibility existed that microfilaments were necessary for cholesterol transport to mitochondria in response to LH stimulation. However, mitochondrial cholesterol content was unchanged in response to LH in the presence or absence of aminoglutethimide (an inhibitor of cholesterol side-chain cleavage enzyme activity) as determined by uptake of ³H-cholesterol or total content determined by gas-liguid chromatography. Further, treatment with cytochalasin B had no effect on mitochondrial cholesterol content. These results suggest a role for microfilaments in LH-induced progesterone production at a point prior to the conversion of cholesterol to pregnenolone.     

Induction of fertile oestrus in seasonally anoestrous ewes with low doses of Gn-RH

Oestrus, conception and lambing performance were assessed in progesterone-primed seasonally anoestrous ewes induced to ovulate with gonadotrophin-releasing hormone (Gn-RH), which was administered intravenously for 48 h as either injections of 250 ng at 2-h intervals (n = 15) or as a continuous infusion at the rate of 125 ng/h (n = 12) or 250 ng/h (n = 12).In $\text{14}\text{15}$ of the ewes injected with Gn-RH, a preovulatory LH peak was recorded at a mean time interval of 33.9 ± 1.8 h after the start of treatment. All ewes displayed oestrus and all ovulated, with a mean ovulation rate of 1.67 ± 0.13. Eleven ewes were diagnosed as pregnant and subsequently lambed. Following infusion of Gn-RH, preovulatory LH peaks were recorded in $\text{21}\text{24}$ ewes at a mean time of 36.1 ± 2.9 h (125 ng/h) and 34.7 ± 2.0 h (250 ng/h). All but two of the ewes displayed oestrus and $\text{23}\text{24}$ ovulated. The group mean ovulation rates of 1.27 ± 0.14 (125 ng/h) and 1.75 ± 0.22 (250 ng/h) were not significantly different. Eleven of the 22 ewes mated were diagnosed as pregnant and produced live lambs.These results suggest that fertility of Gn-RH-induced ovulations in seasonally anoestrous ewes is comparable to that apparent in ewes ovulating spontaneously during the breeding season.     

Reproductive studies of Brahman cattle V. The effect of various dose levels of estradiol-17β upon serum luteinizing hormone in ovariectomized Brahman cows

Twelve mature chronically-ovariectomized Brahman cows were randomly assigned to receive three of six different dosages of estradiol-17_b (E2) at three different time periods such that at the termination of the trial six animals received each E2 dosage. The E2 was suspended in 0, 1, 2.5, 5, 10 and 20 milligrams. A two week period was maintained between injections. The cows were bled via coccygeal vessel puncture immediately before E2 injection, every 2 hr from 0 to 18 hr, every hr from 18 to 42 hr and every 2 hr from 42 to 48 hr postinjection. Blood was processed to yield serum and stored at ?20 Celsius. Serum luteinizing hormone (LH) was quantitated by validated radioimmunoassay. An LH surge was defined as a sustained elevation of LH at least two standard deviations above the level of LH prior to the rise and was observed in $\text{0}\text{6}$, $\text{3}\text{6}$, $\text{5}\text{6}$, $\text{5}\text{6}$, $\text{5}\text{6}$, and $\text{6}\text{6}$ cows administered 0, 1, 2.5, 5, 10, and 20 mg of E2, respectively. All animals injected with E2 responded with a significant initial decrease (independent of E2 dosage) in LH that persisted from 2 through 12 hr post E2 injection. No significant decrease in LH levels was recorded in animals injected with the corn oil vehicle. The time to the LH surge differed (P<.05) between 1 mg E2 (10 hr) vs 20 mg E2 (19.5 hr), 1 mg E2 vs 10 mg E2 (16.2 hr), and 2.5 mg E2 (12.4 hr) vs 20 mg estradiol-17_β. Luteinizing hormone concentrations at the onset of the surge did not differ (P>.10) between E2 dosages. The elapsed time from E2 injection to the peak LH value differed (P<.05) between 1 mg E2 (20.3 hr) vs 20 mg E2 (26.8 hr), and 2.5 mg E2 (21.2 hr) vs 20 mg estradiol-17_β. The peak LH value, the area under the LH curve and the duration of the LH surge did not differ (P>.10) with E2 dosage. The time to the end of the LH surge differed (P<.05) between 1 mg E2 (25.3 hr) vs 2.5 mg E2 (31.6 hr), 1 mg E2 vs 5 mg (34.4 hr), 1 mg E2 vs 10 mg E2 (34.8 hr), 1 mg E2 vs 20 mg E2 (37.3 hr), and 2.5 mg E2 vs 20 mg estradiol-17_β. Luteinizing hormone values at the termination of the surge did not differ (P>.10) between dosages nor did the LH values at the termination of the surge differ (P>.10) from LH concentrations observed at the onset of the LH surge.     

Avermectin B1a: an irreversible activator of the gamma-aminobutyric acid-benzodiazepine-chloride-ionophore receptor complex

Avermectin B_1a, an antihelminthic macrocyclic lactone, has been previously shown to reduce muscle membrane resistance by stimulating γ-aminobutyric acid-mediated chloride conductance. Since the benzodiazepine receptor is coupled to a receptor for γ-aminobutyric acid and related chloride ionophore, the effects of Avermectin B_1a on [³H]diazepam binding to the benzodiazepine receptor were studied. In well-washed membrane fragments from rat cerebral cortex, Avermectin B_1a markedly increased the binding of [³H]diazepam to benzodiazepine receptors. This effect was qualitatively similar to that observed with either γ-aminobutyric acid or chloride ion and was partially reversed by the γ-aminobutyric acid receptor antagonist, bicuculline. In contrast to the effects of γ-aminobutyric acid and chloride, the enhanced binding of [³H]benzodiazepine elicited by Avermectin B_1a was $\text{not}$ reversed by extensive washing of the membrane preparation. Avermectin B_1a appears to irreversibly modify benzodiazepine receptors at a γ-aminobutyric acid-chloride recognition site and may be valuable in biochemical studies of the regulation of benzodiazepine receptor function.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p > 0.10). PRL levels in ovariectomized rats in which the NIL had been removed surgically (NIL-X) or only visualized (NIL-C) were 3–7 ng/ml 4, 7, and 28 days after surgery (p > 0.10); however, they were slightly higher in NIL-X $\text{vs.}$ NIL-C rats 14 days after surgery (p < 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p < 0.05); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p < 0.05). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either $\text{via}$ secreting hypophysiotropic hormones or $\text{via}$ conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.