Red mold rice prevents the development of obesity, dyslipidemia and hyperinsulinemia induced by high-fat diet.

OBJECTIVE: To investigate the influences of red mold rice (RMR) on obesity and related metabolic abnormalities. METHODS AND RESULTS: The 3T3-L1 cell line was used to examine the effects of RMR extracts on preadipocytes and on mature adipocytes. Both water and ethanol extracts of RMR had inhibitory effects on 3T3-L1 preadipocyte proliferation and differentiation. Water extracts of RMR enhanced the lipolysis activity in mature adipocytes, which negatively correlated with the triglyceride content within cells. RMR treatment did not affect heparin-releasable lipoprotein lipase activity in mature adipocytes. Furthermore, animal studies were carried out to explore the antiobesity effects of RMR. The control group of male Wistar rats were fed regular laboratory feed, whereas the other groups were fed the high-fat (HF) diet supplemented with lovastatin, rice or RMR (0.4 and 2%, w w(-1)). The relative caloric intakes of the control and HF groups were 3.34 and 4.85 kcal g(-1), respectively. After 6 weeks, rats treated with RMR at the 0.4 and 2% doses had lower weight gain and less fat pads mass accompanied with smaller fat cells than did the HF-diet rats. These effects probably resulted from an increase in the lipolysis activity of adipose tissue and a reduction in food/energy consumption. On the other hand, the RMR supplement significantly reduced serum total cholesterol, serum low-density lipoprotein (LDL) cholesterol, the ratio of LDL to high-density lipoprotein (HDL) cholesterol and serum insulin in the HF group. Moreover, the 2% RMR treatment significantly increased serum HDL cholesterol. CONCLUSION: This study reveals for the first time that RMR can prevent body fat accumulation and improve dyslipidemia. The antiobesity effects of RMR mainly derive from the lipolytic activity and mild antiappetite potency of RMR. In addition, extracts of RMR suppressed the proliferation and differentiation in 3T3-L1 preadipocytes, which might have contributed to the inhibition of new adipocyte formation or hyperplasia in adipose tissue.

Cannabinoid CB1 receptor-mediated inhibition of prolactin release and signaling mechanisms in GH4C1 cells.

The GH4C1 cell line was used to study the cellular mechanisms of cannabinoid-mediated inhibition of PRL release. Cannabinoid CB1 receptor activation inhibited vasoactive intestinal polypeptide- and TRH-stimulated PRL release, but not its basal secretion. The cannabinoid-mediated inhibition of TRH-stimulated PRL release was reversed by the CB1 receptor-specific antagonist, SR141,716A, and was abolished by pertussis toxin pretreatment, indicating that G alpha subunits belonging to the G(i)alpha and G(o)alpha family were involved in the signaling. Photoaffinity labeling using [alpha-32P] azidoaniline GTP showed that cannabinoid receptor stimulation in cell membranes produced activation of four G alpha subunits (G(i)alpha2, G(i)alpha3, G(o)alpha1, and G(o)alpha2), which was also reversed by SR141,716A. The CB1 receptor agonists, WIN55,212-2 and CP55,940, inhibited cAMP formation and calcium currents in GH4C1 cells. The subtypes of calcium currents inhibited by WIN55,212-2 were characterized using holding potential sensitivity and calcium channel blockers. WIN55,212-2 inhibited the omega-conotoxin GVIA (Conus geographus)- and omega-agatoxin IVA (Aigelenopsis aperta)-sensitive calcium currents, but not the nisoldipine-sensitive calcium currents, suggesting the inhibition of N- and P-type, but not L-type, calcium currents. Taken together, the present findings indicate that CB1 receptors can couple through pertussis toxin-sensitive G alpha subunits to inhibit adenylyl cyclase and calcium currents and suppress PRL release from GH4C1 cells.

Coupling of the expressed cannabinoid CB1 and CB2 receptors to phospholipase C and G protein-coupled inwardly rectifying K+ channels.

Signaling of the cannabinoid CB1 and CB2 receptors through phospholipase C (PLC) and G protein-coupled inwardly rectifying K+ channels (GIRK) was studied after their expression in COS7 cells and Xenopus oocytes. The CB1 or CB2 receptor was co-expressed with alpha subunits of the Galphaq family (Galphaq, Galpha11, Galpha14, Galpha15 and Galpha16) in COS7 cells. Receptor-dependent activation of PLC was observed after co-expressing the CB1 receptor with Galpha14, Galpha15 or Galpha16 but not with Galphaq or Galpha11. Co-expression of Gbeta1 and Ggamma2 abolished the activation, indicating that the activation was mediated by Galpha. PLC activation was not observed when the CB2 receptor was expressed alone or co-expressed with any of the above Galpha subunits. Coupling to GIRK was observed with both CB1 and CB2 receptors after expression in Xenopus oocytes. Significantly larger currents were induced when the receptor was co-expressed with both GIRK1 and GIRK4 than with either GIRK alone. Co-expression of Galpha transducin with the receptor significantly reduced the K+ currents, indicating that GIRK activation was mediated by Gbetagamma but not by Galpha. These findings suggest two new signaling pathways for the cannabinoid receptors.

Gastro-oesophageal reflux during day case gynaecological laparoscopy under positive pressure ventilation: laryngeal mask vs. tracheal intubation.

This study aimed to evaluate whether or not the use of intermittent positive pressure ventilation via the laryngeal mask airway is associated with a higher risk of gastro-oesophageal reflux when compared with intermittent positive pressure ventilation via a tracheal tube in patients undergoing day case gynaecological laparoscopy in the head down position. Sixty healthy women were randomly allocated to receive either the laryngeal mask or cuffed tracheal tube for intra-operative airway maintenance. Using continuous oesophageal pH monitoring, four patients in the tracheal tube group and none in the laryngeal mask group had evidence of gastro-oesophageal reflux (as indicated by a decrease in oesophageal pH to below 4). The difference in the incidence of reflux did not achieve statistical significance (p = 0.11). In conclusion, we found no evidence to suggest that the use of intermittent positive pressure ventilation via the laryngeal mask increases the risk of gastro-oesophageal reflux in patients undergoing elective day case gynaecological laparoscopy.

Functionally active catalytic domain is essential for guanylyl cyclase-linked receptor mediated inhibition of human aldosterone synthesis.

An aspartate-to-alanine point mutation in the catalytic domain (D853A) of guanylyl cyclase-C (GC-C), the heat-stable enterotoxin (STa) receptor, rendered the enzyme catalytically inactive. Mn2+/Triton X-100-stimulated guanylyl cyclase activity was detected in membranes from COS7 cells overexpressing GC-C but not GC-CD853A. STa treatment of paired cells resulted in cGMP production in those transiently expressing GC-C but not GC-CD853A. GC-C and GC-CD853A showed similar Bmax and Kd values for [125I]STa binding in these cells, indicating that the lack of catalytic activity in the latter was not due to differing expression levels or reduced binding affinity. The involvement of the catalytic domain in aldosteronogenesis was studied in human adrenocortical H295R cells. COS7 and H295R cells infected with vaccinia virus-expressing GC-C and GC-CD853A (VVGC-CD853A) had [125I]STa-binding characteristics akin to those in transfected cells. Immunoblot confirmed that both GC-C and GC-CD853A formed similar higher order oligomers in infected cells. Virus-mediated expression of GC-C in H295R cells revealed concentration-dependent STa-stimulated cGMP formation that was undetectable in VVGC-CD853A-infected cells. STa decreased angiotensin II-stimulated human aldosterone generation in a concentration-dependent manner in vaccinia virus-expressing GC-C-infected cells but not in those infected with VVGC-CD853A. These results demonstrate that a catalytically active guanylyl cyclase is required for the inhibition of aldosteronogenesis.

Gastro-oesophageal reflux with the laryngeal mask during day-case gynaecological laparoscopy.

We have evaluated if breathing spontaneously via a laryngeal mask airway is associated with a higher risk of gastro-oesophageal reflux compared with positive pressure ventilation via a laryngeal mask airway in 40 patients undergoing day-case gynaecological laparoscopy. Patients were allocated randomly to receive either positive pressure ventilation or breathe spontaneously via a laryngeal mask airway. Using continuous oesophageal pH monitoring, three patients in the ventilated group and one in the spontaneous breathing group had gastrooesophageal reflux (P = 0.29). We found no evidence to suggest that breathing spontaneously via a laryngeal mask airway increased the risk of gastro-oesophageal reflux compared with positive pressure ventilation in this group of patients.

Determination of delta-opioid in NG108-15 cells.

The delta-opioid receptors in mouse neuroblastoma x rat glioma NG108-15 cells were characterized by receptor binding and cAMP assays. Saturation binding assays using [3H][D-Pen5]enkephalin (DPDPE) or [3H][D-Ser2, Leu5, Thr6]enkephalin (DSLET) gave similar binding capacities (Bmax). Competition binding assays showed that DPDPE and DSLET have similar affinity for the [3H]DPDPE or 3[H]DSLET binding sites. The rank order of potency of competition with [3H]DPDPE and [3H]DSLET was similar: naltriben approximately DSLET > or = DPDPE > 7-benzylidenenaltrexone (BNTX). Both DPDPE and DSLET were found to decrease cAMP formation. The action of DSLET was antagonized by naltriben but not BNTX, while the action of DPDPE was reversed by both antagonists. Therefore, the delta-opioid receptor in NG108-15 cells has similar affinity for the agonists DPDPE and DSLET, and a higher affinity for the antagonist naltriben than BNTX.

Determination of the cannabinoid receptors in mouse x rat hybridoma NG108-15 cells and rat GH4C1 cells.

The cannabinoid receptors expressed in the mouse neuroblastoma X rat glioma NG108-15 cell and the rat pituitary tumor GH4C1 cell were determined by polymerase chain reaction, dideoxysequencing and pharmacologically. The CB1 but not the CB2 or CB1A cannabinoid receptor was found in both cell lines. The cDNA identified in GH4C1 cells corresponds to the rat CB1 receptor. Interestingly, NG108-15 cells express two distinct cDNAs, one corresponds to the rat and the other to the mouse CB1 receptor. The newly developed CB1 receptor selective antagonist SR141716A was found to reverse cannabinoid agonist (WIN55212-2 or CP55940)-induced adenylyl cyclase inhibition. These results provide more direct evidence that the CB1 receptor is mediating the pharmacological actions of cannabinoids in NG108-15 and GH4C1 cells.

The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor: a site-directed mutation study.

Wild-type and mutant serotonin 1A receptors were transiently expressed in COS-7 cells using the infection-transfection variant of the vaccinia virus/T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82-->Asn82, Asp116-->Asn116, and Ser198-->Ala198 all resulted in a decrease in affinity for 5-HT by 60-100-fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199-->Ala199, was too weak to be measured, 5-HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.

Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system.

The co-infection or infection-transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5-HT1ARs in COS-7, BSC-40 and GH3 cells, with co-infection giving ca. 3-fold higher level than infection-transfection. Binding affinities were similar to those of the endogenous 5-HT1AR, with highest affinities for 5-HT and 8-OH-DPAT. Functional properties were demonstrated by assays of agonist-stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.

Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.

Release by U-50,488H of [3H]serotonin from brain slices and spinal cord synaptosomes of U-50,488H-tolerant and nontolerant mice.

The effect of U-50,488H [trans-(+/-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl) benzeneacetamide] on the release of [3H] serotonin from mouse brain slices and spinal cord synaptosomes was studied using a superfusion system. Release was stimulated with 30 mM K+ while superfusing with Krebs-Ringer buffer, in the presence or absence of U-50,488H (3 nM to 1 microM). In both tissues, U-50,488H resulted in a concentration-related increase in release. The estimated EC50 values were found to be 14 and 42 nM in brain and spinal tissues, respectively. The increase in release was antagonized by 3 x 10(-7) M naloxone. The induction of release was found to be a Ca+(+)-dependent process which was significantly reduced in Ca+(+)-free medium containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Tolerance to U-50,488H was induced in mice using a slow release method. They were tested with the tail-flick assay to ensure tolerance had developed. The U-50,488H-induced release of [3H] serotonin was observed to decrease from both tissues of the U-50,488H-tolerant animals. These findings suggest that tolerance to the antinociceptive action of U-50,488H is accompanied by a decrease in its ability to stimulate serotonin release from brain and spinal tissues.

Attenuation of the antinociceptive action of the selective kappa-opioid receptor agonist, U-50,488H by ICS-205-930.

The antinociceptive action of s.c. administered U-50,488H was antagonized by s.c. administered ICS-205-930, a selective 5-HT3 receptor antagonist. To characterize the site of interaction, U-50,488H and ICS-205-930 were administered either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). When U-50,488H was administered i.c.v., its antinociception action was antagonized by ICS-205-930 given either i.c.v. or i.t., increasing the ED50 values of U-50,488H by approximately twofold from 48 to 98 and 90 nmol/mouse, respectively. However, when U-50,488H was administered i.t., its antinociception action was not antagonized by ICS-205-930 given either i.c.v. or i.t. These findings suggest that i.c.v., but not i.t. administered U-50,488H may release serotonin both supraspinally and spinally to interact with 5-HT3 receptors to produce antinociception.

Schedule dependent potentiation of antitumor drug effects by alpha-difluoromethylornithine in human gastric carcinoma cells in vitro.

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with alpha-difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.

Serotonergic involvement in the antinociceptive action of and the development of tolerance to the kappa-opioid receptor agonist, U-50, 488H.

The antinociceptive action of U-50,488H [( trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide]), a selective kappa-opioid receptor agonist, was antagonized by serotonergic (5-HT) but not by noradrenergic receptor antagonists. U-50,488H (i.c.v.) was antagonized by more than 2-fold by i.c.v. administered pindolol, methysergide, mianserin, ketanserin or pirenperone. A similar degree of antagonism of U-50,488H (i.c.v.) was found after intrathecal (i.t.) treatments with pindolol or methysergide but not with mianserin, ketanserin or pirenperone. When U-50,488H was given i.t., its antinociceptive action was antagonized by pindolol or methysergide administered i.t. but potentiated by mianserin, ketanserin or pirenperone administered i.t. Tolerance to the antinociceptive action of U-50,488H was induced in mice using slow release preparations of U-50,488H. A smaller degree of cross-tolerance to the antinociceptive action of 5-HT also developed. 5-HT receptor antagonists (pindolol or ketanserin) were coadministered with U-50,488H to test for their effect on the development of tolerance to U-50,488H. A greater degree of tolerance was observed after ketanserin administration whereas the development of tolerance was partially blocked by the coadministration of pindolol. Changes in the properties of the kappa-opioid, 5-HT1 and 5-HT2 receptor binding sites in the cortical, striatal and spinal tissues of control and U-50,488H-tolerant mice were investigated. There were no significant changes in the maximum binding values of [3H]ethylketazocine, [3H]-5-HT or [3H]ketanserin. The Kd values of all ligands increased approximately 2-fold in most of the regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in drug sensitivity of a human astrocytoma clone previously treated with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea in vitro.

We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3-4) to MeCCNU (10 micrograms/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 micrograms/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 micrograms MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.

Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays.

Previously, we reported on an opioid antagonist, nor-binaltorphimine (nor-BNI), that had high selectivity for kappa opioid receptors in smooth muscle preparations. In this study, nor-BNI administered either s.c. or i.c.v. was shown to antagonize significantly the antinociceptive effects of the kappa opioid agonists, ethylketazocine and U-50,488H at doses that had no effect on the antinociceptive effect of mu agonists, morphine and [D-Ala3, MePhe4, Gly-ol5]enkephalin and the delta agonist, [D-Pen3, D-Pen5]enkephalin. Nor-BNI and U-50,488H were used to demonstrate that kappa opioid receptors in the spinal cord were more important than those located supraspinally for kappa-mediated analgesia. Nor-BNI also possessed high affinity and high selectivity for kappa opioid receptors in the receptor binding assay. However, the comparatively low selectivity of BNI in receptor binding studies did not correlate with the high pharmacologic selectivity for kappa receptors.

Treatment-induced changes in sensitivity in a multiclonal human tumor mixture model in vitro.

An in vitro model has been devised so that mixtures of human tumor cells can be grown together for studies related to drug-induced or -selected changes in sensitivity. In the studies reported here, two human astrocytoma clones, one sensitive and one resistant to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU), were carefully matched for doubling times, cell cycle phase distributions, and colony-forming efficiencies. The clones were mixed and grown together, and after only three weekly treatments with MeCCNU (10 micrograms/ml for 1 h each week) the sensitive cells in the mixture were killed, leaving behind a population that was almost 100% resistant to further exposures to MeCCNU. The loss of the sensitive cells from the mixture each week was easily detected by visual observation of flow microfluorometry histograms since the clones had different DNA indices. Repeated weekly exposures of the unmixed resistant clone (AST 1-1) to MeCCNU caused very little accumulated cell kill. Similar exposures of the unmixed sensitive clone (AST 3-4) produced a linear decrease in survival over the first three weekly treatments with 10 micrograms MeCCNU/ml, but after that time these cells became progressively more resistant to MeCCNU. It is unlikely that the change to resistance in the AST 3-4 clone occurred because of contamination with the resistant AST 1-1 cells, because their DNA index remained stable. These data show that repeated treatments with a single agent can cause a tumor cell population to become more resistant. It remains to be tested whether this resistance was the result of cellular interactions, drug-induced changes in sensitivity, or selection for resistant cells already present in the populations. This mixture model may be useful in studies on how cellular interactions influence growth and drug sensitivity in tumor and normal cell populations.