Subcellular distribution of dynorphin-like immunoreactivity in rat adenohypophysis in comparison with luteinizing hormone and follicle-stimulating hormone

Dynorphin and other proenkephalin B-derived peptides exist in the rat adenohypophysis in high concentrations and may have important roles in endocrine function. At the cellular level, dynorphin peptides are colocalized with the gonadotropins in at least a subpopulation of gonadotrophs. In this study dynorphin-containing particles were compared with secretory granules containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by means of differential centrifugation and sucrose density gradient centrifugation. When anterior pituitary homogenate of male rats was subjected to differential centrifugation, about 70% of both dynorphin- and LH-containing particles sedimented at 30,000 x g. LH granules and dynorphin-containing particles comigrated in continuous sucrose density gradients both under nonequilibrium conditions as well as when equilibrium was attained. FSH storage granules were found to sediment in slightly denser fractions, with substantial overlap. Hence, dynorphin-containing particles and gonadotropin-containing granules exhibit similar characteristics. These hormones may, therefore, be colocalized also at the subcellular level or stored in separate but similar vesicles.     

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.     

Evidence for a Selective Processing of Proenkephalin B into Different Opioid Peptide Forms in Particular Regions of Rat Brain and Pituitary

The distribution of five major products of proenkephalin B [dynorphin1-17, dynorphin B, dynorphin1-8, alpha-neo-endorphin and beta-neo-endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of ir-dynorphin1-17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1-17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1-32 on HPLC. In contrast with all other tissues, anterior pituitary ir-dynorphin B and ir-dynorphin1-17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1-8 were several times higher than those of dynorphin1-17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1-8 was detected in the anterior pituitary. Concentrations of beta-neo-endorphin were similar to those of alpha-neo-endorphin in the posterior pituitary. In contrast, in all brain tissues alpha-neo-endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of beta-neo-endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids -Lys-Arg- as the "typical" cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.     

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10(-8) M GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 microM PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mM Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mM Ca2+ than at 1.2 mM Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mM. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.     

Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was analyzed in cultured pituitary gonadotrophs during treatment with gonadotropin-releasing hormone (GnRH). In pituitary cells purified by centrifugal elutriation, the extent of protein kinase C redistribution during GnRH stimulation was correlated with the enrichment of gonadotrophs. GnRH-stimulated release of luteinizing hormone (LH) from gonadotroph-enriched cells was accompanied by a rapid and dose-dependent decrease in cytosolic protein kinase C and by a corresponding increase in protein kinase C activity in the particulate fraction. Retinal directly inhibited the activity of cytosolic protein kinase C and also attenuated the release of LH from GnRH-stimulated gonadotrophs. These findings, and the ability of GnRH to cause rapid translocation of cytosolic protein kinase C to a membrane-associated form, suggest that hormonal activation of protein kinase C is an intermediate step in the stimulation of pituitary LH secretion by GnRH.     

Binding of estradiol and 5 alpha-androstane-3 beta-17 beta-diol by the male rat enriched gonadotrope cells

Dispersed pituitary cells from 42-day old male rats were separated using centrifugal elutriation. Based on LH and PRL cellular contents, fractionated cells were pooled into two fractions: "Lactotrope++ population" and "gonadotrope++ population". Estradiol and 5 alpha-androstane-3 beta, 17 beta-diol binding was measured in these fractions. Results revealed that: (1) The steroid receptors are not destroyed by cell dispersion and elutriation. (2) The estradiol receptor content is higher in gonadotrope++ cells than in lactotrope++ cells. (3) The number of binding sites for the two steroids changes in the different fractions: whereas it is exactly similar in "lactotrope++ population", it is much higher for estradiol than for 5 alpha-androstane-3 beta, 17 beta-diol in "gonadotrope++ population". These results suggest two different species--or conformations--of receptor binding sites for estradiol in the male rat pituitary; the first one could link both steroids, the second one would be specific for estradiol.     

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

Chromatographic characterization of dynorphin and [Leu5]enkephalin immunoreactivity in guinea pig and rat testis

Tissues of the reproductive tract have been shown to contain mRNAs coding for pro-opiomelanocortin (POMC), pro-enkephalin and pro-dynorphin. However, the amounts of immunoreactive opioid peptides in these tissues are low, and in the case of the enkephalins and dynorphin, the molecular species responsible for the immunoreactivities have not been characterized. The chromatographic properties of dynorphin and enkephalin immunoreactivities in extracts of guinea pig and rat testis have therefore been determined. Dynorphin A and dynorphin B immunoreactivity was heterogeneous, with a significant amount attributable to high-molecular-weight forms. About 20% of the dynorphin A immunoreactivity, and about 40% of the dynorphin B immunoreactivity, in guinea pig testis extracts behaved as authentic dynorphin A or B, respectively during fractionation by ion exchange, gel filtration and high-performance liquid chromatography. Both high- and low-molecular-weight forms of [Leu5]enkephalin immunoreactivity were also present, with roughly 50-70% of the immunoreactivity attributable to low-molecular-weight forms. In extracts of guinea pig testis only a small part of this immunoreactivity eluted as authentic [Leu5]enkephalin during high-performance liquid chromatography. In rat testis most of the low-molecular-weight [Leu5]enkephalin immunoreactivity behaved as the authentic peptide. These results confirm that opioid peptides are produced in guinea pig and rat testis, and demonstrate that immunoreactive forms of the peptides similar to those found in brain and pituitary are present in the tissue.     

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.     

GnRH相关肽在大鼠垂体前叶的细胞学定位

本研究应用特异性抗GnRH相关肽(GAP)N端11个氨基酸的抗血清和六种垂体前叶激素的抗血清,通过免疫组织化学双重染色技术观察GAP在大鼠垂体前叶细胞的定位。结果发现,GAP样免疫反应性物质存在于LH细胞和FSH细胞,而未见于GH、PRL、TSH和ACTH细胞。本文首次证明GAP存在于正常大鼠垂体促性腺激素细胞,为GAP调节LH和FSH的分泌提供了形态学证据;也支持GAP的功能序列在其分子的N端,或GAP进一步裂解出N端片段而发挥作用。     

Immunohistochemical evidence for the presence of glucokinase in the gonadotropes and thyrotropes of the anterior pituitary gland of rat and monkey.

A recent report provides new evidence for the presence of glucokinase (GK) in the anterior pituitary. In the present study, immunohistochemistry was used to identify the cells containing GK in the pituitary of rats and monkeys. In rats, GK was detected as a generalized cytoplasmic staining in a discrete population of cells in the anterior pituitary. In colocalization experiments, the majority of cells expressing follicle-stimulating hormone (FSH) or luteinizing hormone (LH) also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. GK was not detected in cells expressing growth hormone or prolactin. In monkeys, GK was also observed in a discrete population of cells. Intracellular distribution differed from the rat in that GK in most cells was concentrated in a perinuclear location that appeared to be associated with the Golgi apparatus. However, similar to rats, colocalization experiments showed that the majority of cells expressing FSH or LH also contained GK. In addition to the gonadotropes, GK was observed in a subpopulation of corticotropes and thyrotropes. In the monkey, only a few cells had generalized cytoplasmic staining for GK. These experiments provide further evidence for the presence of GK in the anterior pituitary. Although some corticotropes and thyrotropes contained GK, the predominant cell type expressing GK was gonadotropes. In view of the generally accepted role of GK as a glucose sensor in a variety of cells including the insulin-producing pancreatic beta-cells as the prototypical example, it is hypothesized that hormone synthesis and/or release in pituitary cells containing GK may be directly influenced by blood glucose.     

Immunohistochemistry of beta-neoendorphin and dynorphin in the endocrine pancreas of rat and man

Serial sections from araldite-embedded rat and man pancreata were investigated immunohistochemically for the presence of prodynorphin-related peptides and alpha-endorphin. Immunoreactivities were visualized by the avidin/biotin-peroxidase complex (ABC) technique. In the human pancreas, none of the endocrine cells could be immunostained for prodynorphin-, proopiomelanocortin-related peptides and enkephalins. In the rat pancreas, however, all glucagon cells exhibited immunoreactivities for both beta-neoendorphin and dynorphin A. In addition, these cells contain alpha-endorphin-like immunoreactivity but no immunoreactivities for corticotropin, melanotropin, 16 K-fragment, alpha-N-acetyl-alpha-endorphin and enkephalins. All specificity controls confirmed that the rat endocrine pancreas might be an other source of dynorphin and endorphin with a biosynthetic pathway different from that in the pituitary or in other locations. However, concerning synthesis or degradation of peptide precursor substances interspecies differences may exist.     

A change in basal FSH release accompanies the onset of the second or selective phase of increased serum FSH in the cyclic rat

Experiments were undertaken to investigate if changes occur at the level of the anterior pituitary gland to result in selective follicle-stimulating hormone (FSH) release during late proestrus in the cyclic rat. At 1200 h proestrus, prior to the preovulatory luteinizing hormone (LH) surge in serum and the accompanying first phase of FSH release, serum LH and FSH concentrations were low. At 2400 h proestrus, after the LH surge and shortly after the onset of the second or selective phase of FSH release, serum LH was low, serum FSH was elevated about 4-fold, pituitary LH concentration was decreased about one-half and pituitary FSH concentration was not significantly decreased. During a two hour $\text{in}$$\text{vitro}$ incubation, pituitaries collected at 2400 h released nearly two-thirds less LH and 2.5 times more FSH than did pituitaries collected at 1200 h. Addition of luteinizing hormone releasing hormone (LHRH) to the incubations caused increased pituitary LH and FSH release. However, the LH and FSH increments due to LHRH in the 2400 h pituitaries were not different from those in the 1200 h pituitaries. The results indicate that a change occurs in the rat anterior pituitary gland during the period of the LH surge and first phase of FSH release which results in a selective increase in the basal FSH secretory rate. It is suggested that this change is primarily responsible for the selective increase in serum FSH which occurs during the second phase of FSH release.     

Inhibition of release of a novel pituitary polypeptide, 7B2, follicle-stimulating hormone, and luteinizing hormone from rat anterior pituitary cells in vitro by human beta-inhibin

We have recently purified a novel pituitary polypeptide designated 7B2. By raising polyclonal antibodies to a synthetic 7B2 fragment in rabbits, we have developed a sensitive and specific radioimmunoassay for this novel polypeptide, and it has been used for the study of the release of immunoreactive 7B2 from rat anterior pituitary cells in vitro. In addition, immunocytochemical study shows that 7B2 is present in the gonadotropin cells of rat anterior pituitary. The aim of the present studies is to investigate the effect of human beta-inhibin, testosterone, and combined testosterone plus human beta-inhibin on the induced release of immunoreactive 7B2, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in rat anterior pituitary cell culture in vitro. Our results show that both human beta-inhibin and testosterone effectively suppress the stimulatory effect of luteinizing hormone-releasing hormone (LHRH) on immunoreactive 7B2, FSH, and LH release. The present data indicate that the regulation of secretion of 7B2 and pituitary gonadotropins may be under a similar type of feedback mechanism.     

Immunohistochemical localization of carboxypeptidases D, E, and Z in pituitary adenomas and normal human pituitary.

Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.     

Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.     

Brain endopeptidase generates enkephalin from striatal precursors

An enzyme capable of converting putative opioid peptide intermediates to free enkephalin has been purified 300-fold from washed rat brain membranes. The action of this enzyme, an enkephalin-generating endopeptidase (EGE), was compared with the action of carboxypeptidase B after trypsin treatment on enkephalin precursor peptides present in rat striata. After Sephadex G-100 gel filtration of striatal material, fractions were radioimmunoassayed for enkephalin content using an antiserum specific for the carboxyl terminal of enkephalin. Additionally, aliquots of the column fractions were treated with either trypsin and carboxypeptidase B, trypsin and EGE, or EGE alone. The peak of enkephalin immunoreactivity increased with the enzymes' treatment indicating the conversion of the low molecular weight proenkephalin precursor peptides to enkephalin. Trypsin and EGE generated almost as much enkephalin as trypsin and carboxypeptidase B in the conditions of the experiment. Thus EGE is capable of processing precursors to enkephalin after the action of trypsin-like enzyme(s) in the brain. The gel filtration fractions containing enkephalin and its low molecular weight precursors were pooled and one-half treated with EGE. The contents were analyzed by HPLC and the increase in immunoreactivity co-eluted with enkephalin and Leu-enkephalin. Small peptides found to be the most potent competitive inhibitors of this enzyme are Met-Arg-Phe-Ala, and Met-Arg-Phe.