Auto-inhibition of adenylyl cyclase 9 (AC9) by an isoform-specific motif in the carboxyl-terminal region.

Trans-membrane adenylyl cyclase (tmAC) isoforms show markedly distinct regulatory properties that have not been fully explored. AC9 is highly expressed in vital organs such as the heart and the brain. Here, we report that the isoform-specific carboxyl-terminal domain (C2b) of AC9 inhibits the activation of the enzyme by Gs-coupled receptors (GsCR). In human embryonic kidney cells (HEK293) stably overexpressing AC9, cAMP production by AC9 induced upon the activation of endogenous ß-adrenergic and prostanoid GsCRs was barely discernible. Cells expressing AC9 lacking the C2b domain showed a markedly enhanced cAMP response to GsCR. Subsequent studies of the response of AC9 mutants to the activation of GsCR revealed that residues 1268-1276 in the C2b domain were critical for auto-inhibition. Two main species of AC9 of 130 K and ≥ 170 K apparent molecular weight were observed on immunoblots of rodent and human myocardial membranes with NH2-terminally directed anti-AC9 antibodies. The lower molecular weight AC9 band did not react with antibodies directed against the C2b domain. It was the predominant species of AC9 in rodent heart tissue and some of the human samples. There is a single gene for AC9 in vertebrates, moreover, amino acids 957-1353 of the COOH-terminus are encoded by a single exon with no apparent signs of mRNA splicing or editing making it highly unlikely that COOH-terminally truncated AC9 could arise through the processing or editing of mRNA. Thus, deductive reasoning leads to the suggestion that proteolytic cleavage of the C2b auto-inhibitory domain may govern the activation of AC9 by GsCR.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation.

Nursing has important physiological and psychological consequences on mothers during the postpartum period. Tuberoinfundibular peptide of 39 residues (TIP39) may contribute to its effects on prolactin release and maternal motivation. Since TIP39-containing fibers and the receptor for TIP39, the parathyroid hormone 2 receptor (PTH2 receptor) are abundant in the arcuate nucleus and the medial preoptic area, we antagonized TIP39 action locally to reveal its actions. Mediobasal hypothalamic injection of a virus encoding an antagonist of the PTH2 receptor markedly decreased basal serum prolactin levels and the suckling-induced prolactin release. In contrast, injecting this virus into the preoptic area had no effect on prolactin levels, but did dampen maternal motivation, judged by reduced time in a pup-associated cage during a place preference test. In support of an effect of TIP39 on maternal motivation, we observed that TIP39 containing fibers and terminals had the same distribution within the preoptic area as neurons expressing Fos in response to suckling. Furthermore, TIP39 terminals closely apposed the plasma membrane of 82% of Fos-ir neurons. Retrograde tracer injected into the arcuate nucleus and the medial preoptic area labeled TIP39 neurons in the posterior intralaminar complex of the thalamus (PIL), indicating that these cells but not other groups of TIP39 neurons project to these hypothalamic regions. We also found that TIP39 mRNA levels in the PIL markedly increased around parturition and remained elevated throughout the lactation period, demonstrating the availability of the peptide in postpartum mothers. Furthermore, suckling, but not pup exposure without physical contact, increased Fos expression by PIL TIP39 neurons. These results indicate that suckling activates TIP39 neurons in the PIL that affect prolactin release and maternal motivation via projections to the arcuate nucleus and the preoptic area, respectively.

Nesfatin-1/NUCB2 may participate in the activation of the hypothalamic-pituitary-adrenal axis in rats.

Nesfatin-1 is an anorexigenic peptide originating from nucleobinding-2 (NUCB2) protein. Nesfatin-1/NUCB2-immunoreactive neurons are present in the hypothalamic paraventricular nucleus, the center of the stress-axis, and in the medullary A1 and A2 catecholamine cell groups. The A1 and A2 cell groups mediate viscerosensory stress information toward the hypothalamic paraventricular nucleus. They contain noradrenaline, but subsets of these neurons also express prolactin-releasing peptide acting synergistically with noradrenaline in the activation of the hypothalamic paraventricular nucleus during stress. We investigated the possible role of nesfatin-1/NUCB2 in the stress response. Intracerebro-ventricular administration of nesfatin-1 elevated both plasma adrenocorticotropin and corticosterone levels, while in vitro stimulation of the hypophysis was ineffective. Single, long-duration restraint stress activated (Fos positivity) many of the nesfatin-1/NUCB2-immunoreactive neurons in the parvocellular part of the hypothalamic paraventricular nucleus, evoked nesfatin-1/NUCB2 mRNA expression in the parvocellular part of the paraventricular nucleus and in the A1, but not in the A2 cell group. Nesfatin-1/NUCB2 was shown to co-localize in a high percentage of prolactin-releasing peptide producing neurons, in both medullary catecholamine cell groups further supporting its involvement in the stress response. Finally, bilateral adrenalectomy evoked an increasing nesfatin-1/NUCB2 mRNA expression, indicating that it is under the negative feedback of adrenal steroids. These data provide the first evidence for possible participation of nesfatin-1/NUCB2 in the stress-axis regulation, both at the level of the brainstem and in the hypothalamus.

Demonstration of autoantibody binding to muscarinic acetylcholine receptors in the salivary gland in primary Sjögren's syndrome.

A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögren's syndrome (pSS) has been suggested. However, the binding of these antibodies to the receptors in the target tissues has not yet been demonstrated. In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studied with light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivo deposited antibodies in pSS salivary glands was also investigated. Light microscopic immunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membrane of acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelial cell membranes. With electron microscopy, the autoantibody binding was observed to be localised to the junctions of epithelial cell membranes with nerve endings, both in normal and pSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivary glands.

Non-NMDA glutamate receptor antagonist injected into the hypothalamic paraventricular nucleus blocks the suckling stimulus-induced release of prolactin.

The aim of the present investigations was to test the involvement of the glutamatergic innervation of the hypothalamic paraventricular nucleus in the prolactin response to the suckling stimulus. A non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-dione disodium (CNQX), or an NMDA receptor antagonist, dizocipine hydrogen malate (MK-801), was injected bilaterally into the hypothalamic paraventricular nucleus of lactating freely moving rats before the end of a 4-h separation of the dams from their pups. The litters were then returned. Blood samples for prolactin were taken at different time points. The effect of the non-NMDA receptor antagonist was also tested in animals receiving the drug bilaterally into the dorsomedial nucleus area or the arcuate nucleus. Bilateral injection of CNQX into the paraventricular nucleus blocked the elevation in plasma prolactin concentration induced by the suckling stimulus. In contrast, bilateral administration of the NMDA receptor antagonist MK-801 into the paraventricular nucleus or bilateral injection of CNQX into the dorsomedial nucleus area or the arcuate nucleus did not interfere with the prolactin response to the suckling stimulus. The findings indicate that the glutamatergic innervation of the paraventricular nucleus is involved in the mediation of the neural signal of the suckling stimulus inducing prolactin release.

Immobilization stress-induced increase in plasma catecholamine levels is inhibited by a prolactoliberin (salsolinol) administration.

Catecholamines (CAs) are significantly involved in the regulation of homeostasis of the organism at rest and especially during stressful situations. Stress induces increases in plasma CA (epinephrine and norepinephrine) levels and prolactin (PRL) release from the adenopituitary. We have recently observed that salsolinol, which is produced by the neuro-intermediate lobe of the pituitary gland and by the hypothalamus, can selectively release PRL. Salsolinol is therefore considered to be a putative endogenous PRL-releasing factor. Based on the similarity of CA and PRL responses to stressors, we investigated whether salsolinol plays a role in the regulation of plasma CA levels at rest and of CA release induced by immobilization stress (IMO). Salsolinol did not affect CA baseline levels; however, it did inhibit IMO-induced CA release. Thus, the present study shows for the first time that salsolinol is not only a PRL-releasing factor but is also a potent inhibitor of stress-induced release of epinephrine and norepinephrine.

Physiological role of salsolinol: its hypophysiotrophic function in the regulation of pituitary prolactin secretion.

We have recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) produced by hypothalamic neurons can selectively release prolactin from the anterior lobe (AL) of the pituitary gland. Moreover, high affinity binding sites for SAL have been detected in areas, like median eminence (ME) and the neuro-intermediate lobe (NIL) that are known terminal fields of the tuberoinfundibular DAergic (TIDA) and tuberohypophysial (THDA)/periventricular (PHDA) DAergic systems of the hypothalamus, respectively. However, the in situ biosynthesis and the mechanism of action of SAL are still enigmatic, these observations clearly suggest that sites other than the AL might be targets of SAL action. Based on our recent observations it may be relevant to postulate that an "autosynaptocrine" regulatory mechanism functioning at the level of the DAergic terminals localized in both the ME and NIL, may play a role in the hypophyseotrophic regulation of PRL secretion. Furthermore, SAL may be a key player in these processes. The complete and precise mapping of these intra-terminal mechanisms should help us to understand the tonic DAerg regulation of PRL secretion. Moreover, it may also give insight into the role of pre-synaptic processes that most likely have distinct and significant functional as well as pathological roles in other brain areas using DAergic neurotransmission, like striatonigral and mesolimbic systems.