Expression of vasoactive intestinal peptide receptor messenger RNA in the hypothalamus and pituitary throughout the turkey reproductive cycle

Vasoactive intestinal peptide (VIP) has been implicated in the regulation of avian reproductive activity and appears to act at the level of the hypothalamus and pituitary. This in situ hybridization histochemistry study describes the distribution of VIP receptor mRNA expression in the hypothalamus and the pituitary of reproductively active (laying) and quiescent (nonphotostimulated, incubating, and photorefractory) female turkeys and characterizes the differences observed in VIP receptor gene expression. VIP receptor mRNA, while expressed throughout the hypothalamus, was specifically expressed in areas known to contain GnRH-I neurons in the chicken, i.e., the lateral septum, medial preoptic area, anterior hypothalamus, and paraventricular nucleus. Significant differences in VIP receptor mRNA expression between different reproductive states was observed only within the infundibular nuclear complex. VIP receptor mRNA was markedly less in nonphotostimulated and photorefractory hens as compared with laying and incubating hens. The most dense VIP receptor mRNA was found in the anterior pituitary, where it was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that in nonphotostimulated ones. Hens that stopped incubating and became photorefractory displayed pituitary VIP receptor mRNA levels similar to those of nonphotostimulated birds. The changes in pituitary VIP receptor mRNA expression were positively correlated with known changes in pituitary prolactin (PRL) mRNA expression and PRL content and release. These findings indicate that the variations in PRL secretion observed across the turkey reproductive cycle are, in part, regulated by changes in VIP receptors at the pituitary level.     

Stimulation of prolactin secretion from turkey anterior pituitary cells in culture

Prolactin (PRL) secretion by monolayer cultures of turkey anterior pituitary cells was significantly increased (up to 44-fold) by vasoactive intestinal peptide (VIP), arginine vasotocin (AVT), and by an extract of turkey hypothalami (HE). Several other neuropeptides (including thyrotropin-releasing hormone) and neurotransmitters were ineffective in influencing PRL secretion at doses up to 10(-6) M. The dynamic PRL response to HE and VIP was studied using superfused pituitary cells attached to microcarrier beads. HE, administered in 30-min pulses, resulted in a significant, dose-related increase in PRL secretion from a basal secretion rate of 2.32 ng/min/10(7) cells to a peak secretion rate of 127.13 ng/min/10(7) cells at the highest dose of HE tested (1 mg tissue-equivalent weight/ml). VIP significantly increased PRL secretion at all doses studied (from 10(-10) to 10(-6) M), with 10(-8) M VIP producing a response similar to that observed with 1 mg/ml HE. A highly significant (P less than 0.001) linear relationship was demonstrated between the log-dose of VIP administered and peak PRL secretion rate. These studies suggest that VIP, but not TRH, may be a physiological stimulus for PRL release in the turkey.     

Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man

Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.     

Pituitary luteinizing hormone and prolactin messenger ribonucleic acid levels are inversely related in laying and incubating turkey hens.

The relationships of prolactin (PRL) and LH messenger (m) RNA to serum and pituitary content were determined for turkey hens at different phases of the reproductive cycle. In the nonphotostimulated, reproductively inactive hen, serum and pituitary PRL content and pituitary PRL mRNA levels were low. All three PRL values rose after photostimulation and peaked during the incubation phase. Relative to nonphotostimulated hens, hyperprolactinemic incubating hens showed 220-, 11-, and 57-fold increases in serum PRL, pituitary PRL content, and pituitary PRL mRNA levels, respectively. These peak levels declined 80-, 3-, and 6-fold, respectively, in photorefractory hens. In contrast to PRL levels, serum LH, pituitary LH, and pituitary LH beta-subunit mRNA levels did not change as dramatically. Serum LH showed no significant changes for the different reproductive phases. Pituitary LH peaked after photostimulation and declined to its lowest level in incubating hens. Pituitary LH-beta mRNA abundance was highest in photostimulated and laying hens and lowest in incubating and photorefractory hens. These results demonstrate that the abundance of LH-beta and PRL mRNA shows an inverse relationship in photostimulated/laying and incubating turkey hens.     

Differential involvement of protein kinase C in basal versus acetylcholine-regulated prolactin secretion in rat anterior pituitary cells during aging

Although it is well known that plasma concentration of prolactin (PRL) increases during aging in rats, how the anterior pituitary (AP) aging per se affects PRL secretion remains obscure. The objectives of this study were to determine if changes in the pituitary PRL responsiveness to acetylcholine (ACh; a paracrine factor in the AP), as compared with that to other PRL stimulators or inhibitors, contribute to the known age-related increase in PRL secretion, and if protein kinase C (PKC) is involved. We also determined if replenishment with aging-declined hormones such as estrogen/thyroid hormone influences the aging-caused effects on pituitary PRL responses. AP cells were prepared from old (23-24-month-old) as well as young (2-3-month-old) ovariectomized rats. Cells were pretreated for 5 days with diluent or 17beta-estradiol (E(2); 0.6 nM) in combination with or without triiodothyronine (T(3); 10 nM). Then, cells were incubated for 20 min with thyrotropin-releasing hormone (TRH; 100 nM), angiotensin II (AII; 0.2-20 nM), vasoactive intestinal peptide (VIP; 10(-9)-10(-5) M), dopamine (DA; 10(-9)-10(-5) M), or ACh (10(-7)-10(-3) M). Cells were also challenged with ACh, TRH, or phorbol 12-myristate 13-acetate (PMA; 10(-6) M) following PKC depletion by prolonged PMA (10(-6) M for 24 h) pretreatment. We found that estrogen priming of AP cells could reverse the aging-caused effects on pituitary PRL responses to AII and DA. In hormone-replenished cells aging enhanced the stimulation of PRL secretion by TRH and PMA, but not by AII and VIP. Aging also reduced the responsiveness of cells to ACh and DA in suppressing basal PRL secretion, and attenuated ACh inhibition of TRH-induced PRL secretion. Furthermore, ACh suppressed TRH-induced PRL secretion mainly via the PMA-sensitive PKC in the old AP cells, but via additional mechanisms in young AP cells. On the contrary, basal PRL secretion was PKC (PMA-sensitive)-independent in the old AP cells, but dependent in the young AP cells. Taken together, these results suggest differential roles of PMA-sensitive PKC in regulating basal and ACh-regulated PRL responses in old versus young AP cells. The persistent aging-induced differences in AP cell responsiveness to ACh, DA, TRH, and PMA following hormone (E(2)/T(3)) replenishment suggest an intrinsic pituitary change that may contribute, in part, to the elevated in vivo PRL secretion observed in aged rats.     

Antagonists of epithelial chloride channels inhibit cAMP synthesis.

In past studies we observed that the chloride channel blocker, diphenylamine-2-carboxylate (DPC) and chemically related drugs (Hoechst compounds 131, 143, 144) inhibited cAMP formation in mouse pituitary tumor cells. The object of this study was to determine whether these drugs inhibited chloride transport in human T-84 colonic carcinoma cells through an effect on cAMP metabolism. Chloride secretion (measured as 125I efflux from isotope-preloaded cells) was stimulated in a concentration-dependent manner by vasoactive intestinal polypeptide (VIP) (EC50 = 1.5 x 10(-10) M) which similarly increased cAMP synthesis (EC50 = 1.6 x 10(-8) M). The cAMP response to VIP was inhibited 17, 52, 55, and 78% maximally by DPC and compounds 144, 143, and 131, respectively. In untreated T-84 cells, 125I secretion fell by 66% after 3 min; VIP (10(-7) M) increased secretion about fivefold over the same period. Both basal and VIP-stimulated 125I secretion were inhibited up to 60% by compound 131. Pretreatment of cells with pertussis toxin did not attenuate the inhibitory effect of channel blockers on either VIP-stimulated cAMP synthesis or 125I secretion. The cationophore, A-23187, which had no effect on cAMP formation, and 8-Br-cAMP both stimulated 125I secretion from T-84 cells. These secretory responses were inhibited by compound 131. The mechanism by which phenylanthranilic acids antagonize cAMP synthesis and its significance is not known; however, the data suggest that this family of drugs may inhibit chloride transport by both cAMP-dependent and independent mechanisms.     

Integrin expression and integrin-mediated adhesion in vitro of human multipotent stromal cells (MSCs) to endothelial cells from various blood vessels

Estradiol (E2) stimulates not only secretion of prolactin (PRL) and proliferation of PRL-producing cells (PRL cells) in the anterior pituitary, but also the expression of growth factors. In insulin-like growth factor-I (IGF-I) knockout (KO) mice, the number of PRL cells is decreased and administration of IGF-I does not increase either the number of PRL cells or plasma PRL levels, indicating that IGF-I plays a pivotal role in PRL cells. The effect of E2 on PRL cells in KO mice was investigated by immunohistochemistry and real-time RT-PCR. The number of PRL cells in KO mice was significantly lower than in the wild-type (WT) control mice. E2 increased the PRL mRNA in WT and KO mice; however, an increase of PRL mRNA in KO was less than that in WT. In addition, no vasoactive intestinal peptide (VIP)-immunoreactive cells were found in KO mice, therefore IGF-I is essential for VIP expression. To investigate the roles of IGF-I on PRL cells in the postnatal development, double-immunostaining with PRL and BrdU was performed in WT and KO mice from days 5–20. The percentages of PRL cells and BrdU-labeled cells in the anterior pituitary of KO mice were lower than in WT mice. Thus, IGF-I may be responsible for proliferation and differentiation of PRL cells in this postnatal period. Differentiation and the proliferation of PRL cells are controlled by IGF-I during the postnatal development, and IGF may be a mediator of E2 action through VIP induction in PRL cells of adults.     

Increased proliferative activity and programmed cellular death in the turkey hen pituitary gland following interruption of incubation behavior

Incubation behavior or broodiness in turkey hens is characterized by ovarian regression, hyperprolactinemia, and persistent nesting. Nest-deprivation of incubating turkey hens results in disruption of broodiness accompanied by a precipitous decline in plasma prolactin (PRL) concentrations. The objective of the present study is to examine cellular changes in the pituitary gland associated with nest-deprivation for 0, 1, 2, 3, 4, or 7 days. Bromodeoxyuridine (BrdU) was administered prior to kill to study proliferative activity. Pituitary tissue sections were immunostained using turkey growth hormone (GH) antibody, and/or chicken PRL peptide antibody, and BrdU antibody. Plasma PRL concentrations declined significantly following nest-deprivation for 1 or more days. The midsagittal pituitary area immunoreactive (ir) to GH was significantly increased while that of PRL was significantly decreased following nest-deprivation for 2 or more days. Terminal deoxy-UTP nick end labeling and PRL-immunostaining revealed an abundance of apoptotic nuclei in both cephalic and caudal lobes of the anterior pituitary gland, suggestive of programmed cellular death of lactotrophs in the pituitary gland of hens nest-deprived for 2 or more days. Mammosomatotrophs were abundant in hens nest-deprived on Day 0 but were absent in hens nest-deprived for 1 or more days. Proliferating (BrdU-ir) cells were significantly abundant in the pituitary cephalic and caudal lobes following nest-deprivation for 1 or more days but were absent on Day 0 or in laying hens. Dual-labeling studies indicated that most of the BrdU-ir nuclei in the caudal lobe were not colocalized in somatotrophs in hens nest-deprived for 1-4 days but did colocalize with GH following 7 days of nest-deprivation. In conclusion, nest-deprivation of incubating turkey hens results in 1) a precipitous decline in plasma PRL concentration, 2) programmed cell death of lactotrophs, 3) disappearance of mammosomatotrophs, 4) increased proliferative activity of pituitary cells, and 5) recruitment of somatotrophs arising primarily from mitosis of nonsomatotrophic cells.     

Antagonistic analogs of growth hormone releasing hormone (GHRH) inhibit cyclic AMP production of human cancer cell lines in vitro.

Antagonistic analogs of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers both in vivo and in vitro. GHRH, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide stimulate cyclic AMP (cAMP) release from various human cancer cell lines in vitro. Thus, in the present study, we investigated the effects of antagonistic analogs of GHRH on the GHRH- and VIP-induced cAMP release from cultured human cancer cells in a superfusion system. Various human cancer cell lines were exposed to human GHRH(1-29)NH2 (2-20 nM) or VIP (0.1-5 nM) repeatedly for 12 min or continuously for 96 min. GHRH antagonist MZ-5-156 at 100 to 200 nM concentration inhibited the GHRH- or VIP-induced cAMP release from mammary (MDA-MB-468), prostatic (PC-3), and pancreatic (SW-1990 and CAPAN-2) cancer cells. These results show that antagonistic analogs of GHRH suppress the stimulatory effects of GHRH and VIP on the cAMP production of various cancer cells. Because cAMP is a potent second messenger controlling many intracellular functions, including the stimulation of cell growth, an inhibition of autocrine/paracrine action of GHRH by the GHRH antagonists may provide the basis for the development of new methods for cancer treatment.     

Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.

Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.