Cultured fish epithelial cells are a source of alarm substance.

In various species of fishes, the importance of visual cues in the determination of environmental threat and subsequent predator avoidance is clear. Chemical cues also play an essential role facilitating predator avoidance. Among fish in the superorder Ostariophysi, club cells in the epidermis produce an alarm substance. Damage to the skin during a predation event releases an alarm substance (AS), which diffuses through the water column and binds to olfactory receptors of conspecifics. Fish then engage in a number of anti-predator behaviors that may include darting, schooling, or hiding. Behavioral responses to AS and physiological mechanisms that underlie those responses is an active area of study. However, because the precise chemical composition of the alarm substance is unknown, AS is not commercially available. Thus, when fish are challenged alarm substance in various experiments and assays it is obtained from skin extracts or via perfusion of shallow cuts in the epidermis. Both procedures are effective but require the animal to be sacrificed. In this manuscript, we report: •A non-invasive primary cell culture protocol to obtain alarm substance and does not require the model organism to be killed.•The demonstration of anti-predatory behaviors in fish exposed to alarm substance collected by this method.

Stimulation of the novel estrogen receptor-alpha intronic TERP-1 promoter by estrogens, androgen, pituitary adenylate cyclase-activating peptide, and forskolin, and autoregulation by TERP-1 protein.

The estrogen receptor-alpha (ERalpha) pituitary-specific variant, TERP-1, is regulated dramatically by physiological status. We examined hormonal regulation of the TERP-1 promoter in transient transfection assays in GH3 somatolactotrope cells. We found that 17beta-estradiol (E2), genistein, androgen, pituitary adenylate cyclase-activating peptide, and forskolin (FSK) all stimulated TERP-1 promoter activity, whereas progesterone had no effect. ERalpha bound to a palindromic estrogen response element (ERE) and two half-site EREs; mutation of any of these sites decreased basal expression and completely obliterated E2 stimulation. In contrast, mutation of an activator protein-1 site decreased basal and FSK-stimulated promoter activity, but not E2 or androgen stimulation. The pure antiestrogen ICI 182,780 suppressed E2 and genistein, but not FSK or androgen, stimulation. Similarly, mutation of the ERE palindrome or half-site EREs suppressed promoter stimulation by E2 and genistein, but not by androgen or FSK. Because TERP-1 levels regulate ERalpha function on model promoters, we tested TERP-1 modulation of its own and other physiological promoters. TERP-1 suppressed basal and E2-stimulated expression of its own promoter. TERP-1 suppression required the ERE regions of the promoter, and the dimerization domain of TERP-1. TERP-1 overexpression also suppressed E2 stimulation of the progesterone receptor and prolactin promoters. Thus, estrogens, androgen, and FSK can stimulate TERP-1 promoter activity, and increased TERP-1 expression modulates E2 stimulation of physiological promoters. These data suggest that TERP-1 regulation may play a significant role in modifying pituitary ERalpha responses.

Lack of involvement of dopamine and serotonin during the orphanin FQ/Nociceptin (OFQ/N)-induced prolactin secretory response.

The purpose of these studies was to examine possible mechanisms of Orphanin FQ/Nociceptin (OFQ/N)-induced prolactin release. We investigated the involvement of the dopaminergic neurons by quantifying DOPAC:DA levels in the median eminence and neurointermediate lobe following central administration of OFQ/N to female Sprague-Dawley rats. To specifically determine the involvement of the tuberoinfundibular dopaminergic neurons, immunocytochemical studies were conducted to visualize c-fos protein expression in the arcuate nucleus following central administration of OFQ/N. In addition, the role of serotonergic activation was examined in dose response studies using the selective serotonin antagonist ritansarin and the nonselective antagonist metergoline. Finally, the pharmacological specificity of the prolactin response was examined by pretreating animals with [Nphe1] NC (1-13)NH2, a drug reported to antagonize OFQ/N effects. The results of these studies indicate that the increase in prolactin release following central administration of OFQ/N does not inhibit tuberoinfundibular, tuberohypophyseal or periventricular hypophysial dopaminergic neuronal activity at 10 min after drug administration, a time when prolactin levels were significantly elevated. Furthermore, serotonergic activation is not involved since pharmacological blockade of serotonergic receptors did not alter the prolactin secretory response to OFQ/N. NC (1-13)NH2 did not antagonize the stimulatory effects of OFQ/N on prolactin secretion. The neural effects of OFQ/N on dopaminergic neuronal activity may occur following a different time course than that of the prolactin increase.

The estrogen receptor (ER)alpha variant Delta5 exhibits dominant positive activity on ER-regulated promoters in endometrial carcinoma cells.

Estrogen receptor (ER)alpha is a ligand-inducible transcription factor that mediates the physiological effects of 17beta-estradiol (E2). In the uterus, E2 is involved in tissue growth, maintenance, and differentiation. Delta5ERalpha (Delta5) is an ERalpha variant protein expressed in uterine tumors but not in normal tissue. We examined the transcriptional activity of Delta5 and its modulation of human ERalpha basal and E2-stimulated activity in Ishikawa cells, an endometrial cancer cell line. In transient transfection assays, Delta5 increased basal activity of an estrogen response element-containing promoter in the absence or presence of ERalpha but lessened stimulation by ERalpha and E2. Effects of Delta5 were not limited to model reporters, given that cyclin D1 and complement 3 promoters were similarly affected. Increases in basal transcription required dimerization and DNA binding of Delta5, whereas decreased E2 stimulation with ERalpha required only DNA binding. Decreased ligand stimulation was not unique to E2 but also applied to the selective ER modulators tamoxifen and genistein. However, promoter stimulation by epidermal growth factor is retained with Delta5. The ERalpha coactivator small nuclear ring finger protein is expressed in Ishikawa cells and uterine tumors, and it enhances effects of Delta5 alone and with ERalpha on basal activity of an estrogen response element reporter. Thus, in the presence of Delta5 plus ERalpha, there is a lower transcriptional response to E2 and SERMS, but stimulation by epidermal growth factor is retained. The expression of Delta5 in uterine carcinoma may provide a mechanism by which tumors could maintain expression of E2-responsive genes in the absence of E2.

[Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) does not antagonize orphaninFQ/nociceptin-induced prolactin release.

The specificity of the orphaninFQ (OFQ)/nociceptin (N)-induced prolactin increase was determined in male and female rats by pretreating animals with different doses of [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2), a compound originally reported to be a specific OFQ/N antagonist. In addition, the effect of naloxone pretreatment on OFQ/N-induced prolactin release was examined to determine if OFQ/N's effects were mediated by opiate receptors. Furthermore, dose response studies using [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) only were performed to determine potential agonist activity of this drug. Finally, growth hormone (GH) levels were determined as an index of specificity of the prolactin response. Our results confirm previous findings that OFQ/N potently stimulates prolactin release and that a gender difference exists in the magnitude of the response, with females showing a much greater response than male rats. The endocrine response is specific because OFQ/N potently stimulated prolactin, but not GH secretion. The prolactin response is not mediated by actions at opiate receptors because naloxone did not inhibit OFQ/N's effects on prolactin release. However, [Phe(1)Psi(CH(2)-NH) Gly2]NC(1-13) NH(2) did not antagonize OFQ/N's effects on prolactin release. Indeed, this drug acted as a potent agonist. Demonstrating pharmacological specificity of OFQ/N's effects on prolactin release awaits the development of more selective, specific antagonists.