Suppressed heat shock protein response in the kidney of exercise-trained diabetic rats.

Impaired expression of heat shock proteins (HSPs) and increased oxidative stress may contribute to the pathophysiology of diabetes by disrupted tissue protection. Acute exercise induces oxidative stress, whereas exercise training up-regulates endogenous antioxidant defenses and HSP expression. Although diabetic nephropathy is a major contributor to diabetic morbidity, information regarding the effect of HSPs on kidney protection is limited. This study evaluated the effects of eight-week exercise training on kidney HSP expression and markers of oxidative stress at rest and after acute exercise in rats with or without streptozotocin-induced diabetes. Induction of diabetes increased DNA-binding activity of heat shock factor-1, but decreased the expression of HSP72, HSP60, and HSP90. The inflammatory markers IL-6 and TNF-alpha were increased in the kidney tissue of diabetic animals. Both exercise training and acute exercise increased HSP72 and HSP90 protein levels only in non-diabetic rats. On the other hand, exercise training appeared to reverse the diabetes-induced histological changes together with decreased expression of TGF-beta as a key inducer of glomerulosclerosis, and decreased levels of IL-6 and TNF-alpha. Notably, HSP72 and TGF-beta were negatively correlated. In conclusion, impaired HSP defense seems to contribute to kidney injury vulnerability in diabetes and exercise training does not up-regulate kidney HSP expression despite the improvements in histopathological and inflammatory markers.

A rhodopsin-based model for melatonin recognition at its G protein-coupled receptor.

The recent elucidation of the primary structures of different melatonin receptors as well as the deduction of the secondary structure of rhodopsin has allowed us to construct a model for melatonin recognition at its G protein-coupled receptor. To achieve this, we have used the quantum mechanics method Austin model 1 to fully optimize the structures of melatonin and several analogs. We also synthesized three compounds and used the three-dimensional analysis comparative molecular field analysis (CoMFA) to generate a model for the structure-activity relationships of melatonin and 27 melatonin-like compounds. This model predicted with good accuracy the affinities of the synthesized compounds for the melatonin receptor. We propose that recognition of the functional moieties of melatonin occurs through specific interaction of these moieties with fully conserved amino acid residues present in transmembrane helices V, VI and VII of the melatonin receptor. These residues are not found in other members of the G protein-coupled receptor family. The rhodopsin-based model can explain the importance of some structural features of melatonin and related active compounds.

Mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces circulating melatonin levels in the rat.

We have previously shown that the prototype for halogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), diminishes serum melatonin concentration at the same dose in both the most TCDD-susceptible (Long-Evans, Turku AB; L-E) and the most TCDD-resistant (Han/Wistar, Kuopio; H/W) rat strain. The change developed within 24 h and persisted for at least 28 days after TCDD exposure; was independent of the time of day and was not associated with any morphological damage to the pineal gland. In the present study, we investigated the mechanism of this endocrine effect. Despite a 40-50% decrease in circulating melatonin levels, the pineal content of melatonin, serotonin and 5-hydroxyindole acetic acid remained unaltered and the rate-limiting enzyme of pineal melatonin biosynthesis, N-acetyltransferase, displayed only a relatively minor suppression in activity (30%) in TCDD-treated L-E rats. Likewise, TSDD did not influence the ability of pineal glands from L-E rats to synthesize and secrete melatonin in ex vivo or in vitro experiments. TCDD accelerated the disappearance of exogenous melatonin from the serum in both rat strains. This enhancement probably did not originate in the liver, because liver perfusion studies revealed that even control rat livers were capable of total melatonin clearance in spite of the fact that the melatonin concentration far exceeded physiological levels. Urine excretion of the normal main metabolite of melatonin, 6-hydroxymelatoninsulfate, was reduced by TCDD treatment in both strains. This was accompanied by an altered HPLC pattern of metabolites, especially in H/W rats. We conclude that TCDD decreases serum melatonin levels in rats by enhancing the peripheral, evidently extrahepatic, metabolism of the hormone.

Melatonin receptor genes.

Melatonin is produced rhythmically by the pineal gland and the retina with increased synthesis during darkness. Pineal melatonin serves as the 'chemical expression of darkness' conveying information on the ambient light-dark cycle into rhythmic bodily functions. On-going debate on modes and sites of action ranges from views of melatonin affecting each and every cell ('cure-all') to those of melatonin having restricted actions through specific high-affinity receptors. The present review deals with the latter view. The use of 2-[125I]-iodomelatonin has allowed the exact localization and characterization of high-affinity melatonin receptors that signal through the G(i/o) class of G proteins. Molecular cloning of melatonin receptor genes has confirmed that most, if not all, high-affinity melatonin-binding sites represent the G-protein-coupled melatonin receptors. Based on sequence dissimilarities, melatonin receptors are classified into three subtypes, Mel1a, Mel1b and Mel1c. A distribution wider than originally thought of melatonin receptors in the human brain and peripheral sites has brought these receptors into focus of several drug companies, promising exciting times for research on melatonin and new therapeutic possibilities.

Cholinergic signaling in the rat pineal gland.

1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotonin N-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion. 2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland. 3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs). 4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.

Mutagenesis of human Mel1a melatonin receptor expressed in yeast reveals domains important for receptor function.

A yeast functional colorimetric assay was employed to test the effects of site-directed point mutations on the function of the human Mel1a melatonin receptor. Seven mutants were created in transmembrane domains III, V, and VII of the receptor to test the rhodopsin-based model of melatonin recognition. Two mutants in transmembrane domains III and VI were created to investigate the mechanisms of G protein activation in the melatonin receptor. Mutations in transmembrane domain V either potentiated agonist efficiencies (H195A) or totally abolished all responses to tested compounds (V192T+H195A). Mutation N124A in the conserved NRY motif in the end of transmembrane domain III seriously impaired receptor activation. Several mutants were found to have decreased ability to activate functional responses, reflecting the importance of these residues for receptor function. These data also suggest that activation of the receptor involves interaction of the 5-methoxy group of melatonin with the conserved histidine H195 in transmembrane domain V.