Effect of novel motilide ABT-229 versus erythromycin and cisapride on gastric emptying in dogs.

ABT-229 (8,9-anhydro-4"-deoxy-3'-N-desmethyl-3'-N-ethylerythromycin B-6,9-hemiacetal), a synthetic derivative of erythromycin (ERY) with no antibiotic activity, has been shown to bind to motilin receptors and stimulate contractile activity of the antrum and small intestine. The objective of this study was to determine the effect of ABT-229 on canine gastric emptying (GE) and contractile activity of the antrum and duodenum in response to a solid meal. Six beagles were used to determine GE of a solid meal and contractile activity in response to either vehicle, ABT-229 (0.17, 0.83, 2.5, or 5.0 microg/kg/min), ERY (33.3 microg/kg/min), or cisapride (CIS) (10 microg/kg/min). Lag (t(lag)), half-emptying (t(1/2)), and complete emptying (t(full)) times were determined. Contractile data were analyzed for motility index and gastroduodenal coordination. Compared with vehicle, ABT-229 dose dependently accelerated GE, t(lag) was decreased at the two highest doses, t(1/2) was decreased compared with vehicle at the three highest doses, and t(full) was decreased at all doses compared with vehicle. ERY also decreased t(1/2) and t(full), whereas CIS decreased all GE parameters. The slopes of the linear phase of GE curves for all drugs and doses were greater than those for vehicle. ABT-229 dose dependently increased the motility index as well as gastroduodenal coordination. ABT-229 (two highest doses) and CIS accelerated GE of a solid meal by decreasing the lag phase and increasing the rate of GE, whereas ERY only increased the rate of GE. The data suggest that ABT-229 is 7- to 40-fold more potent than ERY in accelerating GE.

Neuropeptide Y Y1-receptor stimulation is required for physiological amplification of preovulatory luteinizing hormone surges.

Neuropeptide Y (NPY) has been shown to potentiate the actions of LHRH during the generation of preovulatory LH surges. It is not yet known, however, if activation of a specific subtype of NPY receptors in the anterior pituitary gland is an obligatory event in the stimulation of spontaneous LH surges. A battery of NPY receptor agonists, as well as the specific NPY Y1 receptor antagonist BIBP3226, were used to assess the role of Y1 receptors in the amplification of LH surges. In Exp 1, the potencies of a number of NPY agonists in facilitating LHRH-induced LH surges were assessed in pentobarbital (PB)-blocked, proestrous rats. The rank-ordered potencies of these compounds were determined to be PYY = [Leu31Pro34]NPY > NPY >> hPP = rPP = NPY(13-36), which most closely reproduces the known rank-ordered affinties of these compounds for the Y1 receptor. In Exp 2, a Y1 subtype- specific antagonist, BIBP3226, was administered to unanesthetized, proestrous rats to assess the involvement of the Y1 receptor in the stimulation of spontaneous LH surges. The BIBP3226 compound strongly attenuated the endogenous proestrous LH surge, reducing the integrated value of LH secretion during the proestrous surge by more than 70%. In Exp 3, we assessed the ability of the Y1 receptor antagonist to block exogenous NPY effects on LHRH-induced LH surges. Treatment with BIBP3226 was found to completely prevent NPY amplification of LHRH-induced LH surges in pentobarbital-blocked, proestrous rats, thus confirming a pituitary locus of action of the drug. Taken together, these data clearly demonstrate that activation of neuropeptide Y receptors of the Y1 subtype is required for the physiological amplification of the spontaneous preovulatory LH surge in rats.

Pituitary follistatin regulates activin-mediated production of follicle-stimulating hormone during the rat estrous cycle.

Follistatin, an activin-binding protein, plays a key role in the modulation of activin-dependent functions. In the anterior pituitary, activin stimulates the synthesis and secretion of FSH. In the current study, we assessed the roles of locally produced activin and follistatin in the control of FSH gene expression and secretion. The anterior pituitary gland follistatin content was measured at frequent intervals during the rat estrous cycle. Follistatin protein levels were high before the primary gonadotropin surges, decreased by 50% on proestrous evening, and rebounded to a peak at midnight on proestrus before returning to presurge levels on estrus morning. Changes in pituitary follistatin protein content were preceded by parallel changes in pituitary follistatin messenger RNA (mRNA). The trough in follistatin protein content on proestrus coincided with a peak in circulating levels of free activin A (not bound to follistatin) and a sharp rise in FSHbeta mRNA levels, suggesting that decreased pituitary follistatin leads to increased available activin. To quantitate the contribution of pituitary free activin to pituitary expression of FSHbeta mRNA and the primary and secondary serum FSH surges, rats were infused through carotid catheters with saline or recombinant human follistatin (288-amino acid isoform; rhFS-288) at different times during the estrous cycle. Infusion of rhFS-288 on diestrus did not affect FSH production. In contrast, infusion of rhFS-288 during the secondary FSH surge decreased the peaks in FSHbeta mRNA and serum FSH by 63% and 47%, respectively, relative to those in saline-infused control animals. Infusion of rhFS-288 during the primary FSH surge decreased serum FSH to a lesser degree (24%). These data indicate a physiological role for pituitary follistatin in the control of activin-mediated FSH synthesis and secretion during the rat estrous cycle.

Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner.

Inhibin A and inhibin B are related dimeric protein hormones and endocrine regulators of the reproductive axis. Specifically, inhibin inhibits FSH secretion from the anterior pituitary. The inhibins are synthesized by the gonads and are themselves modulated by FSH. Although the activity of these ligands has been well characterized, the circulating concentrations of dimeric inhibin A and dimeric inhibin B have not previously been reported for the rat. Our group examined the serum concentration of inhibin A and inhibin B in normally cycling female rats, male rats, and in gonadectomized animals. Both inhibin isoforms are detected in intact female rat serum. Interestingly, inhibin B, but not inhibin A, is detected in intact male rat serum. Neither inhibin isoform is detected in long-term castrate female or male rats. In normally cycling female rats, inhibin A was low on the morning of metestrus and rose steadily to a peak on proestrus. In contrast, inhibin B was elevated on the mornings of metestrus, diestrus, and proestrus. Both ligands persisted in the serum until proestrus evening. Serum inhibins then declined beginning at 2100 h (inhibin A) or 1800 h (inhibin B) on proestrus, and the concentrations reached a nadir on the morning of estrus (0600 h). The nadir coincided with the peak of the secondary FSH surge. Both inhibins rebounded later on the morning of estrus. The results of this study demonstrate that dimeric, ovarian-derived inhibin A and inhibin B circulate in the female rat. The inverse relationship of the inhibins during the secondary FSH surge is consistent with the hypothesis that they participate in the regulation of reproductive cyclicity. The differing patterns of inhibin A and inhibin B during the period of follicular development on metestrus and diestrus suggest different follicle sources or regulation of these molecules during this period. We further demonstrate that inhibin B is the dominant form of FSH regulating protein in the male rat.

Gonadotropin-releasing hormone regulates follicle-stimulating hormone-beta gene expression through an activin/follistatin autocrine or paracrine loop.

The FSH beta gene is stimulated by low frequency pulses of GnRH, but is unaffected or suppressed when GnRH is applied at higher frequencies or continuously. The current studies explored the hypothesis that GnRH frequency-dependent regulation of FSH beta may be mediated by pituitary expression of activin, which stimulates FSH beta messenger RNA (mRNA), and follistatin, which blocks activin. Using a system of perifused male rat pituitary cells, a reciprocal relationship was observed between FSH beta and follistatin mRNAs in response to different patterns of GnRH treatment. Pulses of GnRH (5 min; 10 nM) applied every 60 min stimulated FSH beta mRNA 14.0-fold with no change in follistatin mRNA. Pulses of GnRH applied every 30 and 15 min elicited stepwise increases in follistatin mRNA and decreases in FSH beta mRNA, and continuous GnRH stimulated follistatin mRNA 4.1-fold, with no significant increase in FSH beta mRNA. Stimulation of FSH beta mRNA by hourly GnRH pulses (3.7-fold) was blocked in the presence of 30 ng/ml recombinant follistatin (0.8-fold), suggesting that GnRH stimulation of FSH beta mRNA requires endogenous activin. Treatment of plated pituitary cells with continuous GnRH for 24 h confirmed that secretion of follistatin protein rises (1.5-fold) coincident with follistatin mRNA (1.7-fold) under conditions that suppress FSH beta mRNA (9% of the control value). When male rats were infused through arterial cannulas for 6 h with continuous GnRH (100 nM) or recombinant follistatin (5 micrograms/h), continuous GnRH suppressed FSH beta mRNA levels to 50% of the control value, and follistatin decreased expression to 61% of the control value. We conclude that GnRH stimulation of FSH beta mRNA is activin dependent, and pituitary follistatin production is a major pathway by which higher GnRH pulse frequencies suppress FSH beta mRNA. Changes in activin or follistatin tone, therefore, provide a mechanism by which LH and FSH can be differentially regulated by GnRH in a variety of physiological and pathophysiological conditions.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute increase in responsiveness of luteinizing hormone (LH)-releasing hormone nerve terminals to neuropeptide-Y stimulation before the preovulatory LH surge.

Neuropeptide-Y (NPY) neurons regulate LH secretion in part through facilitation of LHRH release. We tested the hypothesis that responsiveness of LHRH neurons to NPY's facilitatory actions is physiologically regulated during the estrous cycle, and specifically, that it may be increased as a component of the gonadotropin surge-generating process. A dynamic superfusion paradigm was used to examine the role of cycle stage and time of day on LHRH responsiveness to NPY stimulation, using median eminence tissue from animals killed at 0900, 1400, and 1800 h on metestrus and proestrus. Tissue obtained at 0900 and 1800 h on metestrus did not exhibit significant LHRH responses to 10(-7) M NPY, and only moderate responses were seen at 1400 h on metestrus and 0900 h on proestrus. At 1400 h on proestrus, however, median eminence responsiveness to the same concentration of NPY was significantly increased, with LHRH responses to NPY being 2- to 5-fold greater than those at 0900 (P < 0.01), 1400 (P < 0.05), and 1800 h on metestrus (P < 0.01) and at 0900 h on proestrus (P < 0.05). Neither cycle-related changes in basal LHRH release nor changes in the releasability of LHRH in response to depolarization could account for the accentuated responses in the 1400 h proestrous group. These data clearly demonstrate that the responsiveness of LHRH terminals and/or their afferents to the actions of NPY is acutely enhanced during a brief window of time on proestrus, viz. immediately before generation of gonadotropin surges. Our findings are consistent with the hypothesis that the preovulatory endocrine milieu permits an acute increase in the responsiveness of LHRH nerve terminals to the actions of NPY, perhaps by prompting increases in the number and/or affinity of NPY receptors.

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.