Sertoli cells, proximal convoluted tubules in the kidney, and neurons in the brain contain cyclic protein-2.

We analyze by immunocytochemistry the in vivo distribution in rat Sertoli cells of Cyclic Protein-2 (CP-2), which is maximally synthesized and secreted in vitro at stages VI and VII of the cycle of the seminiferous epithelium. This analysis demonstrates that CP-2 staining is strongest in Sertoli cells in stage VI and VII tubules. Additionally, we demonstrate that the staining for CP-2 within a stage VII tubule differs from the staining of another Sertoli cell secretory product, androgen-binding protein. CP-2 is not detected by immunocytochemistry in any other tissues of the reproductive tract, though immunoblot analysis demonstrates the presence of CP-2 in rete testis and epididymal fluids. CP-2 was immunocytochemically detected in only three other organs: the kidney, the brain (with greatest concentration in the supraoptic and paraventricular nuclei), and the posterior pituitary. The presence of CP-2 in the kidney was confirmed by metabolic radiolabeling, immunoprecipitation, and peptide analysis. The presence of CP-2 in the brain was confirmed by immunoblot analysis of radioinert protein immunoprecipitated from the anterior hypothalamus.

Estrogen increases galanin immunoreactivity in hyperplastic prolactin-secreting cells in Fisher 344 rats.

Galanin is a widely distributed regulatory peptide which modulates the pituitary secretion of PRL and GH. Estrogen administration strongly stimulates galanin gene expression in the rat anterior pituitary. In adult female Fischer 344 rats, estrogen also induces hyperplasia of lactotropes. We used immunocytochemical analysis to assess the effects of estrogen on galanin-like immunoreactivity (Gal-IR) in the rat pituitary and hypothalamus during sc diethylstilbestrol (DES) implantation and after its removal at 30 days. In the anterior pituitary, DES implantation increased the portion of Gal-IR-containing cells from less than 2% in the control rats to 18.3% after 3 days of DES and 36% after 30 days. These changes paralleled the lactotrope hyperplasia exhibited in response to DES exposure. Ten and 30 days after removal of the DES capsules, the percentage of Gal-IR-containing cells in the anterior pituitary decreased to 6.3% and 1.5%, respectively. Colocalization studies revealed that Gal-IR-containing cells were predominantly lactotropes. Immunoelectron microscopy demonstrated that Gal-IR was concentrated in the Golgi region of these hyperplastic lactotropes and suggests that little of the synthesized galanin is secreted. The distribution of Gal-IR in the hypothalamus, median eminence, and neurohypophysis was unaffected by DES treatment. These data demonstrate that galanin is synthesized by hyperplastic pituitary lactotropes of Fischer 344 rats and that peptide accumulation is dependent on the presence of circulating estrogens. In contrast, neuronal galanin synthesis in the hypothalamus does not appear to be regulated by estrogen.

Prolactin-releasing factor: cellular origin in the intermediate lobe of the pituitary.

Our laboratory has provided substantial evidence for the presence of PRL-releasing factor (PRF) in the posterior pituitary. The objectives of this study were 1) to determine the distribution of PRF activity between the neural and intermediate lobes, and 2) to assess the PRF activity of cultured posterior pituitary cells. Posterior pituitaries from adult male rats were dispersed with trypsin and cultured for 1-7 days. Cultured cells or intact posterior pituitaries were extracted with acid and lyophilized. PRF activity was determined by the ability of reconstituted extracts to increase PRL release from cultured anterior pituitary cells. Upon dissection of the posterior pituitary, PRF activity was primarily present in the intermediate lobe. There was minimal contamination between the two lobes, as indicated by the localization of 90% of the total oxytocin in the neural lobe and 95% of alpha MSH in the intermediate lobe. Extracts from intact posterior pituitaries and posterior pituitary cells cultured for 4 days stimulated PRL secretion in a similar dose-dependent manner. Cultured liver and cerebral cortex cells had very low PRF activity. Both oxytocin and dopamine, two neuronal markers, were reduced to less than 5% of their original values within 1 week of cell culture. There was also a significant reduction in the cell content of alpha MSH. On the other hand, PRF activity was relatively stable during culture. Incubation of posterior pituitary cells for 4 days with either cycloheximide or PRL caused a 55-60% reduction of the PRF activity of the cells. We conclude the following. 1) PRF is localized, almost exclusively, in the intermediate lobe of the pituitary. 2) PRF activity is present within nonneuronal cells, either melanotrophs or a small subpopulation of nonopioid-producing cells. 3) PRF is tissue specific, and its presence in cultured posterior pituitary cells depends at least in part on de novo synthesis. 4) The synthesis and/or release of PRF may be subjected to short loop negative feedback regulation by PRL.

Nerve growth factor receptor in neural lobe of rat pituitary gland: immunohistochemical localization, biochemical characterization and regulation.

Nerve growth factor-receptor immunoreactivity was detected in the neural lobe of the pituitary gland in developing and adult rats of both sexes. The presence of nerve growth factor receptor in the neural lobe was further verified by a quantitative 125I-nerve growth factor/crosslink/immunoprecipitation assay and subsequent visualization by SDS-PAGE autoradiography. Nerve growth factor-receptor immunoreactivity was detected in the neural lobe of postnatal 5-day-old rats, had increased by 2 months and was much higher in 1-year-old rats. In 2-month-old rats, no immunoreactivity was observed in anterior or intermediate lobes. Pituitary stalk transection in young adult rats greatly increased the expression of nerve growth factor-receptor immunoreactivity in the neural lobe, although the staining pattern remained the same. This increase began 3 days after surgery, and reached peak levels at approximately 15 days. Other physiological or non-physiological changes did not alter the nerve growth factor-receptor immunoreactivity in the neural lobe; these changes included dehydration, pregnancy and lactation, castration of male rats, bilateral superior cervical ganglionectomy and intraventricular injection of colchicine. Intravenously injected 125I-nerve growth factor was specifically accumulated in both normal and denervated neural lobe. Nerve growth factor-receptor immunohistochemical electron microscopy showed that the receptor-positive cells are fusiform and found both inside and outside the basal lamina that delimits the neural lobe parenchyma. Based upon the anatomical localization, morphology and response to axotomy, we identify, at least the perivascular component, as microglia. These data suggest a role for nerve growth factor and/or nerve growth factor receptor in microglial function.

Identification of disulfide-containing peptides in endocrine tissue extracts by HPLC-electrochemical detection and mass spectrometry.

A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.

Oxytocin is the major prolactin releasing factor in the posterior pituitary.

Although the posterior pituitary is known to contain the PRL releasing activity or factor (PRF), its chemical identification has been a matter of dispute. In the present study, we purified PRF in porcine posterior pituitary extracts to chemically determine the primary structure. PRF activity was assessed during purification by the release of immunoreactive PRL from superfused rat pituitary cells. Two hundred seventy porcine posterior pituitaries were boiled, homogenized, and extracted with 2 M acetic acid. The acid extract was precipitated with 67% acetone, and the supernatant was absorbed onto a C18 column. The column was eluted step-wise with 10, 20, 30, 40, 50, and 60% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The greatest PRF activity was recovered in the 30% CH3CN/0.1% TFA fraction and was further purified by ion-exchange chromatography on SP-Sephadex, followed by gel-filtration on Sephadex G-50. The Sephadex G-50 fractions with major PRF activity were finally purified by two cycles of reverse phase HPLC, yielding a single peak of PRF. Amino acid, as well as sequence analyses, indicated that the highly purified PRF was oxytocin. Authentic oxytocin showed the same chromatographic behavior and biological activity as those of the isolated peptide. In another experiment, desalted crude extracts of rat and porcine posterior pituitary tissues were directly chromatographed by reverse phase HPLC, and each fraction was assayed for PRF activity. Only two areas showed PRF activity; the largest activity coeluted with oxytocin and the smaller one co-eluted with vasopressin. The fractions which coeluted with oxytocin also showed oxytocin immunoreactivity, as examined by RIA. The results clearly indicated that the major PRF in these posterior pituitary extracts was oxytocin.

Occurrence of hydrin 2 (vasotocinyl-Gly), a new hydroosmotic neurohypophyseal peptide, in secretory granules isolated from the frog (Rana esculenta) neurointermediate pituitary.

Neurohypophyseal secretory granules have been purified from the frog (Rana esculenta) neurointermediate pituitary gland by sucrose gradient centrifugation, and their polypeptide content has been analyzed by reverse-phase high-pressure liquid chromatography. Aside from vasotocin, mesotocin, and their associated neurophysins, hydrin 2 (vasotocinyl-Gly), previously identified in hydrochloric acid extracts, has been recognized. This finding supports the previous suggestion that hydrin 2, a peptide active on the water permeability of frog bladder and frog skin, is a secreted hormone involved in osmoregulation specific to amphibians. Hydrin 2 has not been found in neurosecretory granules of birds such as the goose.

Immunocytochemical demonstration of vasopressin binding in rat kidney.

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.

Neural-renal interactions in the hypertension induced by papillary necrosis: role of dietary salt intake.

The effects of high salt intake (1.0% NaCl in the drinking water) on rats made hypertensive by 2-bromoethylamine hydrobromide (BEA) treatment (200 mg/kg, i.p.) were examined. BEA-induced medullary necrosis resulted in a mild hypertension (146 +/- 5 mm Hg) that was exacerbated by 4 weeks of high salt intake (163 +/- 6 mmHg). BEA-treated rats had significant salt-induced increases in urinary norepinephrine excretion and hypothalamic and brainstem norepinephrine content, that were not present in BEA-treated rats drinking tap water or control rats drinking saline. BEA treatment in combination with increased salt intake produced a decrease (p less than 0.05) in renal dopamine content and adrenal norepinephrine stores relative to BEA treatment alone. BEA treatment also significantly decreased renal norepinephrine stores and dopamine binding sites irrespective of salt intake. Renal alpha 2-adrenergic receptors and central nervous system stores of dopamine and serotonin were unaffected by BEA treatment. Renal function was well preserved as indicated by normal creatinine, glucose and protein excretion; however, significant (p less than 0.05) disruption of the urinary concentrating mechanism was present. These studies suggest that BEA-induced hypertension has a neural component that is exacerbated by high salt intake. The primary defect in BEA hypertension appears to be the lack of circulating antihypertensive lipids that attenuate the ability of salt loads to simulate sympathetic nervous system activity.

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals.

Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland.

Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.

Pituitary pathology in acquired immunodeficiency syndrome.

Pituitary morphology was studied in 49 autopsied patients with acquired immunodeficiency syndrome. Direct infectious involvement was noted in six adenohypophyses (12%), including five cases by cytomegalovirus and one by Pneumocystis carinii. Two cases with neurohypophysial lesions presumably caused by cytomegalovirus and one questionable case of Toxoplasma gondii were also observed. In all instances these changes were associated with generalized and/or cerebral infection by these same agents. Neither Kaposi's sarcoma nor malignant lymphoma was encountered in the pituitary glands. Acute necrotic foci, presumably due to infarction, were noted in four cases. Four pituitary microadenomas (8%) and four hyperplastic nodules were identified. The incidence of such noninfectious lesions, as well as the prevalence and distribution of the various immunoreactive adenohypophysial cell types, were similar to those seen in the pituitary glands of age-matched male control patients.

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva.

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.

The posterior pituitary contains a potent prolactin-releasing factor: in vivo studies.

The posterior pituitary contains a PRL-releasing factor (PRF), a small (less than 5000 mol wt) peptide which is distinct from known PRL secretagogues. The objectives of this study were to determine if posterior pituitary extracts specifically stimulate PRL release in vivo and to assess the relative contributions of oxytocin (OT), arginine vasopressin (AVP), and beta-endorphin (beta END) to the PRF activity of the extract. Rat posterior pituitaries or cerebellar tissue were extracted with 1.0 N acetic acid, boiled, and ultrafiltered through 5000 mol wt cutoff membranes. The eluates were treated with performic acid (which oxidizes disulfide bonds and methionine residues), lyophilized, and reconstituted in saline. Jugular blood was collected from conscious ovariectomized rats before and after intracarotid injection of test substances and was analyzed for PRL, LH, and GH by RIA. Injection of 0.3, 1.0, and 3.0, posterior pituitary equivalents increased plasma PRL levels by 2-, 8-, and 22-fold, respectively. PRL levels peaked within 5 min after the injection and returned to basal levels by 30 min. Plasma LH levels decreased slightly, and GH was unchanged. Cerebellar extracts did not affect plasma hormone levels. Injection of OT induced a 4-fold rise in plasma PRL levels. Oxidation of OT was well as AVP with performic acid abolished any PRL-releasing activity. Injection of beta END increased plasma PRL levels by 7-fold. Treatment of beta END with performic acid caused a 60% loss in its ability to release PRL. Pretreatment of rats with naloxone abolished the PRL-releasing effect of beta END, but did not alter the PRF activity of posterior pituitary extracts. We conclude that posterior pituitary extracts stimulate PRL release in vivo in the presence of an intact dopaminergic inhibition. This stimulation is rapid, dose dependent, and hormone specific. OT, AVP, and beta END do not contribute significantly to the PRF activity in the posterior pituitary extract.

Tyrosine hydroxylase in the stalk-median eminence and posterior pituitary is inactivated only during the plateau phase of the preovulatory prolactin surge.

This study examined changes in the activity of tyrosine hydroxylase (TH) in the stalk-median eminence (SME) and posterior pituitary (PP) during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. Blood PRL levels were low on the morning of day 30, rose to a peak from 1400-1600 h, remained at a lower plateau from 1800-2400 h, and declined to basal levels on the morning of day 31. SME, PP, and striatum were removed from PMSG-treated rats at selected times during the periovulatory period and from age-matched control rats. TH activity was determined in tissue homogenates by a coupled hydroxylation-decarboxylation assay. Apparent Km and maximum velocity values with respect to 6-methyl tetrahydropterine were estimated from substrate saturation curves. The kinetic parameters for TH in either the SME or the PP of control rats were similar at 1100 and 1800 h on day 30. However, the apparent Km in both tissues was significantly lower than that in the striatum. The affinity of TH in the SME and PP was unchanged before and during the peak phase of the PRL surge, reduced significantly during the late plateau, and returned to presurge levels in the morning of day 31. TH activity in the striatum was similar at all times examined. To determine the state of activation of the enzyme, tissue homogenates were preincubated with cAMP, ATP, and magnesium. TH activity in the SME during the peak phase was unchanged by cAMP, and that in the PP was modestly increased. The relatively inactive enzyme in both tissues during the plateau phase was markedly activated by a cAMP-dependent mechanism. The low affinity of striatal TH was greatly increased by cAMP at both times. These data suggest that TH in the SME and PP exists in an activated state most of the time and is transiently inactivated during the plateau phase of the PRL surge. In contrast, TH in the striatum is relatively inactive in the basal state and is not affected by hormonal changes induced by PMSG. We conclude that the peak PRL surge occurs in spite of active dopamine (DA) neurons, suggesting that it is generated by a nondopaminergic mechanism. Decreased TH activity in DA neurons in the SME and PP may prolong the PRL surge during the plateau phase, whereas increased DA activity coincides with the termination of the surge.

The vasopressin-associated glycopeptide is not a prolactin-releasing factor: studies with lactating Brattleboro rats.

We have recently reported that the posterior pituitary contains PRL-releasing factor (PRF), a small (less than 5000 mol wt) peptide which induces a rapid, hormone-specific, and concentration-dependent stimulation of PRL secretion. Although the identity of posterior pituitary PRF is yet unknown, it is distinct from known PRL secretagogues. Recently, the vasopressin-associated glycopeptide (VAG), which is concentrated in the posterior pituitary, was suggested as a PRF. To investigate whether VAG functions as a PRF, we used Brattleboro rats, which are deficient in arginine vasopressin (AVP), AVP-associated neurophysin, and VAG. Homozygous (DI) and heterozygous (HZ) lactating Brattleboro rats were used. The water consumption of pregnant DI rats (greater than 300 ml/day) was 6-fold higher than that of HZ rats. To correct their water imbalance, DI rats were implanted with osmotic minipumps containing the vasopressin analog 1-desamino-8-D-arginine vasopressin. On days 7-8 of lactation, pups were separated for 6 h, and blood was collected from the dams via a jugular cannula. Upon introduction of the pups, plasma PRL levels increased 100-fold in both DI and HZ rats and remained elevated for the duration of suckling. The suckling-induced rises in plasma oxytocin in DI and HZ rats were also superimposable. The weight gains of the pups of DI and HZ mothers were similar. PRF activity was determined using perifused anterior pituitary cells. Posterior pituitaries from DI and HZ rats contained equivalent amounts of PRF activity. Moreover, purified rat VAG (1.5 and 6.0 micrograms) failed to stimulate PRL release from pituitary cells. The posterior pituitary content of immunoreactive AVP was 2500-fold higher in HZ rats, but the contents of dopamine and oxytocin were similar. It is concluded that VAG neither mediates the suckling-induced rise of plasma PRL, nor stimulates PRL secretion from perifused anterior pituitary cells. Furthermore, posterior pituitaries from DI and HZ rats contain equivalent amounts of PRF activity. Collectively, these data indicate that VAG is not the posterior pituitary PRF.

Total translation of vasopressin and oxytocin in neurohypophysis of rats.

There is a paucity of information about total translation rate of vasopressin and oxytocin. Because the site of synthesis of the neurohypophysial hormones is anatomically separate from the site of storage, we were able to measure total translation by blocking transport of newly synthesized hormone and measuring accumulation in the areas of synthesis in the hypothalamus. Colchicine administered into the third ventricle in doses as low as 3.5 micrograms/rat blocked transport for 18 h. The linear increase in vasopressin and oxytocin content over 18 h indicated a stable rate of synthesis, which was 1.2 and 1.9 pmol/h for vasopressin and 1.4-2.5 pmol/h for oxytocin. The molar correlation for synthesis of total neurophysin to total hormone was 1.16. Infusion of oxytocin and vasopressin into rats indicated that this level of synthesis of hormone was essential under steady-state conditions to maintain plasma levels in the low physiological range of approximately 3 pg/ml for oxytocin and 1 pg/ml for vasopressin. The data on total synthesis of the neurohypophysial hormones provide a reference for studies in which physiological replacement is required and also provide the technique and base-line data to determine how translation of vasopressin and oxytocin is regulated when neurohypophysial function is altered.

Regional distribution of neuropeptide Y and its receptor in the porcine central nervous system.

The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200-1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50-200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained less than 50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0-5.87 pmol/mg of protein. Intermediate binding (0.5-1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but less than 0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.

Galanin and vasopressin coexist in the rat hypothalamo-neurohypophyseal system.

Using indirect immunofluorescence methods and antisera raised against galanin (GAL) and vasopressin (VP), we have demonstrated both peptides coexisting in the very same cell bodies in the supraoptic and magnocellular paraventricular nuclei and the magnocellular accessory cells of the lateral hypothalamic area. Furthermore, dehydration and salt loading, which is known to cause release and depletion of VP and oxytocin from the neurohypophysis, also caused a marked reduction of GAL-like immunoreactivity in the posterior lobe of the pituitary but had no effect on hypothalamic GAL immunoreactivity. Systemically administered GAL caused a brief small increase in blood pressure with no effect on heart rate. A thousandfold molar concentration of GAL, compared of VP, was required to induce comparable effects on blood pressure. GAL itself had no modulatory effect on VP-induced pressor response. Systemically administered GAL resulted in mild diuresis whereas VP caused complete and sustained inhibition of diuresis. GAL had no effect on VP-induced anti-diuresis effects. The significance of the coexistence and corelease of GAL and VP remains to be elucidated.

Isolation of neurosecretory granules containing vasotocin, mesotocin, MSEL- and VLDV-neurophysins from goose neurohypophysis.

Neurosecretory granules have been isolated from goose posterior pituitaries and their contents have been analyzed by reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis. Vasotocin and mesotocin have been identified by their biological activities and their retention times compared with those of synthetic peptides. MSEL- and VLDV-neurophysins have been characterized by their N-terminal sequences, their electrophoretic migrations and their retention times, compared with those of purified goose neurophysins. In contrast to the two-step processing of mammalian provasopressin, processing of the vasotocin - MSEL-neurophysin precursor appears to involve only one cleavage giving the hormone and a "big" MSEL-neurophysin homologous to mammalian MSEL-neurophysin extended by copeptin.