Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes.

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Modulation of Ca2+ oscillation and melatonin secretion by BKCa channel activity in rat pinealocytes.

The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 µM paxilline or 100 nM iberiotoxin). Nicotine (100 µM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) oscillations were abolished by BKCa channel opener [3 µM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and ß3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca(2+) oscillations due to depolarization-synchronized Ca(2+) influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism.

Female spontaneously diabetic Torii fatty rats develop nonalcoholic steatohepatitis-like hepatic lesions.

AIM: To investigate the histological features of the liver in spontaneously diabetic Torii (SDT) fatty rats compared with age-matched Sprague-Dawley (SD) rats. METHODS: Female SDT Lepr(fa) (SDT fatty) rats and age-matched SD rats were fed ad libitum. Body weight and biochemical parameters, such as serum glucose, triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels as well as fatty acid and TG accumulation in the liver were evaluated at 8 wk of age in the non-fasting state and at 8-wk intervals from 8 to 40 wk of age. Histopathological examinations of the liver were performed using hematoxylin and eosin and Sirius Red staining as well as double staining for ED-1 and toluidine blue. The expression of genes involved in TG synthesis, inflammation, and fibrosis was examined in the liver. RESULTS: SDT fatty rats showed significantly increased body weight compared with SD rats. Serum glucose, TG, and TC levels were significantly higher in SDT fatty rats compared with SD rats. The serum AST and ALT levels in SDT fatty rats were significantly elevated at 8 wk of age compared with the levels in SD rats. Hepatic TG content was marked in SDT fatty rats from 8 to 32 wk of age. Histopathologically, severe hepatosteatosis accompanied by inflammation was observed at 8 wk of age, and fibrosis started to occur at 32 wk of age. Furthermore, Sirius Red and ED-1 staining were increased in the liver at 32 wk of age. Hepatic gene expression related to TG synthesis, inflammation and fibrosis tended to increase in SDT fatty rats compared with SD rats, and the gene expression related to TG secretion was decreased in SDT fatty rats compared with SD rats. CONCLUSION: Female SDT fatty rats have the potential to become an important animal model of nonalcoholic steatohepatitis with type 2 diabetes and obesity.

Spontaneous and nicotine-induced Ca2+ oscillations mediated by Ca2+ influx in rat pinealocytes.

The pineal gland regulates circadian rhythm through the synthesis and secretion of melatonin. The rise of intracellular Ca(2+) concentration ([Ca(2+)]i) following nicotinic acetylcholine receptor (nAChR) stimulation due to parasympathetic nerve activity downregulates melatonin production. Important characteristics and roles of Ca(2+) mobilization due to nAChR stimulation remain to be clarified. We report here that spontaneous Ca(2+) oscillations can be observed in ∼15% of the pinealocytes in slice preparations from rat pineal glands when this dissociation procedure is done within 6 h from a dark-to-light change. The frequency and half-life of [Ca(2+)]i rise were 0.86 min(-1) and 19 s, respectively. Similar spontaneous Ca(2+) oscillations were recorded in 17% of rat pinealocytes that were primary cultured for several days. Simultaneous measurement of [Ca(2+)]i and membrane potential revealed that spontaneous Ca(2+) oscillations were triggered by periodic membrane depolarizations. Spontaneous Ca(2+) oscillations in cultured pinealocytes were abolished by extracellular Ca(2+) removal or application of nifedipine, a blocker of voltage-dependent Ca(2+) channel (VDCC). In contrast, blockers of intracellular Ca(2+)-release channels, 2-aminoethoxydiphenylborate and ryanodine, have no effect. Our results also reveal that, in 23% quiescent pinealocytes, Ca(2+) oscillations were observed following the withdrawal of nicotine. Norepinephrine-induced melatonin secretion from whole pineal glands was significantly decreased by the coapplication of acetylcholine (ACh). This inhibitory effect of ACh was attenuated by nifedipine. In conclusion, both spontaneous and evoked Ca(2+) oscillations are due to membrane depolarization following activation of VDCCs. This consists of VDCC α1F subunit, and the associated Ca(2+) influx can strongly regulate melatonin secretion in pineal glands.

[Characteristics of reactions of target enzymes of drugs containing new active ingredients (NAIs) and changes of the NAIs approvals over three decades from 1980 to 2009 in Japan].

We analyzed the target enzymes of drugs containing new active ingredients (NAIs) focusing on the characteristics of the reaction of the enzymes, and attempted to prepare a comprehensive overview of the changes in the approval of the drugs over a 3 decade period from 1980 to 2009 in Japan. Fifty-eight enzyme therapeutic targets of 235 NAIs were found to be primarily distributed to EC1, EC2 and EC3 classes, and the enzymes were unevenly distributed to 21 of 64 sub-classes and 34 of 264 sub-subclasses, respectively, classified by the enzyme nomenclature of the International Union of Biochemistry and Molecular Biology (IUBMB). Though the number of NAIs approved that targeted EC1 class enzymes decreased over the time frame studied, the number of NAIs which targeted EC2 class enzymes increased over the same time. There was no clear change in the number of NAIs which targeted other classes of enzymes. More than 80% of NAIs targeted one enzyme as a primary target, and less than 20% of NAIs targeted more than two enzymes as secondary targets. NAIs which primarily targeted EC2 and EC3 class enzymes had more secondary target enzymes than other classes. The results of this study revealed the characteristics of the reactions of the target enzymes of NAIs and the changes in NAI approvals in Japan during the three decades studied, and provided information regarding possible future trends of NAIs.

[New drug approvals over three decades from 1980 to 2009 in Japan--their therapeutic targets and biochemical properties].

We analyzed the therapeutic target molecules and biochemical properties of 893 new drugs with new molecular entities approved over three decades from 1980 to 2009 in Japan. According to our analysis of the therapeutic targets, 26.2% of new drugs were enzymes. Membrane receptors were found to be the second-most frequent molecular targets of the new drugs, representing 25.3% of the new drugs approved. The biochemical properties of the drugs were found to have changed over time. Though the total number of new drugs approved from the year 2000 was smaller than that in the 1980s and 1990s, the number of new protein drugs approved in the 2000s, largely recombinant bioactive substances and monoclonal antibodies, increased significantly compared with those approved in the 1980s and 1990s. The results obtained in this study indicated changes in the therapeutic targets, biochemical properties and therapeutic areas of new drugs approved over the last 30 years and suggest the aspects of the future development of new drugs.

Porogen approach for the fabrication of plasma-polymerized nanoporous polysiloxane films.

Nanoporous polysiloxane films were fabricated by plasma polymerization of hexamethyldisiloxane mixed with cyclohexane under different conditions. The pores were generated through the elimination of carbonaceous aggregates (porogen) by annealing at 600 degrees C. Results of spectroscopic ellipsometry, Fourier transform infrared spectroscopy, and positron annihilation lifetime spectroscopy suggest that not only film porosity but also average pore size depends on the amount of the decomposable porogen. The pore size was controllable in a range between 0.6 and 1.0 nm in radius by proper selection of the substrate temperature and precursor composition.