Colocalization of connexin 36 and corticotropin-releasing hormone in the mouse brain.

BACKGROUND: Gap junction proteins, connexins, are expressed in most endocrine and exocrine glands in the body and are at least in some glands crucial for the hormonal secretion. To what extent connexins are expressed in neurons releasing hormones or neuropeptides from or within the central nervous system is, however, unknown. Previous studies provide indirect evidence for gap junction coupling between subsets of neuropeptide-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Here we employ double labeling and retrograde tracing methods to investigate to what extent neuroendocrine and neuropeptide-containing neurons of the hypothalamus and brainstem express the neuronal gap junction protein connexin 36. RESULTS: Western blot analysis showed that connexin 36 is expressed in the PVN. In bacterial artificial chromosome transgenic mice, which specifically express the reporter gene Enhanced Green Fluorescent Protein (EGFP) under the control of the connexin 36 gene promoter, EGFP expression was detected in magnocellular (neuroendocrine) and in parvocellular neurons of the PVN. Although no EGFP/connexin36 expression was seen in neurons containing oxytocin or vasopressin, EGFP/connexin36 was found in subsets of PVN neurons containing corticotropin-releasing hormone (CRH), and in somatostatin neurons located along the third ventricle. Moreover, CRH neurons in brainstem areas, including the lateral parabrachial nucleus, also expressed EGFP/connexin 36. CONCLUSION: Our data indicate that connexin 36 is expressed in subsets of neuroendocrine and CRH neurons in specific nuclei of the hypothalamus and brainstem.

In vitro metabolism of the opioid tilidine and interaction of tilidine and nortilidine with CYP3A4, CYP2C19, and CYP2D6.

Tilidine is one of the most widely used narcotics in Germany and Belgium. The compound's active metabolite nortilidine easily penetrates the blood-brain barrier and activates the mu-opioid receptor. Thus far, the enzymes involved in tilidine metabolism are unknown. Therefore, the aim of our study was to identify the cytochrome P450 isozymes (CYPs) involved in N-demethylation of tilidine in vitro. We used human liver microsomes as well as recombinant CYPs to investigate the demethylation of tilidine to nortilidine and quantified nortilidine by liquid chromatography-tandem mass spectrometry. Inhibition of CYPs was quantified with commercial kits. Moreover, inhibition of ABCB1 and ABCG2 was investigated. Our results demonstrated that N-demethylation of tilidine to nortilidine followed a Michaelis-Menten kinetic with a K(m) value of 36 +/- 13 microM and a v(max) value of 85 +/- 18 nmol/mg/h. This metabolic step was inhibited by CYP3A4 and CYP2C19 inhibitors. Investigations with recombinant CYP3A4 and CYP2C19 confirmed that the demethylation of tilidine occurs via these two CYPs. Inhibition assays demonstrated that tilidine and nortilidine can also inhibit CYP3A4, CYP2C19, CYP2D6, ABCB1, but not ABCG2, whereas inhibition of CYP2D6 and possibly also of CYP3A4 might be clinically relevant. By calculating the metabolic clearance based on the in vitro and published in vivo data, CYP3A4 and CYP2C19 were identified as the main elimination routes of tilidine. In vivo, drug-drug interactions of tilidine with CYP3A4 or CYP2C19 inhibitors are to be anticipated, whereas substrates of CYP2C19, ABCB1, or ABCG2 will presumably not be influenced by tilidine or nortilidine.

Two forces for arousal: Pitting hunger versus circadian influences and identifying neurons responsible for changes in behavioral arousal.

The mechanisms underlying CNS arousal in response to homeostatic pressures are not known. In this study, we pitted two forces for CNS arousal against each other (circadian influences vs. restricted food availability) and measured the neuronal activation that occurs in a behaviorally defined group of animals that exhibited increased arousal in anticipation of feeding restricted to their normal sleeping time. The number of c-FOS+ neurons was significantly increased only in the ventromedial nucleus of the hypothalamus (VMH) in these mice, compared with control animals whose feeding was restricted to their normal active and feeding time (P < 0.01). Because the activation of VMH neurons coincides with the earliest signs of behavioral arousal preceding a change in meal time, we infer that VMH activation is involved in the increased arousal in anticipation of food.