Mechanisms of PTHrP-induced inhibition of smooth muscle contractility in the guinea pig gastric antrum.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) that causes hypercalcemia of malignancy appears to function as an endogenous smooth muscle relaxant. For example, PTHrP released upon bladder wall distension relaxes detrusor smooth muscle to accommodate urine. Here, we explored mechanisms underlying PTHrP-induced suppression of the smooth muscle contractility in the gastric antrum that also undergoes a passive distension. METHODS: Effects of PTHrP on phasic contractions and electrical slow waves in the antral smooth muscle of the guinea pig stomach were studied using isometric tension and intracellular microelectrode recordings, respectively. Fluorescent immunohistochemistry was also carried out to identify the distribution of PTH/PTHrP receptors. KEY RESULTS: Parathyroid hormone-related protein (1-100 nM) reduced the amplitude of phasic contractions and the basal tension. Nω -nitro-l-arginine (L-NA, 100 µM), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 10 µM), a guanylate cyclase inhibitor, diminished the PTHrP (10 nM)-induced reduction in the amplitude of phasic contractions. SQ22536 (300 µM), an adenylate cyclase inhibitor, attenuated the PTHrP-induced reduction in basal tension. The combination of ODQ (10 µM) and SQ22536 (300 µM) inhibited the PTHrP-induced reductions in both phasic contractions and basal tension. PTHrP (100 nM) had no inhibitory effect on the electrical slow waves in the antral smooth muscle. PTH/PTHrP receptors were expressed in cell bodies of PGP9.5-positive neurons in the myenteric plexus. CONCLUSIONS & INFERENCES: Parathyroid hormone-related protein exerts its inhibitory actions on the antral smooth muscle via both nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and cyclic adenosine monophosphate (AMP) pathways. Thus, PTHrP may act as an endogenous relaxant of the gastric antrum employing the two complementary signaling pathways to ensure the adaptive relaxation of stomach.

Effects of transglucosidase on diabetes, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes: a 12-week, randomized, double-blind, placebo-controlled trial.

In this 12-week, randomized, double-blind, placebo-controlled trial, the efficacy and safety of transglucosidase (TGD) were compared with placebo in patients with type 2 diabetes mellitus (T2DM). At 12 weeks, TGD 300 mg/day and TGD 900 mg/day significantly reduced HbA1c (0.18 and 0.21%) and insulin concentration (19.4 and 25.0 pmol/l), respectively, vs. placebo. TGD 300 mg/day and TGD 900 mg/day also significantly reduced low-density lipoprotein cholesterol (0.22 and 0.17 mmol/l, respectively). TGD 900 mg/day significantly reduced triglyceride by 0.24 mmol/l and diastolic blood pressure by 8 mmHg. Placebo was associated with a significant increase from baseline in body mass index, alanine aminotransferase and aspartate aminotransferase (0.17 kg/m(2) , 3 and 2 U/l, respectively), whereas TGD was not. TGD 300 mg/day significantly increased high-molecular-weight adiponectin by 0.6 µg/ml. Adverse events did not differ significantly between the groups. TGD resulted in lowering of HbA1c and blood insulin level and improvements in metabolic and cardiovascular risk factors in T2DM.

Glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through the PI3K/Akt/mTOR/GCLc/redox signaling pathway.

Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. Oxidative stress-induced neuronal cell apoptosis is a contributing factor. Glucagon-like peptide (GLP)-1 has recently become an attractive treatment modality for patients with diabetes. It also readily enters the brain, prevents neuronal cell apoptosis, and improves the cognitive impairment characteristic of Alzheimer's disease. Therefore, we investigated whether GLP-1 could protect against oxidative stress-induced neuronal cell apoptosis in pheochromocytoma (PC12) cells. PC12 cells were exposed to 1 mM methylglyoxal (MG) or MG plus 3.30 microg/ml GLP-1. Cell apoptosis, expression and phosphorylation of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/gamma-glutamylcysteine ligase catalytic subunit (GCLc), and redox balance were then determined. The data showed that MG induced PC12 apoptosis in accordance with the redox (glutathione (GSH) and GSH/glutathione disulfide [GSSG]) imbalance. GLP-1 protected against this MG-induced apoptosis, which corresponded to the phosphorylation of PI3K, Akt, and mTOR, as well as the upregulation of GCLc and the restoration of the redox imbalance. Inhibitors of PI3K (LY294002), Akt (Akt-I), and mTOR (rapamycin) reduced the GLP-1-induced GCLc upregulation and its protection against MG-induced PC12 apoptosis. The GLP-1-induced redox restoration was also attenuated by rapamycin. In conclusion, the neuroprotective effect of GLP-1 is due to an enhancement of PI3K/Akt/mTOR/GCLc/redox signaling.

Clinical evaluation of pioglitazone in patients with type 2 diabetes using alpha-glucosidase inhibitor and examination of its efficacy profile.

AIM: Pioglitazone is considered to reduce insulin resistance. This study was conducted to evaluate the efficacy, safety and clinical profile of pioglitazone in patients whose type 2 diabetes were poorly controlled with alpha-glucosidase inhibitor alone or alpha-glucosidase in combination with sulfonylurea. METHODS: Twenty patients with type 2 diabetes were treated with pioglitazone (30 mg q.d.) orally for 16 weeks. RESULTS: There were significant reductions in HbA1C, FPG and postprandial plasma glucose at week 16. As adverse events, oedema, hypoglycaemia-like reaction, increases in LDH, CPK, etc. were noted. There was no significant change in TNF-alpha. Leptin levels increased significantly at week 16 and were still increasing 4 weeks after the treatment. Per cent body fat was almost constant throughout the study period. When efficacy was classified by demographic variables, pioglitazone was found to be more effective in the subjects who had a higher postprandial 2-h plasma glucose level, leptin level or per cent body fat value. CONCLUSION: Pioglitazone was considered to be effective when used in patients whose type 2 diabetes were poorly controlled with alpha-glucosidase inhibitor alone or alpha-glucosidase in combination with sulfonylurea.

Mechanical responses evoked by nerve stimulation in gastric muscles of mouse lacking inositol trisphosphate receptor.

Alteration of mechanical responses elicited by transmural nerve stimulation (TNS) was investigated in pylorus muscle of stomach isolated from mutant mice lacking expression of IP, type-1 receptor. In wild and mutant mice. TNS inhibited spontaneous contractions and generated an off-response at the cessation. The effects of inhibitors of neurotransmission revealed that in wild mice, acetylcholine and nitric oxide were involved as excitatory and inhibitory mediators, respectively. In mutant mice, a lack of nitroxidergic component with associated attenuation of cholinergic transmission was found. The off-response was inhibited by apamin in both mice. In mutant mice, spantide-sensitive excitatory response appeared in the presence of apamin. Acetylcholine and substance P enhanced while noradrenaline and sodium nitroprusside inhibited spontaneous contractions, in both wild and mutant mice; the actions were weaker in mutant mice than in wild mice for any agonists. The results indicate that pylorus smooth muscles receive cholinergic excitatory and nitroxidergic and non-adrenergic non-cholinergic inhibitory projections, and a lack of IP, type-1 receptor results in an impairment of cholinergic and nitroxidergic components, with no alteration of non-adrenergic non-cholinergic inhibitory projections. In addition, the mutation induces a substance P projection which is not detected in wild mice.

Single-component organic semiconductors based on novel radicals that exhibit electrochemical amphotericity: preparation, crystal structures, and solid-state properties of N,N'-dicyanopyrazinonaphthoquinodiiminides substituted with an N-alkylpyridinium unit.

N,N'-Dicyanonaphthoquinodiimines fused with a pyrazine ring 1 were prepared from the corresponding quinones 4. The new acceptors 1 have a planar pi-system and undergo reversible two-stage 1e-reduction. Quaternization of the pyridyl substituent in 1d-f gave pyridinium derivatives 2d+, 2e+, and R-3+, respectively, which are stronger acceptors that undergo three-stage 1e-reduction. Upon electrochemical reduction of these cations, novel radicals 2d., 2e., and R-3. were generated and isolated as stable solids. The molecular geometries determined by X-ray analysis indicated that these radicals adopt a zwitterionic structure, in which the unpaired electron is located on the quinodiimine unit but not on the pyridyl group. These novel radicals undergo facile and reversible 1e-oxidation as well as two-stage 1e-reduction. The observed amphotericity endows the radicals with electrical conductivities (10(-5) to 10(-9) S cm-1), and these thus represent a new motif for single-component organic semiconductors.

Hyperpolarization-induced dilatation of submucosal arterioles in the guinea-pig ileum.

1. The effects of inhibition of acetylcholine (ACh)-induced hyperpolarization on dilatation of submucosal arterioles were investigated in the guinea-pig ileum. 2. In smooth muscles of the arterioles depolarized by Ba(2+) (0.5 mM) to about -40 mV, ACh (3 microM) repolarized the membrane to about -65 mV (hyperpolarization), irrespective of the absence or presence of L-N(omega)-nitroarginine (L-NOARG, 0.1 mM) and diclofenac (1 microM), and increased the diameter (dilatation). 3. Combined application of charybdotoxin (CTX, 50 nM) and apamin (0.1 microM), inhibitors of some types of K(+)-channels, abolished the ACh-induced hyperpolarization and dilatation. 4. 18 beta-Glycerrhetinic acid (18 beta-GA, 30 microM), a known inhibitor of gap junctions, depolarized the membrane to about -36 mV, either in the absence or in the presence of Ba(2+), with no associated contraction of the arterioles. In the presence of 18 beta-GA, ACh-induced hyperpolarization was abolished, however the dilatation was inhibited only partially, with associated inhibition of constriction produced by Ba(2+) and NA. 5. 18 beta-GA inhibited the dilatation produced by sodium nitroprusside, an NO donor. 6. The ACh-induced hyperpolarization and dilatation were abolished in the presence of 2-aminoethoxydiphenyl borate (30 microM), an inhibitory modulator of inositol trisphosphate receptor-mediated Ca(2+) release from intracellular stores. 7. It is concluded that in submucosal arterioles, hyperpolarizations produced by ACh have causal relationship to the arteriolar dilatation. 18 beta-GA did not induce parallel relationship between hyperpolarization and dilatation produced by ACh. 18 beta-GA may have unidentified inhibitory effects on agonist-mediated actions, in addition to the inhibition of gap junctions.

Endothelium-dependent hyperpolarization and intercellular electrical coupling in guinea-pig mesenteric arterioles.

1. Using the conventional whole-cell clamp method, the electrical responses of individual smooth muscle and endothelial cells to acetylcholine (ACh) were observed in multicellular preparations where the two types of cells remained in close apposition. 2. In both types of cells, ACh induced similar hyperpolarizing responses which, when recorded in current clamp mode, had two phases (an initial fast and a second slower phase). 3. After blocking gap junctions, including myoendothelial junctions, with 18beta-glycyrrhetinic acid, ACh induced an outward current with two phases in voltage-clamped endothelial cells. The outward current appeared around -90 mV and increased linearly with the membrane depolarization. 4. In smooth muscle cells, ACh failed to induce a membrane current after gap junctions had been blocked with 18beta-glycyrrhetinic acid. The inhibition of ACh-induced response by 18beta-glycyrrhetinic acid was observed using either sharp or patch electrodes. 5. Nominally Ca2+-free solution reduced the initial phase and abolished the second phase of ACh-induced responses of endothelial cells. Both phases were also reduced by charybdotoxin (CTX). 6. Our results indicate that in guinea-pig mesenteric arterioles, ACh hyperpolarizes endothelial cells by activating Ca2+-activated K+ channels which are sensitive to CTX. On the other hand, hyperpolarizing responses detected in smooth muscle cells seem to originate in endothelial cells and conduct to the muscle layer via myoendothelial gap junctions.

Electrical properties of colonic smooth muscle in spontaneously non-insulin-dependent diabetic rats.

Electrical properties of colonic smooth muscle were investigated in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model animal for spontaneous non-insulin-dependent diabetes mellitus (NIDDM), and the results were compared with those obtained from the Long-Evans Tokushima Otsuka (LETO) rat, a control of OLETF rat. At experiments (aged 60-80 weeks), blood glucose level was about 171 mg/dl in LETO rats and 370 mg/dl in OLETF rats. Feces in the colon were restricted to the proximal region in LETO rats and distributed widely in the whole colon in OLETF rats. In both LETO and OLETF rats, the circular smooth muscle strips of the isolated distal colon revealed two types of spontaneous electrical response, slow wave and transient hyperpolarization. The resting membrane potential was smaller in OLETF rats than in LETO rats by about 3 mV, but it was not positively related with the blood glucose level. The amplitude of hyperpolarization produced by noradrenaline (NA) was smaller in OLETF rats than in LETO rats. Transmural nerve stimulation evoked a non-adrenergic, non-cholinergic (NANC) inhibitory junction potential (i.j.p.) in both LETO and OLETF rats; the amplitude of the i.j.p. was smaller in OLETF rats than in LETO rats, while the latency of the i.j.p. was longer in OLETF rats than in LETO rats. Thus, in the distal colon, NIDDM may cause a depolarization of the membrane, an attenuation of NANC inhibitory transmission and a reduction in reactivity of adrenoceptors to NA. These results suggest that the constipation appearing with diabetes mellitus involves dysfunction of both the enteric autonomic nerves and the smooth muscles in the colon.

Alteration of the properties of gastric smooth muscle in the genetically hyperglycemic OLETF rat.

Membrane responses were recorded from isolated gastric smooth muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats, using microelectrode techniques. At the age of 68-76 weeks, the blood sugar level was 181 mg/dl in LETO rats and 350 mg/dL in OLETF rats. In both rats, the membrane potential was stable in fundus muscle and spontaneously active with generation of slow waves in antrum muscle. The resting membrane potential was about - 46 mV in fundus and - 55 mV in antrum muscles of LETO rats, and the values were 3-7 mV lower in OLETF rats. The slow waves were generated regularly in LETO rats, while they were irregular and of small amplitude in OLETF rats. Transmural nerve stimulation evoked a cholinergic excitatory junction potential and following inhibitory junction potential in LETO rats, and only an inhibitory junction potential of smaller size was generated in most of OLETF rats. The acetylcholine-induced depolarization was greater in OLETF than in LETO rats. The level of hyperpolarization produced by noradrenaline was similar between OLETF and LETO rats. Thus, the reduction of the resting membrane potential, weakening of spontaneous activity, impairment of cholinergic transmission and cholinergic supersensitivity were associated with hyperglycemia. These alterations were considered due to the development of diabetes mellitus.

Properties of inhibitory junctional transmission in smooth muscle of the guinea pig lower esophageal sphincter.

Inhibitory neurotransmission in guinea pig lower esophageal sphincter (LES) muscles was investigated by using electrophysiological methods. Transmural nerve stimulation (TNS) initiated an inhibitory junction potential (i.j.p.); the amplitude increased 35% by atropine (10(-6) M) and converted to a muscarinic excitatory junction potential (e.j.p.) by apamin (10(-7) M) plus Nomega-nitro-L-arginine (L-NNA, 10(-5) M). In atropinized tissue, the i.j.p. amplitude was reduced 58% by guanethidine (5 x 10(-6) M), 41% by L-NNA (10(-5) M), 57% by suramin (10(-4) M), and it was abolished by apamin (10(-7) M), suggesting that this potential was produced by ATP and nitric oxide (NO) released from adrenergic and nitrergic nerves, respectively, through the activation of Ca2+-sensitive K+ channels. Hyperpolarizations produced by ATP and NO were inhibited by apamin. The i.j.p. amplitude was reduced after desensitizing the membrane with ATP. In atropinized tissue, TNS produced a relaxation that was reduced 15% by guanethidine (5 x 10(-6) M), 50% by L-NNA (10(-5) M), and 30% by apamin (10(-7) M). Thus the LES receives cholinergic excitatory and adrenergic and nitrergic inhibitory innervations; the latter two components contribute evenly to the i.j.p. generation. The relaxation is mainly produced by NO in a membrane potential-independent way.

Effects of suramin on electrical and mechanical activities in antrum smooth muscle of the guinea-pig stomach.

The effects of suramin on electrical and mechanical responses produced by adenosine triphosphate (ATP), acetylcholine and transmural nerve stimulation were observed in antrum smooth muscle isolated from the guinea-pig stomach. Suramin (>10(-6) M) inhibited the non-adrenergic non-cholinergic inhibitory junction potential, with no alteration of the resting membrane potential, slow wave and the ATP-induced responses (hyperpolarization and inhibition of slow waves). The amplitude, but not the frequency, of spontaneous rhythmic contraction was inhibited by suramin (>10(-5) M), with no alteration of electrical responses of the membrane. Transmural nerve stimulation elicited cholinergic excitatory and non-adrenergic non-cholinergic inhibitory responses on the spontaneous contraction, and suramin inhibited only the latter. Suramin did not alter the ATP-induced inhibition of spontaneous contraction. The contractions produced by low concentrations (<10(-7) M), but not high concentrations (10(-6) - 10(-5) M), of acetylcholine were inhibited by suramin. It is concluded that in smooth muscle of the guinea-pig antrum, suramin inhibits contractions produced spontaneously and by low concentrations of acetylcholine, with no relation to the electrical responses of the membrane. Parallel inhibition by suramin of the electrical and mechanical responses elicited by excitation of non-adrenergic non-cholinergic inhibitory nerves may not be causally related to the inhibition of ATP-receptors.

Multiple parapapillary choledochoduodenal fistulas with ampullary carcinoma.

Only very rarely do multiple parapapillary choledochoduodenal fistulas occur concurrently with ampullary carcinoma. The following presents just such a case, which occurred in a 51 year-old Japanese female hospitalized for epigastralgia. Gastrointestinal fiberscopy (GIF) showed abnormal swelling of Vater's papilla. She was diagnosed as having ampullary carcinoma and choledochoduodenal fistulas, as determined by hypotonic duodenography (HDG), endoscopic retrograde cholangiopancreatography (ERCP) and from the histopathology of the ampullary mucosal biopsy. Pancreatoduodenectomy was performed. We postulated that the multiple fistulas were formed on the longitudinal fold of Vater's papilla by an ampullary carcinoma, and that the fistulas played a major role in bile drainage. As a result, jaundice was not seen throughout the entire clinical course. We report on the mechanism of fistula formation, with a review of the recent literature.

Effects of DQ-2511, a novel prokinetic agent, on electrical activities of smooth muscle in the guinea pig stomach.

Electrophysiological experiments were carried out to investigate the prokinetic actions of DQ-2511 on isolated smooth muscle of the guinea pig stomach. DQ-2511 enhanced the myogenic gastric slow waves and cholinergic excitatory junction potential and reduced the frequency of slow waves and the nonadrenergic, noncholinergic, and nonnitrergic inhibitory junction potential, with no significant alteration of the resting membrane potential. The results suggest that the prokinetic actions of DQ-2511 involve excitatory actions directly on smooth muscle and indirectly on cholinergic transmission.

Oxygen dependence of lipid peroxidation in mice.

Peroxidation of lipids in serum and tissues of mice placed in low or high levels of oxygen was examined. After exposure to 100% oxygen for 3 h, no significant differences were observed between control and exposed mice. However, exposure to 100% oxygen for 6 h resulted in a decrease in oxygen consumption, an increase in lipid peroxides in tissues and serum, and the formation of hydroxyl radicals in tissues and serum. At low concentrations of oxygen (14% or 16%), a decrease in oxygen consumption, peroxidation of lipids and formation of hydroxyl radicals also were observed. Damage to mice was great with the lower oxygen concentrations of oxygen. There was a close correlation between the consumption of oxygen lipid peroxidation, formation of hydroxyl radicals.

The effects of oxygen toxicity on the pregnant rat using an apparatus designed to measure oxygen consumption method.

To evaluate the effects of oxygen toxicity in the pregnant rat at high and low concentrations of oxygen, an oxygen-consumption apparatus was devised to measure the consumption of oxygen continuously over long periods. Oxygen consumption, partial oxygen pressure on the skin (tcPo2), and lipid peroxide levels in the serum were measured. There was a close correlation between oxygen consumption and body weight of rats that weighed between 150 g and 450 g. Oxygen consumption during the later stages of pregnancy increased by 1.8 ml/day, an increase of 8%. Oxygen consumption by rats during parturition increased markedly for up to 1 h and then reached and remained at a plateau value until the end of delivery. Exposure of pregnant rats to low concentrations of oxygen resulted in a marked depression in oxygen consumption and tcPo2 during the exposure time. A significant increase in the lipid peroxide level in serum was observed in the mother rat after birth and in the newborn offspring of pregnant rats exposed to 16% oxygen for 3 h.

[Effect of sex-hormone on composition of rat skin surface lipid].

The optimum analytical conditions for studying the composition of skin surface lipid were examined by high-performance liquid chromatography equipped with a photo-diode array detector. Optimum conditions were as follows: ULTRON N-C18 (150 x 4.6 mm) as stationary phase, acetonitrile/tetrahydrofuran/water (55/35/10, V/V) as eluent at the flow rate of 1.0 ml/min, and column temperature of 40 degrees C. The peaks were detected by monitoring the absorbance at 210 nm. Effect of sex-hormone on composition of skin surface lipids was examined. Gonadectomized Sprague-Dawley rats were injected with either testosterone (50 mg/kg, s.c.) or estradiol (5 mg/kg, s.c.) for 12 days. Amount of crude lipid from the skin surface was decreased at 8 days after castration; estradiol dosing to castrated rats also decreased the amount. The other hand, in ovariectomized rats, testosterone injection increased skin surface lipids. It is recognized that sex-hormone dosing after gonadectomy changes the percentage composition of squalene and cholesterol in male rats, but does not charge them in females.