Alan T. Lim

Atrial Natriuretic Factor is Released into Hypophysial Portal Blood: Direct Evidence that Atrial Natriuretic Factor may be a Neurohormone Involved in Hypothalamic Pituitary Control.

Atrial natriuretic factor (ANF) is produced in atriomyocytes (1, 2), released as a 28 amino‐acid peptide into the systemic circulation and probably plays an important role in fluid and electrolyte balance (3, 4). The facts that immunoreactive ANF (ir‐ANF) is also present in nerve terminals in the external layer of the median eminence (ME) (5), ir‐ANF can be released from the hypothalamus in vitro by potassium depolarization (6) and specific ANF binding sites are present at a high concentration in rat pituitary tissue (7) suggest that ANF may be involved in hypothalamic‐pituitary regulation. We report here that ir‐ANF concentrations in hypophysial portal blood are about two to four times greater than in peripheral plasma from hypophysectomized as well as pituitary‐intact adult female rats. These results show for the first time that ir‐ANF is secreted from the hypothalamus into the hypophysial portal circulation at a concentration (≈10−9 M) consistent with a role for ANF as a hypothalamic‐pituitary regulator or modulator.

Glucocorticoids potentiate the adenylyl cyclase—cAMP system mediated immunoreactive β-endorphin production and secretion from hypothalamic neurons in culture

Beta-endorphin(beta EP)1-31, a potent opioid peptide of proopiomelanocortin (POMC) derivatives, is produced and released from neurons at arcuate nuclei of the rat hypothalamus. Although dexamethasone (DM) suppresses the production and secretion of POMC related peptides from rat pituitary corticotrophs, the effect of glucocorticoids on the function of hypothalamic beta EP neurons remains unclear. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 day treatment with 10 microM of forskolin increased ir-beta EP levels in cell content and culture media by approximately 1.7 (P < 0.05) and 4.1 times (P < 0.01) above vehicle treated control cultures (mean +/- S.E.M., 47.3 +/- 2.6 pg/well and 40.4 +/- 3.0 pg/well; n = 3) respectively. Although 4 day treatment with DM alone had little effect on the release and the cell content of ir-beta EP, it significantly enhanced forskolin-induced elevation of ir-beta EP levels in cell content and in culture media. The effect of DM was dose-related and time-dependent, with an EC50 of about 1 nM; at this concentration DM enhanced ir-beta EP secretion about 2.1 times (P < 0.01) above that induced by 10 microM of forskolin alone. Furthermore, the potentiating effect of DM was specifically suppressed by 100 nM of RU38486 (P < 0.01), a glucocorticoid receptor antagonist, but not by an equivalent dose of RU28318, a mineralocorticoid receptor antagonist. In addition, Northern blot analysis showed that forskolin (10 microM) increased the abundance of POMC mRNA 1.4 fold above that of vehicle treated control cultures. Whereas by itself, DM (10 nM) had little effect on the level of POMC mRNA, it enhanced forskolin-stimulated increase of the abundance of POMC mRNA approximately 2.6 times. Moreover, DM also augmented 1.6 times (P < 0.05) forskolin-induced but not 3-isobutyl-1-methylxanthine (IBMX)-induced increase of cAMP production (5.5 +/- 0.4 pmol/well; mean +/- S.E.M., n = 3) in the cultures. Taken together, our findings suggest that in contrast to the inhibitory effect on pituitary corticotrophs, glucocorticoids enhance the production and secretion of beta EP from rat hypothalamic neurons by facilitating the stimulatory effect mediated, in part, through the adenylyl cyclase-cAMP system.

Hypothalamic Atrial Natriuretic Peptide Secretion Plasticity: Differential Modulation of Alpha and Beta Adrenoceptors

Increasing evidence suggests that atrial natriuretic peptide (ANP), a 28 amino acid peptide with biologically active 4-28 and 5-28 congeners, modulates salt-water homeostasis at both peripheral and central levels. In rats, immunoreactive (ir) ANP is found in hypothalamic (HT) neurons of preoptic and paraventricular regions rich in aminergic innervation. Employing a well-characterized perifusion model of rat HT explants, the acute effects of norepinephrine (NE) on HT release of irANP were examined. Pulsatile administration (20 min) of NE (10(-7) to 10(-5) M) induced a dose-related release of irANP. The stimulatory effect of 10(-5) M NE (2.66 +/- 0.54 pg/ml/HT, means +/- SE, n = 12) was abolished in the presence of 10(-7) M propranolol, a beta-antagonist, but was 50% higher when administrated with 10(-5) M phentolamine, an alpha-antagonist. Administration of equivalent doses of propranolol or phentolamine alone, consistently suppressed (40% below basal secretion rate, BSR) or stimulated (50% above BSR) irANP release, respectively. In addition, infusion of isoprenaline (10(-5) M), a beta-agonist, enhanced BSR by 45%, whilst phenylephrine (10(-5) M), an alpha-agonist, suppressed it by 25%. We conclude that in rat hypothalami (1) occupancy of the beta-adrenoceptor by its agonist stimulates irANP release, (2) alpha- and beta-adrenoceptors modulate irANP secretion in an opposing manner, and (3) the basal release of irANP is a product of the activation of alpha- and beta-adrenoceptors by their endogenous ligands.

The adenylyl cyclase-cyclic AMP system modulates morphological and functional development of hypothalamic β-endorphin neurons in culture

In rats, opioidergic β‐endorphin (βEP1–31) is produced and released from neurons of arcuate nuclei in the hypothalamus. Although the neuropeptide has been implicated in sexual maturation and stress‐induced reproductive dysfunction, the intra‐hypothalamic regulation of βEP neurons remains unclear. Employing long‐term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 days of treatment with 10 μM forskolin increased approximately 3‐fold (P<0.01) the proportion of immunoreactive (ir)‐ βEP positive neurons bearing neurites. In addition, treatment of forskolin also enhanced ir‐βEP release (634 ± 59 pg/well; mean β SE, n = 4, P<0.01) by 14‐fold and ir‐βEP content (119±13 pg/well; P<0.01) by 2‐fold above that of vehicle‐treated cultures; in both instances, the EC50 and the Emax, of forskolin were approximately 10μM and 100 μM, respectively. The forskolin‐stimulated release of ir‐βEP was mimicked by cholera toxin and (Bu)2cAMP treatment in a dose‐related manner, but not by pertussis toxin. Although by itself 3‐isobutyl‐1‐methyl‐xanthine (100μM) only doubled ir‐βEP secretion, it markedly potentiated the stimulatory effect of forskolin. This forskolin‐induced stimulation was reversible and in cultures re‐exposed to the same drug within the first 24 h period, there was a marked increase in the stimulated release of ir‐βEP (P < 0.05); re‐challenge of forskolin at later stages, however, induced a smaller but significant secretion of ir‐βEP (P<0.01) compared to that of vehicle‐treated control cultures. Sephadex G‐50 gel Chromatographie profile of the media prepared from forskolin‐treated cultures revealed a major ir‐βEP peak of 3 K M. Highperformance liquid chromatography analysis showed that ir‐βEP of the 3 K M, species was eluted with a retention time similar to that of synthetic rat βEP1–31. We thus conclude that the adenylyl cyclase‐cAMP system plays an important role in the modulation of βEP1–31 production and release from hypothalamic βEP neurons in culture. Furthermore, the functional responsiveness and the orphological development of these neurons are affected, at least in part, by the intrinsic activity of the adenylyi cyclase‐cAMP system.

ANP(5–28) is the major molecular species in hypophysial portal blood of the rat

We have previously demonstrated that the concentrations of immunoreactive atrial natriuretic peptide (IR-ANP) are significantly higher in hypophysial portal compared with peripheral blood of the rat, and that ANP suppresses the pituitary release of ACTH and beta-endorphin in vitro and in vivo. Using HPLC, we have now shown that the predominant species of IR-ANP in extracts of portal blood from adult male and female rats is ANP(5-28), whereas in peripheral blood, ANP(1-28) predominates. The ratio of ANP(5-28) in portal compared with peripheral blood was 4.2 in male and 4.8 in female animals.

D2 Receptors Mediate Dopamine Suppression of irANF Release and Pro-ANF mRNA Expression of Rat Hypothalamic Neurons in Culture

Although N-terminal truncated forms of atrial natriuretic factor (ANF) are produced and released from rat hypothalamic neurons, the intrahypothalamic regulation of these processes remains unclear. Employing a well-characterized hypothalamic cell culture system, we report here that dopamine, mediating through D2 receptors, inhibits the synthesis and release of ANF. In long-term cultures of hypothalamic neurons, daily treatment for 4 days with quinpirole, a D2 agonist, significantly suppressed the basal irANF release in a time-related and a dose-dependent manner. The ED50 and Emax of the drugs were 9.1 x 10(-8) M and 10(-5) M, respectively. This effect of quinpirole was mimicked by 10(-7) M of dopamine, a physiological ligand for D2 receptor. Furthermore, the suppressing effects of both quinpirole and dopamine were abolished by sulpiride, a D2 antagonist. Whereas 10(-6) M of forskolin treatment consistently enhanced the release of irANF through activating the adenylyl cyclase-cAMP system, this stimulatory effect was suppressed by quinpirole in a dose-related manner. In addition, the application of pertussis toxin, a bacterial toxin which inactivated G1 protein activity, reversed the suppressing effect of quinpirole or dopamine on irANF release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures as determined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D2 receptor was colocalized with irANF at a single cell level by double fluorescent staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Colocalization of atrial natriuretic factor and β-endorphin in rat thymic macrophages

Recent demonstration of immunoreactive (IR) atrial natriuretic factor (ANF) and beta-endorphin (beta-EP) in the thymus prompted a reexamination of the distribution and cellular localization of the two peptides within that tissue. Double labeling immunohistochemistry was carried out on gelatin-embedded cryostat thymic sections of adult male Sprague-Dawley rats. Cells stained positive with antiserum (S118), raised against rANF(1-28), were colocalized in > 95% of cases with immunofluorescent staining of IR-beta-EP(1-31). The cells were found sparsely distributed along the corticomedullary junction and in subcapsular regions. In 1- or 5-day monolayer cultures of adherent thymic cells, 15-20% of the cells stained positive for either IR-ANF or IR-beta-EP. Under these conditions, > 95% of IR-ANF or IR-beta-EP positive cells were also fluorescence stained for the rat macrophage marker ED-1. Thus, taken together with previous reports, our present findings suggest that, in the rat thymus, both ANF and beta-EP are produced by the same population of macrophages. To further investigate their presence in the thymus, the contents and molecular species of the two peptides were compared over the developmental period of the animal using well-characterized radioimmunoassays (RIA). Both peptides significantly increased their contents between day 2 and day 60. However, in terms of concentration, IR-ANF at day 2 was approximately 50% higher than day 16 and five times greater that at day 60; in comparison the concentration of IR-beta-EP remained relatively constant and the only significant difference from day 2 being a slight increase in the day 16 animals.(ABSTRACT TRUNCATED AT 250 WORDS)