Effects of zeranol on in vitro growth hormone release by lamb and rat pituitary cells

A series of experiments was conducted to evaluate the effect of zeranol on release and synthesis of growth hormone (GH) by anterior pituitary cells established in either static or continuous flow cultures. Young adult male rats, slaughter-age lambs and juvenile lambs were used as sources of pituitary cells. In static primary cell cultures, no consistent effect of zeranol at 10(-7), 10(-9) or 10(-11) M was demonstrated by either rat or ovine cells. Rat pituitaries established in perifusion culture chambers showed no repeatable response to zeranol. Dissociated cells from lambs established in perifusion culture, however, had significant increases in release of GH in response to 37% of zeranol pulse exposures. When dissociated cells from juvenile lamb pituitaries were used, up to 10-fold increases in GH release consistently were measured within minutes of exposure to zeranol.     

Effects of VIP and GRF on the release of growth hormone in perifused bovine adenohypophysis

The effects of vasoactive intestinal polypeptide (VIP) and growth hormone releasing factor (GRF:hpGRF(1–29)-NH₂) on the release of growth hormone (GH) from anterior pituitaries from cows were examined by using an in vitro superfusion system. The pituitaries were excised randomly from cycling cows, dissected to obtain medial portions, and minced to obtain cubes with approximate dimensions of 1.5mm on a side. For each perifusion setup, 5 pieces of pituitary tissues were chambered and flushed with modified KRB solution saturated with 95% O₂-5% CO₂ at 38C. Perifusion with media containing 10⁻⁶ and 10⁻⁷M VIP for 30 min induced a significant release of GH during the treatments (P<0.05). VIP (10⁻⁸M) increased GH levels significantly (P<0.05), but to a minor degree. Perifusion with the media containing 10⁻⁶, 10⁻⁷ and 10⁻⁸M GRF for 30 min markedly increased the GH concentration and the effects continued up to 90 min after termination of the perifusion of the peptide (P<0.05, P<0.01). The GH releasing effects of GRF could be seen at doses as low as 10⁻¹¹M GRF (P<0.05, P<0.01).

These findings indicate that the GH releasing effect of VIP is less potent that that of GRF in cows.     

Effects of Ghrelin on growth hormone secretion from cultured adenohypophysial cells in pigs

To clarify the direct effects of Ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in pigs, GH-releasing effects of human Ghrelin (hGhrelin) and rat Ghrelin (rGhrelin) on porcine AP cells were compared with GHRH in vitro. The AP cells were obtained from 6-month-old pigs and the cells (2 x 10(5) cells per well) were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses of 10(-8) and 10(-7)M (P < 0.05). The rates of increase in GH at 10(-8) and 10(-7)M of hGhrelin were 82.7 and 131.9%, while those with rGhrelin were 43.9 and 79.5%, respectively. GHRH significantly stimulated GH release from the cells at a dose as low as 10(-11)M (P < 0.05), and the response to GHRH was greater than that induced by Ghrelins. In time-course experiments, GHRH continued to increase GH concentrations in media until 120 min after incubation; however, those in media treated with hGhrelin reached a plateau 60 min after incubation, and the maximal value was approximately one third that obtained with GHRH. When hGhrelin (10(-8)M) and GHRH (10(-8)M) were added together, additive effects of both peptides on the release of GH were observed (P < 0.05). Somatostatin (SS, 10(-7)M) significantly blunted GH release induced by hGhrelin (10(-8)M) and GHRH (10(-8)M) (P < 0.05). In the presence of SS, additive effects of hGhrelin and GHRH on the release of GH were observed (P < 0.05). These results show that Ghrelin directly stimulates GH release from anterior pituitary cells in pigs; however, the GH-releasing effect is weaker than that of GHRH in vitro. The present results also show that Ghrelin interacts with GHRH and SS to in the release of GH from porcine adenohypophysial cells.     

GABA Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study examines the involvement of GABA_{A or B} receptors in gonadotrophin‐releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates γ‐aminobutyric acid (GABA)–GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)‐α at 4°C; within 2 h, slices were singly perifused at 37°C with oxygenated MEM‐α (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA_{A or B} receptor ligands (0.1–10 mm ). The GABA_{A or B} agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mm GABA_{A or B} antagonists (bicuculline or CGP52432, respectively). The GABA_A antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus‐pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to the GABA_A antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA_A antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA_A receptor inhibitory control and this is attenuated in the presence of oestradiol.     

Noradrenergic Control of GnRH Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study investigates the influence of α₁‐adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates α₁‐adrenoreceptor‐GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media‐α (MEM‐α) at 4°C and within 2 h were singly perifused at 37°C with oxygenated MEM‐α (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4‐h equilibration, 10‐min fractions were collected for 4 h interposed with a 10‐min exposure at 60 min to specific α₁‐adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1–10 mm ). The α₁‐adrenoreceptor agonist (10 mm ) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, α₁‐adrenoreceptor agonist (10 mm )‐induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus–pituitary sequential double‐chamber perifusion. Only after exposure of hypothalamic slices to α₁‐adrenoreceptor agonist (10 mm ), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 ± 12.3–36.2 ± 21.6 ng/ml) confirming that the α₁‐adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.     

The bovine preoptic area and median eminence: sites of opioid inhibition of luteinizing hormone-releasing hormone secretion.

Autoradiography was used to quantify opioid receptors in the median eminence (ME) and preoptic area (POA) of the brain of eight heifers, and in vitro perifusion of ME and POA tissue from seven cows and heifers was used to examine the release of LHRH after administration of naloxone (NAL). For quantitative receptor autoradiography, [3H]NAL was used as the radioligand and NAL or morphine as competitors. Specific binding of [3H]NAL in POA and ME resulted in linear Scatchard plots with similar equilibrium dissociation constants (Kd = 4.2 +/- 1.1 nM) and mean binding site densities in the POA and ME (POA: 80.3 +/- 5.8; ME 67.5 +/- 8.0 fmol/mm2). There were no differences between mean binding site densities of zonas externa and interna of the ME; however, between various regions of the POA within individual animals, binding site densities varied threefold (47.6 to 165.1 fmol/mm2). During in vitro perifusions of isolated POA and ME, basal LHRH secretion from ME decreased (P less than .001) from 15.9 +/- 1.8 to 7.3 +/- .8 pg/10 min fraction (500 microliters) but remained constant for POA (3.1 +/- .4 pg/fraction). Injections of medium alone did not affect LHRH secretion. Although there was no significant dose (10(-9) to 10(-7) M) effect, NAL increased (P less than .05) LHRH efflux from the ME and POA when administered at 110 min from the initiation of perfusion and again at 200 min for ME but not for POA. All tissues responded to KCl (30 mM) administered at 290 min of perifusion with increased (P less than .001) LHRH efflux. Both immunoreactive-LHRH and immunoreactive-beta-endorphin were immunocytochemically localized in neurons from some of these perifused tissues. We suggest that endogenous opioids suppress LHRH secretion by actions on specific opioid receptors located within the POA and ME of the brain.     

Influence of catecholamines, prostaglandins and thyroid hormones on growth hormone secretion by chicken pituitary cells in vitro

In young chickens plasma concentrations of growth hormone (GH) are depressed by prostaglandins (PG) E1 and E2, epinephrine, norepinephrine, alpha 2 and beta agonists or thyroid hormones. A primary culture of chicken adenohypophyseal cells was used to examine the direct effects of these agents at the level of the pituitary as evaluated by GH release in the presence and absence of growth hormone releasing factor (GRF). Following collagenase dispersion and culture (preincubation, 48 hr) cells were exposed (incubation, 2 hr) to test agents, except for thyroid hormones which were added during the preincubation, and incubation period. Growth hormone release was increased (P less than .05) in the presence of PGE1 (10(-8)M by 34%; 10(-7)M by 54%), PGE2 (10(-8)M by 29%; 10(-7)M by 29%), PGF2 alpha (10(-8)M by 28%), and the beta agonist isoproterenol (10(-7)M by 46%). Basal GH release from chicken pituitary cells was not affected by dopamine, norepinephrine, epinephrine, thyroxine (T4), triiodothyronine (T3), or alpha adrenergic agonists. Growth hormone releasing factor stimulated GH release was not affected by the presence of prostaglandins E1, E2 or F2 alpha in the incubation media. However, GRF stimulated GH release was reduced by high doses of catecholamines: dopamine (10(-6)M by 34%), norepinephrine (10(-6)M by 74%), epinephrine (10(-8)M by 47%; 10(-7)M by 41%; 10(-6)M by 89%), and by the alpha 1 adrenergic agonist, phenylephrine (10(-7)M by 52%), the alpha 2 agonist, clonidine (10(-8)M by 34%; 10(-7)M by 83%) and the beta agonist, isoproterenol (10(-7)M by 64%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The chicken pituitary-specific transcription factor PIT-1 is involved in the hypothalamic regulation of pituitary hormones

Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.     

The control of adenohypophysial hormone secretion by amino acids and peptides in swine

Several different amino acids and peptides control secretion of adenohypophysial hormones and this control may be indirect, via the modulation of hypothalamic hormone secretion. Indeed, classical hypothalamic hormones (e.g., gonadotropin-releasing hormone [GnRH], growth hormone-releasing hormone [GHRH], somatostatin, etc.) may be released into the hypothalamo-hypophysial portal vasculature, travel to the adenohypophysis and there stimulate or inhibit secretion of hormones. Alternatively, some amino acids and peptides exert direct stimulatory or inhibitory effects on the adenohypophysis, thereby impacting hormone secretion. In swine, the most extensively studied modulators of adenohypophysial hormone secretion are the excitatory amino acids (ExAA), namely glutamate and aspartate, and the endogenous opioid peptides (EOP). In general, excitatory amino acids stimulate release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL). Secretion of adenohypophysial hormones induced by ExAA is primarily, but perhaps not exclusively, a consequence of action at the central nervous system. By acting primarily at the level of the central nervous system, EOP inhibit LH secretion, stimulate GH release and depending on the animal model studied, exert either stimulatory or inhibitory influences on PRL secretion. However, the EOP also inhibited LH release by direct action on the adenohypophysis. More recently, peptides such as neuropeptide-Y (NPY), orexin-B, ghrelin, galanin, and substance P have been evaluated for possible roles in controlling adenohypophysial hormone secretion in swine. For example, NPY, orexin-B, and ghrelin increased basal GH secretion and modulated the GH response to GHRH, at least in part, by direct action on the adenohypophysis. Secretion of LH was stimulated by orexin-B, galanin, and substance P from porcine pituitary cells in vitro. Because the ExAA and various peptides modulate secretion of adenohypophysial hormones, these compounds may play an important role in regulating swine growth and reproduction.     

Regulation of growth hormone-releasing hormone and somatostatin from perifused,bovine hypothalamic slices. III. Reciprocal feedback between growth hormone-releasing hormone and somatostatin

An in vitro perifusion system for bovine hypothalamic tissue was used to determine if growth hormone-releasing hormone (GHRH) and somatostatin (SRIF) modulate each other's release, and whether SRIF mediates D₁-agonist-induced suppression of GHRH in cattle. Up to three sagittal slices (600 μm) of bovine hypothalamus, immediately parallel to the midline, were cut in an oxygenated balanced salt solution at 4° C, placed in 5 cc syringe barrels, and perifused at 37° C with oxygenated minimum essential medium-α at a flow rate of 0.15 ml/min. Three experiments were conducted, and medium effluent was collected every 20 min before (two samples), during (one or three samples), and after (six samples) treatment. Areas under GHRH and SRIF response curves (AUC), adjusted by covariance for pretreatment values, were calculated from samples collected during the treatment/post-treatment period. Perifusion of SRIF at 10⁻⁶ M and 10⁻⁴ M decreased AUC for GHRH from 86.3 (control) to 65.4 and 59.5 ± 6.3 ng · ml⁻¹ min, but 10⁻⁸ M SRIF was ineffective. Relative to controls, 10⁻⁸, 10⁻⁶, and 10⁻⁴ M GHRH increased release of SRIF 190, 675, and 1,135%, respectively. Activation of D₁ receptors with 10⁻⁶ M SKF 38393 increased AUC for SRIF from 12.5 ng · ml⁻¹ min (control) to 484.9 ng · ml⁻¹ min and decreased AUC for GHRH from 36.4 ng · ml⁻¹ min (control) to 18.2 ng · ml⁻¹ min. Blockade of SRIF action with a SRIF antagonist, cyclo-[7-aminoheptanoyl-phe-d-trp-lys-thr(bzl)], increased release of GHRH 1.9-fold. In addition, the SRIF antagonist blocked SKF 38393-induced suppression of GHRH. We concluded that GHRH and SRIF interact within the bovine hypothalamus/pituitary stalk to modulate the release of the other. Moreover, SRIF mediates the inhibitory effects of activation of D₁ receptors on release of GHRH in cattle.     

γ-Amino Butyric Acid Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims to ascertain the influence of gamma-amino butyric acid (GABA)(A or B) receptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates GABA-AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)-alpha at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to a specific GABA(A or B) receptor agonist or antagonist at various doses (0.1-10 mm). GABA(A) (muscimol; no E(2), n = 7 perifusion chambers, with E(2), n = 11) or GABA(B) (baclofen; no E(2), n = 8, with E(2), n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20-30 min after exposure to 10 mm GABA(A or B) antagonists (bicuculline, no E(2), n = 7: from 4.6 +/- 0.7 to 33.0 +/- 0.4, with E(2), n = 17: from 11.9 +/- 1.4 to 32.8 +/- 6.0; CGP52432, with E(2), n = 14: from 14.0 +/- 2.6 to 28.8 +/- 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABA(B) but not GABA(A) antagonist-stimulated AVP release was enhanced in the presence of E(2). In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E(2) through GABA(B) receptors in vitro.     

Androgens modulate growth hormone-releasing factor-induced GH release from bovine anterior pituitary cells in static culture.

Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10(5) cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17 beta (E2, 10(-11) to 10(-8) M), testosterone (T, 10(-8) to 10(-5) M), dihydrotestosterone (DHT, 10(-9) to 10(-6) M) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol, 10(-11) to 10(-8) M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P > 0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1-44-NH2 (10(-8) M). In non-steroid treated wells, GRF increased (P < 0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P > 0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P < 0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.     

Gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) secretion from perifused equine pituitaries.

In vitro responsiveness of the horse anterior pituitary (AP) gonadotropes to single and multiple GnRH challenges was examined. The pituitaries were collected from reproductively sound mares in estrus (n = 5) and diestrus (n = 5). Uniform 0.5 mm AP slices were subdivided using a 3 mm biopsy punch and then bisected for use in the perifusion chamber. Four bisected sections per chamber were perifused at 0.5 ml/min at 37 C for 560 min in Medium 199 saturated with 95% 0(2)/5% CO2. Ten minute fractions were collected after an initial 2 hr equilibration period. Four different treatment regimes of GnRH (10(-10) M) were evaluated: (A) three consecutive 10 min GnRH pulses separated by 80 and 100 min, respectively; (B) a single 120 min GnRH infusion; (C) a 10 min GnRH pulse followed 80 min later by a 120 min GnRH infusion and (D) two 10 min GnRH pulses separated by 60 min followed 80 min later by a 120 min GnRH infusion. Estimated total pituitary LH content was higher in estrous than diestrus mares (p less than 0.05). The total amount of LH released in response to GnRH tended to be greater in estrus than diestrus (p less than 0.1), whereas the percentage of LH released in estrus and diestrus was similar. An increase in the area under the LH response curve was noted with each successive 10 min pulse of GnRH during both estrus and diestrus (p less than 0.05), demonstrating a self-priming effect of GnRH. In addition, a significant increase in the peak LH amplitude (p less than 0.05) and the slope to peak amplitude (p less than 0.05) were observed for the 120 min GnRH pulse in regime C and D indicating that prior exposure to short-term pulses of GnRH increased the acute LH secretory response. These results suggest that in the cycling mare (1) the responsiveness of the pituitary (amount of LH released as percent of total LH) is similar in both estrus and diestrus, however, the magnitude of the LH response (total microgram amount of LH released) differs with the stage of the estrous cycle, being highest in estrus, and appears to be related, in part, to pituitary LH content and (2) GnRH self-priming occurs independently of the stage of the estrous cycle. Furthermore, we have demonstrated that the pulsatile mode of GnRH can act directly on the anterior pituitary to dictate the pulsatile release pattern of LH in the cycling mare.     

Localization of Neuropeptide Y-like immunoreactive structures in the brain of the pejerrey,Odontesthes bonariensis (Teleostei,Atheriniformes)

The aim of this study was to determine the distribution of Neuropeptide-Y (NPY) immunoreactive neurons and fibres in the brain and pituitary of Odontesthes bonariensis by immunohistochemical methods. A wide distribution of immunoreactive NPY (ir-NPY) cells and fibres in the forebrain and midbrain was observed. A prominent ir-NPY nucleus was found in the ventral telencephalon and other ir-NPY cells groups were recognized at the dorso-medial telencephalon. The diencephalon showed ir-NPY cells in the Nucleus entopeduncularis, the Nucleus preopticus periventricularis and in the Nucleus lateralis tuberis. Ir-NPY fibres were conspicuous in the preoptic region and the hypothalamus. There were also numerous ir-NPY fibres at the epithalamic level running ventrally to the hypothalamus and the pituitary stalk. At the rhomboencephalic level, the ir-NPY neurons were observed in the Locus coeruleus. Double-labelled immunostaining showed a close association between ir-NPY fibres that reach the adenohypophysis and growth hormone (GH)- and gonadotropin (GtH)-expressing cells. Although our results exhibit some relevant differences when compared to other fish groups, they support the existence of a conserved NPY system in teleosts.     

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP), prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) on the release of growth hormone from cultured bovine anterior pituitary cells in vitro

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone (GH) release was compared with that of prostaglandin E₂ (PGE₂) and growth hormone releasing factor (GRF) from cultured bovine anterior pituitary cells in vitro. Both PACAP and PGE₂ stimulated GH release at concentrations as low as 10⁻⁹ and 10⁻⁸ M, respectively, (P<0.01). However, GRF released GH at a concentration as low as 10⁻¹³ M (P<0.01). Percent increases of GH compared with controls were not significantly different among GRF, PACAP, and PGE₂ at 10⁻⁷ M; however, the increases of GH by the 10⁻⁸ M GRF, PACAP and PGE₂ were 196, 118, and 27%, respectively, (P<0.01), and 124, 65, and 1% in the 10⁻⁹ M media, respectively, (P<0.01). When GRF and somatostatin (SS) were added together, the GH releasing effect of GRF was blunted (P<0.01). Similar bluntness were observed in PACAP and PGE₂, when SS was added. The stimulatory effects of GRF and PGE₂ together were similar to that by either GRF or PGE₂ alone. When GRF and PACAP were added together, the GH released by both secretagogues was greater than that by PACAP alone (P<0.01); however, a synergistic effect was not clear when compared with GRF alone.

These findings suggest that PACAP and PGE₂ may modulate the release of GH in cattle.     

Luteinizing hormone in the bovine pars tuberalis: secretion in response to luteinizing hormone releasing hormone and intracellular isoforms

The objective of this study was to examine the physiological characteristics of gonadotropes in the bovine (b) pars tuberalis as assessed by their ability to release Luteinizing Hormone (LH) in response to LH-Releasing Hormone (LHRH) and the intracellular distribution of LH isoforms. At slaughter, the stalk median eminence and associated pars tuberalis as well as the anterior pituitary gland were collected from each of 7 castrate males. Each stalk median eminence and pituitary gland was mid-sagitally sectioned and weighed. One half of each tissue was immediately frozen and subsequently homogenized to determine the intracellular distribution of bLH isoforms. Tissue extracts were desalted by flow dialysis against water and chromatofocused on pH 10.5-7.0 gradients. The remaining half of the pituitary was sliced with a Staddie-Riggs slicer. The pituitary slices and the remaining half of the stalk median eminence were perifused (0.1 ml/min) for a total of 360 min with effluent samples (1.0 ml) collected every 10 min. At 130 min tissues were stimulated with 5 x 10(-8) M LHRH. Concentrations of LH in the effluent samples and the fractions collected from chromatofocusing were determined by radioimmunoassay. The release of LH in response to LHRH was 43.9% and 47.0% above basal secretion for the pars tuberalis and pituitary, respectively, suggesting similar degrees of responsiveness. Pars tuberalis and pituitary extracts resolved into nine LH isoforms during chromatofocusing and were coded with letters beginning with the most basic form. No differences (P greater than .05) were observed in distribution of LH isoforms between the pars tuberalis and the pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid modulation of gonadotropin releasing hormone release from the hypothalamic preoptic area in the pig

Two experiments (Exp) were conducted to examine in vitro the release of gonadotropin releasing hormone (GnRH) from the hypothalamus after treatment with naloxone (NAL) or morphine (MOR). In Exp 1, hypothalamic-preoptic area (HYP-POA) collected from 3 market weight gilts at sacrifice and sagitally halved were perifused for 90 min prior to a 10 min pulse of morphine (MOR; 4.5 × 10⁻⁶ M) followed by NAL (3.1 × 10⁻⁵ M) during the last 5 min of MOR (MOR + NAL; N=3). The other half of the explants (n=3) were exposed to NAL for 5 min. Fragments were exposed to KCl (60 mM) at 175 min to assess residual GnRH releasability. In Exp 2, nine gilts were ovariectomized and received either oil vehicle im (V; n=3); 10 μg estradiol-17β/kg BW im 42 hr before sacrifice (E; n=3); .85 mg progesterone/kg BW im twice daily for 6 d prior to sacrifice (P₄; n=3). Blood was collected to assess pituitary sensitivity to GnRH (.2 μg/kg BW) on the day prior to sacrifice. On the day of sacrifice HYP-POA explants were collected and treated as described in Exp 1 except tissue received only NAL. In Exp 1, NAL increased (P<.05) GnRH release. This response to NAL was attenuated (P<.05) by coadministration of MOR. Cumulative GnRH release after NAL was greater (P<.05) than after MOR + NAL. All tissues responded similarly to KCl with an increase (P<.05) in GnRH release. In Exp 2, pretreatment luteinizing hormone (LH) concentrations were lower (P<.05) in E gilts compared to V and P₄ animals with P₄ being lower (P<.05) than V gilts. LH response to GnRH was lower (P<.05) in E pigs than in V and P₄ animals, while the responses was similar between V and P₄ gilts. NAL increased GnRH release in all explants, whereas, KCl increased GnRH release in 6 of 9 explants. These results indicate that endogenous opioid peptides may modulate in vitro GnRH release from the hypothalamus in the gilt.     

Suppressing effects of short-chain fatty acids on growth hormone (GH)-releasing hormone-induced GH release in isolated anterior pituitary cells of goats.

The involvement of tetrodotoxin-sensitive Na+ channels and receptor-operated nonspecific Ca2+ channels, and the effects of short-chain fatty acids, on growth hormone (GH) release induced by GH-releasing hormone (GHRH) were investigated in cultured and freshly isolated caprine anterior pituitary cells. In 3-d cultured cells in Dulbecco's modified Eagle's medium, an increase in GH release induced by GHRH (10 nmol/l) was moderately, but significantly, reduced by a voltage-sensitive Na+ channel antagonist tetrodotoxin (1 micromol). The GHRH-induced GH increase, which was not affected by a simultaneous addition of a receptor-operated nonspecific Ca2+ channel antagonist tetramethrine (0.1 mmol/l), was significantly reduced by a voltage-sensitive L-type Ca2+ channel antagonist nifedipine (1 micromol/l). Propionate and butyrate at 10 mmol/l, however, not only suppressed basal GH release but also significantly reduced the GH increase induced by 10 nmol/l of GHRH. The inhibitory action of these acids was also reproduced by an addition of beta-hydroxy butyrate (10 mmol/l) and octanoate (10 mmol/l). In freshly isolated and perifused cells, butyrate (10 mmol/l) as well as somatostatin (100 nmol/l) significantly reduced the GH increase induced by GHRH. From these findings we conclude that tetrodotoxin-sensitive Na+ channels and voltage-dependent L-type Ca2+ channels are involved in the cellular mechanism for GHRH-induced GH release, and that short-chain fatty acids such as propionate and butyrate have a direct action on somatotrophs to reduce basal and GHRH-induced GH release, in caprine somatotrophs.     

Relationships among concentrations of four opioid neuropeptides and luteinizing hormone-releasing hormone in neural tissues of beef cows following early weaning

Acute changes associated with removal of the inhibition of estrus caused by suckling were examined in beef cows. Calves were weaned during the fifth week after parturition and cows were slaughtered at 0 (n = 8), 36 (n = 8) or 72 h (n = 8) after calf removal. Tissues of preoptic area (POA), hypothalamus (HYP), pituitary stalk-median eminence (SME) and pituitary neurointermediate lobe (NIL) were obtained for analyses of luteinizing hormone-releasing hormone (LHRH) and four opioid neuropeptides. In addition, one-half of each SME was superfused in vitro for measurement of basal and potassium-induced release of LHRH. The following opioid neuropeptides were quantified: methionine-enkephalin (Met-Enk), beta-endorphin (beta-EP), dynorphin-A, 1-17 (DYN-17) and dynorphin-A, 1-8 (DYN-8). All four opioid neuropeptides were most concentrated in the pituitary NIL. Luteinizing hormone-releasing hormone was most concentrated in the SME tissue, which also contained substantial concentrations of Met-Enk and beta-EP, but very little DYN-17 or DYN-8. In addition, weaning increased the weight of NIL between 0 and 36 h (P less than .05), and the concentrations of LHRH, Met-Enk, and DYN-17 in the combined POA + HYP (P less than .05) tissue between 36 and 72 h. No differences occurred among groups in SME content of LHRH or in vitro release of LHRH from the superfused SME. Although they were not affected by weaning, within-cow correlations among parameters revealed that: 1) concentrations of DYN-17 and DYN-8 were always positively correlated (P less than .05); 2) concentrations of LHRH were positively correlated with Met-Enk (P less than .01), beta-EP (P less than .05) and DYN-17 (P less than .05) in the combined POA + HYP tissue; 3) LHRH concentrations in SME tissue were negatively related to POA + HYP concentrations of Met-Enk (P less than .01) and beta-EP (P less than .05), but not of LHRH or DYN-17 and 4) in vitro release of LHRH from the pituitary SME was correlated with concentrations of DYN-8 in various tissues including the SME (P less than .01). In summary, bovine neural tissues differ widely in concentrations of the four opioid neuropeptides with NIL tissue having the greatest concentrations. Weaning calves at 36 and 72 h before slaughter caused parallel changes in LHRH, Met-Enk and DYN-17 in preoptic and hypothalamic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)