Modulation of the cGMP signaling pathway by melatonin in pancreatic beta-cells.

Melatonin influences the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) signaling pathway in pancreatic beta-cells via a receptor-mediated mechanism. In the present study, it was determined how the regulation of cGMP concentrations by melatonin proceeds. The results provide evidence that melatonin acts via the soluble guanylate cyclase (sGC), as molecular investigations demonstrated that long-term incubation with melatonin significantly reduced the expression levels of the sGC mRNA in rat insulinoma beta-cells (INS1) cells, whereas mRNA expression of membrane guanylate cyclases was unaffected. Incubation with melatonin abolished the S-nitrosoacetyl penicillamine-induced increase of cGMP concentrations in INS1 cells. In addition, the cGMP-inhibitory effect of melatonin was reversed by preincubation with the sGC inhibitors 1H-(1,2,4)oxadiazolo(4,3-alpha)quinoxalin-1-one and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one. Nitric oxide (NO) production was not influenced after 1 hr of melatonin application, but was influenced after a 4 hr incubation period. Preincubation of INS1 cells with the NO synthase inhibitor N(G)-monomethyl-l-arginine did not abolish the cGMP-inhibitory effect of melatonin. Transcripts of cyclic nucleotide-gated (CNG) channels were significantly reduced after melatonin treatment in a dose-dependent manner, indicating the involvement of these channels in mediating the melatonin effect in INS1 cells. The results of this study demonstrate that melatonin mediates its inhibitory effect on cGMP concentrations in pancreatic beta-cells by inhibiting the sGC, but does not influence NO concentration or NO synthase activity in short-term incubation experiments. In addition, it was demonstrated that melatonin is involved in modulation of CNG channel mRNA.

Increased melatonin synthesis in pineal glands of rats in streptozotocin induced type 1 diabetes.

It is well-documented that melatonin influences insulin secretion. The effects are mediated by specific, high-affinity, pertussis-toxin-sensitive, G protein-coupled membrane receptors (MT(1) as well MT(2)), which are present in both the pancreatic tissue and islets of rats and humans, as well as in rat insulinoma cells (INS1). Via the Gi-protein-adenylatecyclase-3',5'-cyclic adenosine monophosphate (cAMP) and, possibly, the guanylatecyclase-cGMP pathways, melatonin decreases insulin secretion, whereas, by activating the Gq-protein-phospholipase C-IP(3) pathway, it has the opposite effect. For further analysis of the interactions between melatonin and insulin, diabetic rats were investigated with respect to melatonin synthesis in the pineal gland and plasma insulin levels. In this context, recent investigations have proven that type 2 diabetic rats and humans display decreased melatonin levels, whereas type 1 diabetic IDDM rats or those with diabetes induced by streptozotocin (STZ) of the present study show increased plasma melatonin levels and elevated AA-NAT-mRNA. Furthermore, the mRNA of pineal insulin receptors and beta1-adrenoceptors, including the clock genes Per1 and Bmal1 and the clock-controlled output gene Dbp, increases in both young and middle-aged STZ rats. The results therefore indicate that the decreased insulin levels in STZ-induced type 1 diabetes are associated with higher melatonin plasma levels. In good agreement with earlier investigations, it was shown that the elevated insulin levels observed in type 2 diabetes, are associated with decreased melatonin levels. The results thus prove that a melatonin-insulin antagonism exists. Astonishingly, notwithstanding the drastic metabolic disturbances in STZ-diabetic rats, the diurnal rhythms of the parameters investigated are maintained.

Melatonin and type 2 diabetes - a possible link?

The aim of the present study was to determine the existence of melatonin membrane receptors and to examine the mRNA expression of nuclear orphan receptors in human pancreatic tissue, in an effort to explain differences between type 2 diabetic and metabolically healthy patients. Molecular and immunocytochemical investigations established the presence of the melatonin membrane receptors MT1 and MT2 in human pancreatic tissue and, notably, also in the islets of Langerhans. Results of a calculation model to determine mRNA expression ratios, as well as subjective analysis of immunoreactions, showed elevated MT1 receptor expression in comparison with MT2 expression. mRNA transcript levels of melatonin receptors appeared to be significantly higher in type 2 diabetic patients than in a control group. An upregulation of receptor expression in type 2 diabetic patients was also observed in immunocytochemical investigations. In addition, transcripts of the nuclear orphan receptors RORalpha, RZRbeta, RORgamma and RevErbalpha were detected in human pancreatic tissue and islets. In correlation with membrane melatonin receptors, data indicate increased mRNA expression levels of RORalpha, RZRbeta, and RORgamma in type 2 diabetic patients. Thus, our data demonstrate the existence of the melatonin membrane receptors MT1 and MT2 as well as mRNA expression of nuclear orphan receptors in human pancreatic tissue, with upregulated expression levels in type 2 diabetic patients.

Characterization of the rhesus monkey superior olivary complex by calcium binding proteins and synaptophysin.

This study was performed in order to characterize the main nuclei of the rhesus monkey superior olivary complex by means of antibodies against the calcium binding proteins parvalbumin, calbindin and calretinin and the synaptic vesicle protein synaptophysin. These markers revealed the neuronal morphology and organization of nuclei located within the rhesus monkey superior olivary complex. The architectural details included the distribution of axonal terminals on neurons. The medial superior olivary nucleus was present as a column of neurons. No clear segregation of calretinin-positive terminals was noticed on the medial and lateral dendritic fields of these neurons. The lateral superior olivary nucleus was characterized by a distinct nuclear shape. Calretinin-, parvalbumin- or calbindin-positive terminals contacted somata and dendrites. The medial nucleus of trapezoid body could be clearly differentiated as a distinct region in the rhesus monkey superior olivary complex. Somata of that nucleus showed calbindin- and parvalbumin-labelling whereas somatic calyces of Held were reavealed by calretinin and synaptophysin labelling. The results are discussed with respect to the processing of acoustic information in primate species and their ability to hear high and low frequencies, which is reflected by anatomical correlates.

Characterization of the human superior olivary complex by calcium binding proteins and neurofilament H (SMI-32).

This study provides a morphologic characterization of the human superior olivary complex as revealed by immunohistochemistry by using antibodies against the calcium binding proteins parvalbumin, calbindin, calretinin, and the nonphosphorylated neurofilament H SMI-32. By combining these markers, it was possible to establish the neuronal architecture and details of the morphologic organization (including axonal terminals) of the different nuclei. The medial superior olivary nucleus is formed by a sheet of parallel-oriented cells. A clear segregation of axon terminals was noticed on the medially and laterally oriented dendrites of the mostly bipolar neurons. The lateral superior olivary nucleus lacked a distinct nuclear shape but was formed by several patches of rather irregularly arranged neurons. Calretinin or parvalbumin immunoreactive afferent terminals were observed which contacted somata or dendrites of these neurons. The immunolabeling also revealed the boundaries of the dorsal periolivary nucleus and morphologic detail of its neurons. A coherent nuclear structure that could be addressed as the medial nucleus of the trapezoid body was not identified by any single one or by combinations of the markers used. The data were also used to establish a three-dimensional-reconstruction of the three major subnuclei of the superior olivary complex. The results are discussed with respect to the possible role of the superior olivary complex in the processing of spatial acoustic information in the azimuthal plane.