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Pituitary adenylate cyclase activating polypeptide (PACAP) and its close relative vasoactive intestinal polypeptide (VIP) were demonstrated in the anterior pituitary gland. The cells which exhibited PACAP immunoreactivity were oval or round shaped. Their distribution was similar to that of gonadotropes but the number of PACAP immunoreactive cells was less. Double labeling revealed that PACAP immunoreactivity partially colocalized with luteinizing and follicle-stimulating hormone; however, colocalization with other pituitary hormone immunoreactivities was not demonstrated. Our results suggest an autocrine or paracrine role of PACAP in the regulation of pituitary functions.

Gonadotropin-releasing hormone (GnRH) and oxytocin both stimulate the secretion of luteinizing hormone (LH), although with different characteristics. Therefore, interaction between oxytocin and GnRH in the control of LH may be postulated. We developed models for investigating whether oxytocin can modulate GnRH action on LH in vivo. Pentobarbitone is known to pharmacologically isolate the pituitary from hypothalamic GnRH. We found that after pentobarbitone anesthesia of female rats at proestrus, oxytocin caused a synergistically enhanced LH response to administered GnRH (p < 0.04). In a second series of experiments, female proestrous rats were anesthetized with althesin. This anesthetic allows transport of endogenous GnRH from the hypothalamus to the pituitary. In control animals, which received no exogenous hormone, there was a surge in the mean LH concentration on the evening of proestrus, indicating the presence of endogenous GnRH activity. Thus, the novel model enables detection of interactions of administered hormones with endogenous GnRH. Administration of GnRH plus oxytocin in the afternoon of proestrus caused a reduction (p < 0.01) in the mean level of LH observed in the evening. The reduction was larger than if GnRH alone was administered. Following althesin anesthesia, rats sometimes had low LH levels on the afternoon of proestrus. There was a statistically significant difference between the number of rats that received oxytocin plus GnRH and had low LH levels and the number with low LH levels in the control group (p < 0.02). Neither of the hormones administered alone had a significant effect. Thus, it appears that oxytocin accentuated the effect of GnRH in reducing LH concentrations in althesin-anesthetized rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of VIP precursor products: prepro-VIP(22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP(111-122), VIP, prepro-VIP(156-170), and prepro-VIP mRNA in the anterior pituitary of estrogen-treated, ovariectomized rats, of ovariectomized controls, and of sham-operated controls was examined. Using radioimmunoassays based on antisera against each of the prepro-VIP sequences, we found that all sequences were expressed and markedly induced by estrogen, except PHI and PHV, which both were undetectable. By immunohistochemistry, it appeared that the number of cells immunoreactive for each of these sequences was increased in the estrogen-treated animals. However, PHI/PHV-immunoreactive cells could not be detected, despite the use of four different PHI antisera with different specificities. Estrogen treatment increased the prepro-VIP mRNA as judged by Northern blotting. In situ hybridization signals for both VIP mRNA and PHI mRNA were observed in few pituitary cells from control animals whereas strong positive signals were observed in a larger number of cells after estrogen treatment. The findings show that estrogen causes activation of the VIP gene expression in anterior pituitary cells, and that the absence of PHI and PHV probably is due to translational or posttranslational events.

Pituitary cells from 14-day-old rats were separated by unit gravity velocity sedimentation, and a highly enriched population of gonadotrophs was established in reaggregate cell culture in serum-free and serum albumin-free defined culture media. The medium conditioned by these aggregates was concentrated, ultrafiltrated, and the concentrated substances consecutively separated by two reversed-phase HPLC steps, a gel filtration step and an additional reversed-phase purification step. Two peptides could be isolated that displayed an N-terminal amino acid sequence identical to fragments of rat presecretogranin II: one was cleaved at the dibasic amino acid residues K182-R183 and the other at the dibasic amino acid residues K569-R570. The latter peptide was completely purified and separated into three variants with the same N-terminal amino acid sequence. From an analysis by electrospray ionization mass spectrometry, it was deduced that the three forms extended up to amino acid residue 612 (A), which is, in presecretogranin II, also flanked by two basic amino acids (K613R614). Two variant forms had a molecular mass that was 17 Da higher. The present data support the hypothesis that secretogranin II is a precursor of putative regulatory peptides.

The anterior pituitary (AP) has been shown to contain a wide variety of bioactive peptides: brain-gut peptides, growth factors, hypothalamic releasing factors, posterior lobe peptides, opioids, and various other peptides. The localization of most of these peptides was first established by immunocytochemical methods and some of the peptides were localized in identified cell types. Although intracellular localization of a peptide may be the consequence of internalization from the plasma compartment, there is evidence for local synthesis of most of these peptides in the AP based on the identification of their messenger-RNA (mRNA). In several cases the release of the peptide from the AP cell has been shown and regulation of synthesis, storage and release have also been described. Because the amount of most of the AP peptides is very low (except for POMC peptides and galanin), endocrine functions are not expected. There is more evidence for paracrine, autocrine, or intracrine roles in growth, differentiation, and regeneration, or in the control of hormone release. To demonstrate such functions, in vitro AP experiments have been designed to avoid the interference of hypothalamic or peripheral hormones. The strategy is first to show a direct effect of the peptide after adding it to the in vitro system and, secondly, to explore if the endogenous AP peptide has a similar action by using blockers of peptide receptors or antisera immunoneutralizing the peptide.

Immunohistochemical analysis of the pituitary of sexually mature axolotls revealed both ACTH(1-39)-related and alpha-MSH-related immunoreactivity present in corticotropic cells located in the rostral anterior pituitary. Gel filtration analysis indicated that the ACTH(1-39)-sized immunoreactivity and the alpha-MSH-sized immunoreactivity detected in acid extracts of the axolotl anterior pituitary were present in a ratio in a range between 1:1 and 1:0.6. Reversed-phase HPLC analyses indicated that the alpha-MSH-sized immunoreactivity had the same retention time as synthetic ACTH(1-13)-NH2. The corticotropic activity of the ACTH(1-39)-sized immunoreactivity and the purified ACTH(1-13)-NH2 was tested in a heterologous, larval bullfrog adrenal bioassay system. As expected, the ACTH(1-39)-sized immunoreactivity stimulated corticosterone release; however, the purified ACTH(1-13)-NH2 lacked glucocorticoid activity. The proteolytic cleavage of ACTH in corticotropes of sexually mature axolotls was identical to the cleavage events observed in neotenic Ambystoma tigrinum that had not reached sexual maturity. These studies indicate that the transient expression of ACTH cleavage activity is not affected by the reproductive state of the animal. Since axolotls do not undergo metamorphosis, it is possible that events associated with metamorphosis may induce the decline in ACTH cleavage activity observed in amphibian corticotropes.

We found that GH3 cells, a rat anterior pituitary tumor cell line, expressed a single class of high-affinity binding sites for radiolabeled cholecystokinin octapeptide (CCK-8) with a Kd of 48 pM. The binding sites had high affinity for CCK-8, CCK-4, gastrin I, and L-365,260 (CCK-B antagonist), and had low affinity for devazepide (CCK-A antagonist), indicating that the binding sites are CCK-B receptors. GTP and its stable analogues inhibited radiolabeled CCK-8 binding to GH3 cell membranes, suggesting a coupling of CCK-B receptors to a G-protein.

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of alpha-melanocyte-stimulating hormone (alpha-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of alpha-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-alpha-MSH, while monoacetyl-alpha-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-alpha-MSH was the major form of alpha-MSH. The profile of alpha-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-alpha-MSH and diacetyl-alpha-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of alpha-MSH predominate in PI tissue, while nonacetylated alpha-MSH is the major form in AL tissue. It appears, however, that acetylation of alpha-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.

The joining peptide (JP) and the N-terminal peptide of proopiomelanocortin (NPP) were isolated from an acid-acetone extract of the distal lobe of the pituitary of the bullfrog, Rana catesbeiana, and purified by gel filtration and reverse-phase high performance liquid chromatography. The amino acid sequence of the bullfrog JP resembled the sequences of the JPs of Rana ridibunda (86% similarity) and Xenopus laevis (54% similarity), as deduced from the nucleotide sequences of their cDNAs. The amino acid sequence of bullfrog NPP showed 100%, 85%, and 50% similarity with those of Rana ridibunda, Xenopus laevis, and human NPPs, respectively. Administration of bullfrog NPP (0.05-5 micrograms/ml) to perifused Rana ridibunda interrenal slices induced a dose-dependent stimulation of corticosterone and aldosterone release. The present results indicate that the primary structure of NPP has been highly conserved during evolution. These data also reveal that NPP, which has no sequence homology with ACTH, exhibits a substantial corticotropic activity.

The effects of neonatal administration of monosodium glutamate (MSG) and castration on hypothalamic and anterior pituitary levels of neurokinin A (NKA) were studied in male and female rats killed at 46 days of age. In male rats treated neonatally with MSG, body, anterior pituitary, testis, ventral prostate, and seminal vesicle weights and serum testosterone levels were significantly lower than in saline-injected controls. Hypothalamic NKA was significantly lower in MSG-treated male rats as compared with the controls, and no apparent changes were recorded in anterior pituitary NKA. Orchidectomy was followed by a significant decrease in hypothalamic NKA in saline controls, but not in MSG-treated rats. In female rats treated with MSG, there was a significant decrease in body, anterior pituitary, and ovarian weights, as compared with saline-injected controls, but no significant differences were observed in uterine weights and serum estradiol levels. Hypothalamic NKA was lower, although not significantly, in MSG-treated rats as compared with the respective controls, and no differences were recorded in anterior pituitary NKA levels. Ovariectomy was followed by a significant decrease in hypothalamic NKA in both MSG-treated and control rats, but NKA in the anterior pituitary was significantly increased after ovariectomy only in saline-treated controls, whereas MSG-treated females failed to show this response. It is concluded that neonatal MSG treatment resulted in a decrease of hypothalamic NKA, which was particularly pronounced in male rats without any significant change in anterior pituitary NKA levels. The response of hypothalamic NKA to castration and the response of anterior pituitary NKA to ovariectomy were also altered in MSG-treated rats; this may reflect a functional block of some neuroendocrine functions of the hypothalamus that resulted from the neuronal lesions induced by MSG.