Transcription factor CREB3L1 regulates vasopressin gene expression in the rat hypothalamus.

Arginine vasopressin (AVP) is a neurohypophysial hormone regulating hydromineral homeostasis. Here we show that the mRNA encoding cAMP responsive element-binding protein-3 like-1 (CREB3L1), a transcription factor of the CREB/activating transcription factor (ATF) family, increases in expression in parallel with AVP expression in supraoptic nuclei (SONs) and paraventicular nuclei (PVNs) of dehydrated (DH) and salt-loaded (SL) rats, compared with euhydrated (EH) controls. In EH animals, CREB3L1 protein is expressed in glial cells, but only at a low level in SON and PVN neurons, whereas robust upregulation in AVP neurons accompanied DH and SL rats. Concomitantly, CREB3L1 is activated by cleavage, with the N-terminal domain translocating from the Golgi, via the cytosol, to the nucleus. We also show that CREB3L1 mRNA levels correlate with AVP transcription level in SONs and PVNs following sodium depletion, and as a consequence of diurnal rhythm in the suprachiasmatic nucleus. We tested the hypothesis that CREB3L1 activates AVP gene transcription. Both full-length and constitutively active forms of CREB3L1 (CREB3L1CA) induce the expression of rat AVP promoter-luciferase reporter constructs, whereas a dominant-negative mutant reduces expression. Rat AVP promoter deletion constructs revealed that CRE-like and G-box sequences in the region between -170 and -120 bp are important for CREB3L1 actions. Direct binding of CREB3L1 to the AVP promoter was shown by chromatin immunoprecipitation both in vitro and in the SON itself. Injection of a lentiviral vector expressing CREB3L1CA into rat SONs and PVNs resulted in increased AVP biosynthesis. We thus identify CREB3L1 as a regulator of AVP transcription in the rat hypothalamus.

Effect of hyperosmotic stress on the gene expression and activity of neuronal nitric oxide synthase (nNOS) in the preoptic-hypothalamic neurosecretory system of the euryhaline fish Oreochromis mossambicus.

We examined the effects of hyperosmotic stress on the gene expression and activity of neuronal nitric oxide synthase (nNOS) in the preoptic/hypothalamic neurosecretory system of the euryhaline tilapia Oreochromis mossambicus (Mozambique tilapia) by means of semiquantitative RT-PCR and NADPHd histochemistry. Expression of nos1 was rapidly and transiently up-regulated in the preoptic region and hypothalamus in response to a salinity change (70% seawater, SW). Expression levels increased 4 h after the salinity change and then returned to basal levels within 8 h of the hyperosmotic challenge. NADPHd histochemistry revealed that positive magnocellular and gigantocellular preoptic neurons increased in number 4 h after the salinity change, while the number of parvocellular preoptic neurons reactive for NADPHd showed no significant change. These results indicate that the nNOS gene expression and NOS activity are stimulated in the preoptic/ hypothalamic neurosecretory system in response to hyperosmotic stress and suggest that NO influences neuronal responses to short-term osmotic stimulation in euryhaline fish.

Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus.

A constitutive NOS complementary DNA (cDNA) was partially cloned by RT-PCR from the brain of a teleost, the Nile tilapia (Oreochromis niloticus), using degenerate primers against conserved regions of NOS. The predicted 206-long amino acid sequence showed a high degree of identity with other vertebrate neuronal NOS (nNOS) protein sequences. In addition, phylogenetic analysis revealed that Nile tilapia NOS clustered with other known nNOS. Using the coupled reaction of semi-quantitative RT-PCR and Southern blotting, the basal tissue expression pattern of the cloned nNOS gene was investigated in discrete areas of the central nervous system (CNS) and in the heart and skeletal muscle tissue. As revealed, expression of nNOS transcripts was detected in all the CNS regions examined, whereas nNOS gene was not expressed in the heart and skeletal muscle. The distribution pattern of nNOS gene expression showed the highest expression levels in the forebrain followed by the optic tectum, the brainstem and the spinal cord, whereas scarce expression was detected in the cerebellum. Cellular expression of nNOS mRNA was analyzed in the CNS by means of in situ hybridization. According to the RT-PCR results, most nNOS mRNA expressing neurons are localized in the telencephalon and diencephalon, whereas in the mesencephalic optic tectum, the brainstem and the spinal cord, nNOS mRNA expressing neurons are relatively more scattered. A very low hybridization signal was detected in the cerebellar cortex. These results suggest that NO is involved in numerous brain functions in teleosts.

Co-localization of neuronal nitric oxide synthase with arginine-vasotocin in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus.

This study provides evidence that, in the preoptic-hypothalamo-hypophysial system of the teleost Oreochromis niloticus, several sub-populations of arginine-vasotocin (AVT)-producing neurons and neurosecretory fibers terminals express neuronal nitric oxide synthase (nNOS)-like molecules. The co-localization between nNOS and AVT was demonstrated by means of double immunofluorescence staining with the confocal microscope. This study is the first to provide evidence that nNOS may be co-localized with AVT in neurons of a non-mammalian vertebrate.

Expression of nitric oxide synthase in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus.

In the present study, we have analyzed the expression of nitric oxide synthase (NOS) in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus. The assay for enzyme activity demonstrated that a constitutive NOS activity is present both in soluble and particulate fractions of the homogenates of diencephalons. Western blot analysis using an antibody against the N-terminus of human nNOS revealed two bands both in the supernatant and in the pellet. One band co-migrates at approximately 150 kDa with that detected in the rat cerebellum homogenates and presumably corresponds to neuronal NOS (nNOS) of mammals. The additional band, which migrates at approximately 180 kDa, might be attributed to an alternatively spliced nNOS isoform. Using NADPH diaphorase (NADPHd) histochemistry in combination with NOS immunohistochemistry, nNOS expression has been detected in preoptic nuclei, hypophysiotrophic nuclei of the ventral hypothalamus, and the pituitary gland. Various degrees of dissociation of NADPHd activity and nNOS immunoreactivity have been detected that could be attributed to the expression of different subtypes of nNOS in the preoptic/hypothalamo/hypophysial system of tilapia. In this paper, we also investigated the colocalization of nNOS with arginine-vasotocin (AVT) by means of immunolabeling of consecutive sections. Results suggest that NO may be colocalized with AVT in a subpopulation of neurosecretory neurons. Present findings suggest that nitric oxide (NO) is implicated in the modulation of hormone release in teleosts in a similar way to mammals.