In Hoi Huh

Signal transduction mechanism via adenosine A1 receptor in the cat esophageal smooth muscle cells

We investigated what adenosine receptor type exists and the signaling pathways on the contraction of circular muscle cells isolated by enzymatic digestion from the cat esophagus. Adenosine or the selective A1 receptor agonist R-PIA causes a concentration-dependent contraction. After pretreatment with A1 receptor antagonist, DPCPX, adenosine-mediated contraction was abolished. Adenosine-induced contraction was significantly increased when A1 receptors were preserved by pretreatment with DPCPX followed by inactivation of all unprotected receptors with N-ethylmaleimide. Adenosine- or R-PIA-induced contraction was significantly augmented in the preserved cells and the increase was abolished in the presence of the A1 receptor antagonist DPCPX. PTX abolished contraction induced by adenosine or R-PIA, implying that contraction activated by A1 receptor was coupled to a pertussis toxin (PTX)-sensitive G(i) protein. After permeabilization, contraction was inhibited by G(i2), but not by G(i1) and G(i3), antibodies. These data suggest that adenosine-induced contraction of esophagus depends on PTX-sensitive G(i2.) Adenosine- or R-PIA-induced contraction of esophageal smooth muscle cells was not affected by the phospholipase D (PLD) inhibitor rho-chloromercuribenzoic acid (rhoCMB), phospholipase A(2) (PLA(2)) inhibitor DEDA or PKC antagonist chelerythrine, but was significantly abolished by phospholipase C (PLC) inhibitor, neomycin. PLC-beta3 antibody inhibited R-PIA-induced contraction. R-PIA-induced contraction of esophageal muscle cells was inhibited by IP(3) receptor antagonist heparin, which suggests that the contraction of esophageal smooth muscle cells is dependent on phosphatidylinositol-specific phospholipase (PI-PLC) and IP(3). In conclusion, adenosine- and R-PIA-induced contraction in cat esophageal smooth muscle cell was mediated by A1 receptor. A1 receptor is coupled to PTX-sensitive G protein G(i2), which results in the activation of PI-PLC-beta3. PI hydrolysis by PI-PLC forms IP(3), which binds to IP(3) receptor on endoplasmic reticulum, resulting in the release of intracellular Ca(2+).

The role of nitric oxide and prostaglandin E2 on the hyperalgesia induced by excitatory amino acids in rats.

The present study was designed to investigate the role of nitric oxide (NO), N‐methyl‐D‐aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO.

Combination of vitamin C and rutin on neuropathy and lung damage of diabetes mellitus rats

We investigated the role of vitamin C or rutin on neuropathy and lung damage of diabetic mellitus (DM) rats. Norepinephrine content was significantly decreased in sciatic nerves of DM rats compared with non-DM controls but vitamin C had no effect on decreases of norepinephrine. 2,4-dinitrophenylhydrazine (DNPH) incorporation, which is biomarker of protein oxidation, was increased in sciatic nerve of DM rats as compared with normal control. However, vitamin C had no effects on increases of DNPH incorporation. We measured the content of conjugated dienes (CD) as a biomarker of lipid oxidation in sciatic nerve. CD was increased in DM as compared with normal control. Vitamin C or rutin had no effects on increases of CD. However, Rutin plus vitamin C significantly decreased the content of CD as compared with DM rats. In lung of DM rats, DNPH incorporation or CD was increased as compared with normal control. Vitamin C or Rutin had no effects on increases of CD. However, Rutin plus vitamin C significantly decreased the content of DNPH incorporation or CD in lung tissue. Vitamin C caused marked pathological changes such as the increases of parenchyma and the thickening of alveolar septa in the lung of DM. Rutin had protective effects on the pathological changes in the lung of DM rats. In conclusion, Vitamin C had no effects on oxidative parameter, such as DNPH incorporation or CD, and on the decreases of norepinephrine content in DM rats. Vitamin C caused the marked pathological changes in the lung of DM rats but rutin had protective effects against the pathological changes.

Endothelium‐dependent Sensory Non‐adrenergic Non‐cholinergic Vasodilatation in Rat Thoracic Aorta: Involvement of ATP and a Role for NO

The involvement of non‐adrenergic non‐cholinergic (NANC) transmitters, such as adenosine 5′‐triphosphate (ATP) and nitric oxide (NO), in the neurogenic relaxation of rat thoracic aorta was investigated in vessel segments suspended for isometric tension recording by polygraph.

NMDA receptor and NO mediate ET-1-induced behavioral and cardiovascular effects in periaqueductal gray matter of rats

Endothelin-1 (ET-1), a novel and potent vasoconstrictor in blood vessel, is known to have some functions in the rat central nervous system (CNS). In order to investigate the central functions of ET-1, ET-1 was administered to the periaqueductal gray area (PAG) of anesthetized rats to induce barrel rolling and increase the arterial blood pressure (ABP). ET-1 had a modulatory effect on central cardiovascular and behavioral control. The selective N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (3 μmol/kg, i.p.) blocked the ET-1 induced responses, and both the nitric oxide synthase (NOS) inhibitor L-NAME (N-nitro-L-arginine methylester 1 mmol/rat) and the nitric oxide (NO) scavenger hemoglobin (15 nmol/rat) had similar effects in reducing the ET-1 (10 pmol/rat)-induced behavioral changes and ABP elevation. However, NO donor sodium nitroprusside (SNP 10 μg, 1 μg/rat) decreased the ET-1 induced ABP elevation, and recovered the ET-1-induced barrel rolling effect that was reduced by MK-801. These results suggest that ET-1 might have neuromodulatory functions such as ABP elevation and barrel rolling induction in the PAG of the rats via the NMDA receptor and NO.

Increase of [Ca2+]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and Rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.