Improvement of learning and increase in dopamine level in the frontal cortex by methylphenidate in mice lacking dopamine transporter.

The symptoms of attention-deficit/hyperactivity disorder (ADHD) are characterized by inattention and hyperactivity-impulsivity. It is a common childhood neurodevelopmental disorder that often persists into adulthood. Improvements in ADHD symptoms using psychostimulants have been recognized as a paradoxical calming effect. The psychostimulant methylphenidate (MPH) is currently used as the first-line medication for the management of ADHD. Recent studies have drawn attention to altered dopamine-mediated neurotransmission in ADHD, particularly reuptake by the dopamine transporter (DAT). This hypothesis is supported by the observation that DAT knockout mice exhibit marked hyperactivity that is responsive to acute MPH treatment. However, other behaviors relevant to ADHD have not been fully clarified. In the present study, we observed learning impairment in shuttle-box avoidance behavior together with hyperactivity in a novel environment in DAT knockout mice. Methylphenidate normalized these behaviors and enhanced escape activity in the tail suspension test. Interestingly, the effective dose of MPH increased extracellular dopamine in the prefrontal cortex but not striatum, suggesting an important role for changes in prefrontal dopamine in ADHD. Research that uses rodent models such as DAT knockout mice may be useful for elucidating the pathophysiology of ADHD.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

Regulatory mechanisms of ethylene biosynthesis in response to various stimuli during maturation and ripening in fig fruit (Ficus carica L.).

In order to obtain a greater uniformity of maturation, the growth of the fig fruit (Ficus carica L.) can be stimulated by the application of either olive oil, ethrel/ethephon or auxin. The three treatments induce ethylene production in figs. In this study, we investigated the regulatory mechanisms responsible for oil, auxin and ethylene induced ethylene production in figs. The ethylene production in response to olive oil, auxin, and propylene treatments and during ripening were all induced by 1-methylcyclopropene (1-MCP) and inhibited by propylene indicating a negative feedback regulation mechanism. Three 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (Fc-ACS1, Fc-ACS2 and Fc-ACS3) and one ACC oxidase gene (Fc-ACO1) were isolated and their expression patterns in response to either oil, propylene or auxin treatment in figs determined. The expression patterns of Fc-ACS1 and Fc-ACO1 were clearly inhibited by 1-MCP and induced by propylene in oil treated and ripe fruits indicating positive regulation by ethylene, whereas Fc-ACS2 gene expression was induced by 1-MCP and inhibited by propylene indicating negative regulation by ethylene. The Fc-ACS3 mRNA showed high level accumulation in the auxin treated fruit. The inhibition of Fc-ACS3 gene by 1-MCP in oil treated and in ripe fruits suggests that auxin and ethylene modulate the expression of this gene by multi-responsive signal transduction pathway mechanisms. We further report that the olive oil-induced ethylene in figs involves the ACC-dependent pathway and that multiple ethylene regulatory pathways are involved during maturation and ripening in figs and each specific pathway depends on the inducer/stimulus.

Depressive mood is independently related to stroke and cardiovascular events in a community.

By means of a multivariate Cox model, we investigated the predictive value of a depressive mood on vascular disease risk in middle-aged community-dwelling people. In 224 people (88 men and 136 women; mean age: 56.8 +/- 11.2 years) of U town, Hokkaido (latitude: 43.45 degrees N, longitude: 141.85 degrees E), a chronoecological health watch was started in April 2001. Consultations were repeated every 3 months. Results at the November 30, 2004 follow-up are presented herein. 7-day/24-h blood pressure (BP) and heart rate (HR) monitoring started on a Thursday, with readings taken at 30-min intervals between 07:00 h and 22:00 h and at 60-min intervals between 22:00 h and 07:00 h. Data stored in the memory of the monitor (TM-2430-15, A and D company, Japan) were retrieved and analyzed on a personal computer with a commercial software for this device. Subjects were asked to answer a self-administered questionnaire inquiring about 15 items of a depression scale, at the start of study and again after 1-2 years. Subjects with a score higher by at least two points at the second versus first screening were classified as having a depressive mood. The other subjects served as the control group. The mean follow-up time was 1064 days, during which four subjects suffered an adverse vascular outcome (myocardial infarction: one man and one woman; stroke: two men). Among the variables used in the Cox proportional hazard models, a depressive mood, assessed by the Geriatric Depression Scale (GDS), as well as the MESOR of diastolic (D) BP (DBP-MESOR) and the circadian amplitude of systolic (S) BP (SBP-Amplitude) showed a statistically significant association with the occurrence of adverse vascular outcomes. The GDS score during the second but not during the first session was statistically significantly associated with the adverse vascular outcome. In univariate analyses, the relative risk (RR) of developing outcomes was predicted by a three-point increase in the GDS scale (RR = 3.088, 95% CI: 1.375-6.935, P = 0.0063). Increases of 5 mmHg in DBP-MESOR and of 3 mmHg in SBP-Amplitude were associated with RRs of 2.143 (95% CI: 1.232-3.727, P = 0.0070) and 0.700 (95% CI: 0.495-0.989, P = 0.0430), respectively. In multivariate analyses, when both the second GDS score and the DBP-MESOR were used as continuous variables in the same model, GDS remained statistically significantly associated with the occurrence of cardiovascular death. After adjustment for DBP-MESOR, a three-point increase in GDS score was associated with a RR of 2.172 (95% CI: 1.123-4.200). Monday endpoints of the 7-day profile showed a statistically significant association with adverse vascular outcomes. A 5 mmHg increase in DBP on Monday was associated with a RR of 1.576 (95% CI: 1.011-2.457, P = 0.0446). The main result of the present study is that in middle-aged community-dwelling people, a depressive mood predicted the occurrence of vascular diseases beyond the prediction provided by age, gender, ABP, lifestyle and environmental conditions, as assessed by means of a multivariate Cox model. A depressive mood, especially enhanced for 1-2 years, was associated with adverse vascular outcomes. Results herein suggest the clinical importance of repetitive assessments of a depressive mood and the need to take sufficient care of depressed subjects. Another result herein is that circadian and circaseptan characteristics of BP variability measured 7-day/24-h predicted the occurrence of vascular disease beyond the prediction provided by age, gender, depressive mood and lifestyle, as assessed by means of a multivariate Cox model. Earlier, we showed that the morning surge in BP on Mondays was statistically significantly higher compared with other weekdays. Although a direct association between the Monday surge in BP and cardiovascular events could not be demonstrated herein, it is possible that the BP surge on Monday mornings may also trigger cardiovascular events. We have shown that depressive people exhibit a more prominent circaseptan variation in SBP, DBP and the double product (DP) compared to non-depressed subjects. In view of the strong relation between depression and adverse cardiac events, studies should be done to ascertain that depression is properly diagnosed and treated. Chronodiagnosis and chronotherapy can reduce an elevated blood pressure and improve the altered variability in BP and HR, thus reducing the incidence of adverse cardiac events. This recommendation stands at the basis of chronomics, focusing on prehabilitation in preference to rehabilitation, as a public service offered in several Japanese towns.

Characterization of heteromultimeric G protein-coupled inwardly rectifying potassium channels of the tunicate tadpole with a unique pore property.

Two cDNAs that encode the G protein-coupled inwardly rectifying K(+) channel (GIRK, Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly rectifying K(+) channel-A and -B; TuGIRK-A and -B) have been isolated. The deduced amino acid sequences showed approximately 60% identity with the mammalian Kir3 family. Detected by whole mount in situ hybridization, both TuGIRK-A and -B were expressed similarly in the neural cells of the head and neck region from the tail bud stage to the young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein beta(1)/gamma(2) subunits (Gbetagamma) in Xenopus oocytes, an inwardly rectifying K(+) current was expressed. In contrast, coinjection of TuGIRK-B with Gbetagamma did not express any current. When both TuGIRK-A and -B were coexpressed together with Gbetagamma, an inwardly rectifying K(+) current was also detected. The properties of this current clearly differed from those of TuGIRK-A current, since it displayed a characteristic decline of the macroscopic conductance at strongly hyperpolarized potentials. TuGIRK-A/B current also differed from TuGIRK-A current in terms of the lower sensitivity to the Ba(2+) block, the higher sensitivity to the Cs(+) block, and the smaller single channel conductance. Taken together, we concluded that TuGIRK-A and -B form functional heteromultimeric G protein-coupled inwardly rectifying K(+) channels in the neural cells of the tunicate tadpole. By introducing a mutation of Lys(161) to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a higher sensitivity to the Ba(2+) block and a slightly lower sensitivity to the Cs(+) block, and the decrease in the macroscopic conductance at hyperpolarized potentials was no longer observed. Thus, the differences in the electrophysiological properties between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least partly, due to the presence of Lys(161) at the external mouth of the pore of the TuGIRK-B subunit.

Impact of circadian amplitude and chronotherapy: relevance to prevention and treatment of stroke.

The long-acting calcium antagonist nifedipine reduces the incidence of stroke in Eastern Asia, as shown by the Shanghai Trial Of Nifedipine in the Elderly (STONE) and the Systolic Hypertension in China (Syst-China) trials. Recent trials in Japan have shown that benidipine may be more efficient than the former drug in preventing strokes in the elderly. Benidipine, commonly prescribed in Japan for a definite depressor effect, reportedly without causing remarkable fluctuations in blood pressure (BP), is investigated herein from a chronobiological viewpoint. Eighteen subjects (nine women and nine men, 39 to 87 years of age) with essential hypertension (office and ambulatory systolic, S/diastolic, D BP values above 160/95 mm Hg and 130/80 mm Hg, respectively) were enrolled in this investigation. Ambulatory BP was monitored at 30-min intervals for at least 24 h (ABPM-630, Colin Medical) before and after 4 weeks of crossover treatment with nifedipine tablets (twice daily, 20 mg/d) and benidipine (once daily, 4 mg/d, in the morning). The results indicate that: 1) benidipine and nifedipine reduce 24-h daytime (10:00-20:00) and nighttime (00:00-06:00) averages of SBP and DBP (P < 0.001); 2) the circadian double amplitude of BP is decreased after treatment with benidipine (from 28.6 to 21.1 mm Hg SBP and from 19.7 to 15.2 mm Hg DBP; P< 0.05), while the day-night difference in SBP is increased after treatment with nifedipine (18.6 vs 27.9 mm Hg, P< 0.01); and 3) the increase in the day-night difference of heart rate (HR) is significant after treatment with benidipine (13.6 vs 18.8 beats per minute, bpm; P< 0.05), but not with nifedipine. We have previously evaluated the usefulness of the circadian amplitude of BP as a prognostic tool of cardiovascular outcome, and found that an excessive circadian SBP or DBP amplitude was associated with an increased risk of vascular disease. The fact that benidipine reduces the circadian BP amplitude may be one reason for the superiority of this treatment over nifedipine in preventing an adverse outcome. A reduced heart rate variability (HRV) also predicts adverse cardiovascular outcomes in patients with overt cardiovascular disease and in hypertensive subjects. The fact that benidipine increases the day-night difference in HR may be another reason for the positive effects of this treatment.

Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium and pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 binuclear cluster DNA-binding motifs and regulate transcription of melanin biosynthesis genes in a developmentally specific manner.

Colletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichum melanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA-binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1Delta mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.

Flavonols from Heterotheca inuloides: tyrosinase inhibitory activity and structural criteria.

Tyrosinase inhibitory activity of flavonols, galangin, kaempferol and quercetin, was found to come from their ability to chelate copper in the enzyme. In contrast, the corresponding flavones, chrysin. apigenin and luteolin, did not chelate copper in the enzyme. The chelation mechanism seems to be specific to flavonols as long as the 3-hydroxyl group is free. Interestingly, flavonols affect the enzyme activity in different ways. For example, quercetin behaves as a cofactor and does not inhibit monophenolase activity. On the other hand, galangin inhibits monophenolase activity and does not act as a cofactor. Kaempferol neither acts as a cofactor nor inhibits monophenolase activity. However, these three flavonols are common to inhibit diphenolase activity by chelating copper in the enzyme.

Age-related changes in the response to thermal noxious heat and reduction of C-fibers by neonatal treatment with capsaicin.

Developmental changes of the response to nociceptive heat were examined in mice treated with capsaicin (50 mg/kg) on postnatal days 2-15. Tests of hot-plate (55 degrees C) and irradiation by infrared (IR test) were carried out after 10 days of capsaicin administration up to 120 days at intervals of 10 or 20 days. The time until forepaw (hot-plate) and hindpaw (IR test) withdrawal was assessed as the response latencies to suprathreshold and thermal threshold, respectively. Moreover, the numbers of unmyelinated C-fibers and myelinated fibers in the L4 dorsal roots of the animals treated on postnatal days 2 and 5 were counted on electron micrograph montages. Despite the marked reduction of C-fibers (60% mean decrease) in the 4 dorsal roots of the animals treated on postnatal day 2, thresholds were normal compared with those of controls. However, the animals treated with capsaicin on postnatal day 5 showed an apparent delay of thermal threshold latency only in the IR test, although the mean reduction of C-fibers was very likely the same as that of the animals pretreated on day 2. The reduction of C-fibers in mice treated on postnatal days 10 and 15 was lower than the animals treated on days 2 or 5, but their threshold latencies were significantly increased (hypoalgesia). A possible implication of these findings is discussed on the basis of the development of inhibitory systems in the intraspinal and supraspinal dorsal horn and sprouting from the surviving primary afferent neurons in the superficial dorsal horn.

KQT2, a new putative potassium channel family produced by alternative splicing. Isolation, genomic structure, and alternative splicing of the putative potassium channels.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of the testing membrane potential, and development of memory and learning. Eleven mouse cDNA clones homologous to the new human putative K+ channel (designated HNSPC, which we recently reported) were isolated from the brain cDNA libraries. All these proteins coded by the isolated cDNAs were identical from the N-terminal to the sixth transmembrane domain, but exhibited differences in the sequence and length of the C-terminal cytoplasmic region. Analyses of the mouse genomic DNAs showed that these clones originated from a single gene located on mouse chromosome 2H3-4, which proved that these clones were generated by alternative RNA splicing. Since all isoforms showed significant structural identity with KVLQT1 (64% identity in the transmembrane domains), which is known to associate with IsK, they were designated mKQT2.1-mKQT2.11. Northern blot analysis indicated that the mRNAs of the mKQT2 isoforms were exclusively expressed in the brain. In the mouse cerebellum region, the localized expression of these clones in the Purkinje cell layer and Golgi cells was shown by in situ hybridization analysis. These transcripts were also detected in the mouse embryonic developmental stage (11th, 15th and 17th day); and in particular, the mRNAs for shorter forms (mKQT2.9, mKQT2.10 or mKQT2.11) were abundantly found on the 11th day after gestation. Although these mKQT2 isoforms had the characteristic structure of voltage-gated K+ channels, functional expression of K+ currents were not detected in Xenopus oocytes.

RGS8 accelerates G-protein-mediated modulation of K+ currents.

Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signalling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. Here we report the isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, and the determination of its function. We show that RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and that it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerated not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. We conclude that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors.

Antioxidative constituents in Heterotheca inuloides.

Sesquiterpenoids, 7-hydroxy-3,4-dihydrocadalin and 7-hydroxycadalin, and flavonoids, quercetin, kaempferol and their glycosides, isolated from Heterotheca inuloides (Asteraceae), a Mexican medicinal plant known as "arnica", were evaluated as antioxidants. These compounds showed potent scavenging activity on diphenyl-p-picrylhydrazyl (DPPH) radical. Microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH was inhibited by both terpenoids and flavonoids, though only flavonoids possessed superoxide anion scavenging activity in microsome. Flavonoids also scavenged enzymatically and non-enzymatically generated superoxide anion. On the other hand, mitochondrial lipid peroxidation induced by Fe(III)-ADP/NADH was inhibited only by sesquiterpenoids. Furthermore, these terpenes protected mitochondrial enzyme activity against oxidative stress. These results showed that two types of antioxidants existed in the dried flower of H. inuloides and would contribute to protection of tissues against various oxidative stresses.

Prospective and randomized trial of lipiodol-transcatheter arterial chemoembolization for treatment of hepatocellular carcinoma: a comparison of epirubicin and doxorubicin (second cooperative study). The Cooperative Study Group for Liver Cancer Treatment of Japan.

A randomized, controlled clinical trial was conducted to compare the use of epirubicin (EPI) and doxorubicin (DOX) in Lipiodol (Laboratoire Guerbet, Roissy-Charles-de-Gaulle Cedex, France)-transcatheter arterial chemoembolization as a treatment of hepatocellular carcinoma. One hundred ninety-two hospitals participated, and 415 patients were enrolled in the study during the period between October 1989 and December 1990. The patients were randomly allocated to group A (EPI) or group B (DOX) by a centralized telephone registration. The actual doses of EPI and DOX were 72 mg/body and 48 mg/body, respectively. The 1-, 2-, and 3-year survival rates were, respectively, 69%, 44%, and 33% for group A and 73%, 54%, and 37% for group B. There were no statistically significant differences (P = .2296, log-rank test). When each group of patients was classified retrospectively into high-risk and low-risk subgroups based on the severity index calculated by the Cox regression model from the significant prognostic factors (the pretreatment tumor size, the pretreatment serum alpha-fetoprotein level, tumor encroachment, and Child's classification), the survival curve of the low-risk DOX subgroup was significantly superior to that of the low-risk EPI subgroup (P = .0182). However, there was no significant difference between the high-risk subgroups (P = .4606). The change in the serum alpha-fetoprotein level, the extent of Lipiodol accumulation in the tumor, and the extent of tumor reduction after the treatment did not show any significant differences between the groups. The white blood cell count in group B showed a tendency to decrease slightly more than in group A at 3 weeks after Lipiodol-transcatheter arterial chemoembolization. In conclusion, there was no statistically significant difference between the survival curves of the EPI and DOX groups in Lipiodol-transcatheter arterial embolization treatment of hepatocellular carcinoma.

Cytotoxic and antioxidative sesquiterpenoids from Heterotheca inuloides.

Four sesquiterpenoids, beta-caryophyllene, beta-caryophyllene 4,5 alpha-oxide, 7-hydroxy-3,4-dihydrocadalin, and 7-hydroxycadalin, isolated from the dried flower of Heterotheca inuloides Cass. (Asteraceae), have been found to exhibit cytotoxic activity against several solid tumor cell lines. Among them, 7-hydroxy-3,4-dihydrocadalin and 7-hydroxycadalin have also been found to inhibit autoxidative and microsomal lipid peroxidation.

A weakly inward rectifying potassium channel of the salmon brain. Glutamate 179 in the second transmembrane domain is insufficient for strong rectification.

A cDNA encoding for a weakly inward rectifying K+ channel (sWIRK: salmon weakly inward rectifying K+ channel) was isolated from the masu salmon brain by expression cloning. The sWIRK channel exhibited the highest similarity with members of the ROMK1 subfamily, BIR10/KAB-2 (70% amino acid identity) and ROMK1 (46%). An ATP binding motif which is characteristic of this subfamily was also conserved. The sWIRK RNA was detected in the brain, but not in the heart, kidney, skeletal muscle, liver, testis, and ovary. In the brain, the expression was observed in the ependymoglial cells on the surface of the ventricles as well as in the small perineuronal glia-like cells in the midbrain and the medulla. When compared with the strong inward rectifier IRK1 channel, the sWIRK channel showed a much weaker inward rectification property, and the activation kinetics upon hyperpolarization was slower and less voltage-dependent. The slope conductance of the single channel inward current was 37 pS (140 mM K+o), and outward current channel events were also observed. The weak rectification of sWIRK is significant in that it has a negatively charged residue (glutamate) in the M2 region which is reported to cause strong inward rectification. By introducing a point mutation to remove this negative charge (glutamine), the sWIRK E179Q mutant channel lost its inward rectification property completely, and the single channel property (45 pS; 140 mM K+o) was ohmic up to highly depolarized potential, even in the presence of the physiological cytoplasmic blockers such as Mg2+ and polyamines.

Functional characterization and localization of a cardiac-type inwardly rectifying K+ channel.

We cloned inwardly rectifying K+ channel cDNAs from porcine, rat and human, which were structurally almost identical with recently reported CIR(cKATP-1). The expression of CIR alone was low and unstable in Xenopus oocytes. The CIR/GIRK1 co-expression showed an increased current amplitude. Both the CIR and CIR/GIRK1 currents increased by coexpressing G beta gamma. The CIR and CIR/GIRK1 currents displayed two qualitative differences. (1) The conductance of the CIR channel did not saturate, but that of the CIR/GIRK1 channel showed saturation at hyperpolarized potential. (2) The CIR current showed instantaneous activation upon hyperpolarization, whereas the CIR/GIRK1 current exhibited slow activation, which was fitted by the sum of two exponentials. The CIR/GIRK1 current was also different from the GIRK1 current. The activation of the CIR/GIRK1 current was approximately ten times faster than that of the GIRK1 current. The increase in current amplitude and the qualitative differences imply the formation of functional heteromultimer. The CIR/GIRK1 channel showed differences from the native muscarinic K+ channel in that the basal level before m2 receptor activation is significantly large, and that the activation kinetics are much faster. Using anti-CIR antiserum, the CIR was detected in myocardial cells of the atrium and the ventricular subendocardial layer, and in the cardiac ganglion.

New chlorine-containing prenylated C6-C3 compounds increasing choline acetyltransferase (ChAT) activity in culture of postnatal rat septal neurons from Illicium tashiroi.

The structures of two new chlorine-containing C6-C3 compounds isolated from the woods of Illicium tashiroi have been established as 2(R)-12-chloro-2,3-dihydroillicinone E (1) and 12-chloroillicinone E (2) by X-ray crystallographic analysis and spectroscopic data, respectively. Compound 1 has been found to significantly increase ChAT activity in culture of P10 rat septal neurons.

Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel.

Parasympathetic nerve stimulation causes slowing of the heart rate by activation of muscarinic receptors and the subsequent opening of muscarinic K+ channels in the sinoatrial node and atrium. This inwardly rectifying K+ channel is coupled directly with G protein. Based on sequence homology with cloned inwardly rectifying K+ channels, ROMK1 (ref. 11) and IRK1 (ref. 12), we have isolated a complementary DNA for a G-protein-coupled inwardly rectifying K+ channel (GIRK1) from rat heart. The GIRK1 channel probably corresponds to the muscarinic K+ channel because (1) its functional properties resemble those of the atrial muscarinic K+ channel and (2) its messenger RNA is much more abundant in the atrium than in the ventricle. In addition, GIRK1 mRNA is expressed not only in the heart but also in the brain.

Primary structure and functional expression of a mouse inward rectifier potassium channel.

A complementary DNA encoding an inward rectifier K+ channel (IRK1) was isolated from a mouse macrophage cell line by expression cloning. This channel conducts inward K+ current below the K+ equilibrium potential but passes little outward K+ current. The IRK1 channel contains only two putative transmembrane segments per subunit and corresponds to the inner core structure of voltage-gated K+ channels. The IRK1 channel and an ATP-regulated K+ channel show extensive sequence similarity and constitute a new superfamily.