Down-regulation of the two-component system and cell-wall biosynthesis-related genes was associated with the reversion to daptomycin susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus.

Daptomycin (DAP) is widely used in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection. The emergence of DAP non-susceptible MRSA strains during therapy is a major concern in clinical settings. Recent studies revealed that MRSA spontaneously reverts to a subsequent methicillin-susceptible S. aureus (MSSA) strain. However, it is not clear whether DAP non-susceptible MRSA has the ability to revert to a susceptible strain. We obtained an MRSA strain pair, DAP non-susceptible strain and subsequent DAP susceptible strain, from a patient. To understand the underlying mechanism by which DAP non-susceptible MRSA reverts to a susceptible strain, we performed genetic and phenotypic analysis in the strain pair. Although whole-genome analysis revealed four missense mutations, including L826F in mprF, in both strains, the net cell-surface charge was similar between the DAP non-susceptible and susceptible strains. However, the thickness of the cell wall was higher in the DAP non-susceptible strain, which was decreased to the same level as the control after reversion to the DAP susceptible strain. Moreover, the non-susceptible strain showed higher mRNA expression of the two-component system (TCS), such as VraSR, yycG and GraS, with the up-regulated transcription levels of cell-wall biosynthesis-related genes. The expression levels of those genes were decreased after reversion to the susceptible strain. These results indicated that DAP non-susceptibility due to up-regulation of the TCS and cell-wall biosynthesis-related genes may be reversible by the discontinuation of DAP, leading to reversion to the DAP susceptible phenotype.

Tuberculosis of the esophagus.

We report a case of a patient with esophageal tuberculosis, a very uncommon form of extrapulrhonar tuberculosis. Initially, because of constitutional symptomatology and radiological findings of mediastinal lymph node enlargement, lymphoma was considered. However, the endoscopic findings of ulcerative masses and a sinus tract revealed by esophagram were suspicious of tuberculous origin. Diagnosis was achieved after bacterial examination of smear samples from esophageal ulcers that revealed bacillus tuberculous and histological demonstration of caseating granulomas in cervical lymph nodes. Tuberculous mediastinal lymphadenitis was thought to be source of the spread to esophagus.The patient was successfully treated with a three antituberculous drugs regimen. In spite of its rarity, even in patients without risk factors, the diagnosis would be considered in the differential diagnosis of uncertain esophageal lesions.

[Off-pump coronary artery bypass grafting for angina pectoris with coronary artery aneurysm due to kawasaki disease: report of a case].

We report a 27-year-old woman who underwent off-pump coronary artery bypass grafting (OPCAB) for angina pectoris with coronary artery aneurysm due to Kawasaki disease. At the age of 3, she was diagnosed as Kawasaki disease with coronary artery aneurysms in the right coronary artery and the left anterior descending artery. She was medically followed-up since then Because an enlarged aneurysm and a stenotic lesion were recognized in the right coronary artery, operation was indicated. In operation, the right coronary artery was ligated at the inflow and the outflow of the aneurysm. OPCAB was also conducted with the right internal thoracic artery anastomosed to the right coronary artery. Postoperative course was uneventful, and she was discharged at the day 5 after operation. Revascularization using arterial grafts, especially the internal thoracic arteries, may be the choice for young patients to expect a good patency rate in the long-term.

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Indirect action of 5-hydroxytryptamine on the isolated muscularis mucosae of the guinea-pig oesophagus.

1 The site of action of 5-hydroxytryptamine (5-HT) was examined on the isolated muscularis mucosae attached to the submucous plexus of the guinea-pig oesophagus. Isotonic responses of the longitudinal muscularis mucosae were recorded.2 5-HT produced a transient contraction of the muscularis mucosae at concentrations higher than 3 muM. The contraction was rapid in onset, reaching a peak in about 15 s or less, and was restored to the basal level after 20 to 30 s without washing out 5-HT. When the 5-HT-induced contraction faded to the basal tone, successive applications of 5-HT no longer produced any contracture.3 Nicotine (Nic), at concentrations higher than 10 muM, also produced a transient contraction which had a very similar pattern to that induced by 5-HT. Again, the successive application of Nic no longer produced any contracture following prior treatment with Nic itself. However, the 5-HT-induced contraction was not modified by the presence of Nic.4 Exogenously applied acetylcholine (ACh) produced a concentration-dependent contraction of the muscularis mucoase, the 50% effective concentration (EC(50)) was 69 +/- 5.6 nM. The contraction was sustained during incubation with ACh, and was not modified by prior treatment with 5-HT or Nic.5 The 5-HT (100 muM)-induced contraction was completely abolished by tetrodotoxin (0.2 muM) and atropine (0.2 muM). This means that the action is mediated by stimulating cholinergic nerves in the submucous plexus attached to muscularis mucosae. Moreover, the stimulating action of 5-HT does not involve nicotinic receptors, since the action was not blocked by hexamethonium (100 muM).6 Among several 5-MT antagonists examined, methysergide (1 muM), ketanserin (1 muM) and morphine (100 muM) failed to modify the 5-HT (100 muM)-induced contraction significantly. Cinanserin (0.1-3 muM), cyproheptadine (3-100 nM) and phenoxybenzamine (0.1-3 muM) inhibited the 5-HT-induced contraction, in a concentration-dependent manner, and each highest concentration abolished the response. However, none of these antagonists was specific for 5-HT, but the Nic (100 muM) or ACh (0.1 muM)-induced contractions were also inhibited by them.7 The present results indicate that 5-HT contracts the muscularis mucosae of the guinea-pig oesophagus indirectly by stimulating cholinergic nerves in the submucous plexus, and has no direct action on the muscularis mucosae. In addition, the type of 5-HT receptors responsible for the stimulant action may be different from those in other parts of the gastrointestinal tract, blood vessels or brain, because of the different effects of 5-HT antagonists.

Antagonistic effect of compound 48/80 on the inhibitory actions of morphine and methionine-enkephalin on electrically-induced contractions of the guinea-pig ileum.

1 The effect of compound 48/80 was studied on the twitch-like contractions of the longitudinal muscle of guinea-pig ileum induced by electrical stimulation of intramural cholinergic nerves. 2 Compound 48/80 alone, at concentrations up to 30 microgram/ml, had no effect on the twitch contractions. The contraction to exogenously applied acetylcholine was slightly depressed by the compound. 3 At 100 microgram/ml, compound 48/80 caused a weak but long-lasting increase in tone and irregular contractile activity in the ileum, part of which was reduced but not completely abolished by pretreatment with chlorpheniramine (1 muM) or by repeated applications of compound 48/80. 4 The inhibitory effects of morphine and methionine-enkephalin on the twitches were antagonized by the presence of compound 48/80 (3 to 30 microgram/ml), possibly in a competitive manner. The antagonism was not affected by pretreatment with the antihistaminics, chlorpheniramine and/or metiamide. 5 The inhibitory effects of noradrenaline and adrenaline on the twitches were slightly but significantly increased by the presence of compound 48/80 (10 or 30 microgram/ml), whereas that of ATP was not modified. 6 Thesese results indicate that compound 48/80 acts as a selective and competitive antagonist at opiate receptors located in the intramural cholinergic nerves of guinea-pig ileum.

Contractile responses to substance P and related peptides of the isolated muscularis mucosae of the guinea-pig oesophagus.

The site of action of substance P and related tachykinins with respect to isotonic contractions was examined on the isolated muscularis mucosae attached to the submucous plexus of the guinea-pig oesophagus. Substance P (greater than 30 nM) produced a concentration-dependent contraction of the muscularis mucosae (EC50 1.9 +/- 0.5 microM, n = 10). The contractions were rapid in onset (2 min or less), sustained, reversible by washing and the preparation did not show tachyphylaxis. Eledoisin and physalaemin produced similar sustained contraction of the muscularis mucosae. The order of sensitivity was eledoisin greater than substance P greater than physalaemin. Contractions induced by 1 microM of each tachykinin were not significantly modified by incubation of the tissue with substance P or eledoisin (10 microM for 30 min). The contractile responses to tachykinins were unaffected by tetrodotoxin (0.3 microM), atropine (0.3 microM), phentolamine (1 microM), chlorpheniramine (1 microM), methysergide (1 microM), baclofen (100 microM) and verapamil (10 microM), but were abolished by the incubation of the tissue with calcium-free, EGTA (0.1 mM)-containing Tyrode solution. A substance P antagonist, [D-Pro2, D-Trp7,9]-substance P (greater than 0.1 microM), produced a transient contraction of the muscularis mucosae and the smooth muscle regained its original tone within 6 to 10 min. Contractions to the tachykinins were now inhibited by the antagonist (0.1-10 microM) in a concentration-dependent manner, the order of sensitivity being physalaemin greater than substance P = eledoisin. The cholinergically mediated electrically (0.1 Hz, 0.5 ms, supramaximal voltage)-induced twitch contractions of the muscularis mucosae were not significantly modified by substance P (0.01-0.3 microM). 7 The present results indicate that substance P and related tachykinins contract the isolated muscularis mucosae of the guinea-pig oesophagus by a direct action on the smooth muscle, probably by stimulating SP-E receptors.

Pharmacological characterization of the opioid receptor in the submucous plexus of the guinea-pig oesophagus.

1 The cholinergically mediated electrically-induced contractions of the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus were used to study the actions of opioid peptides and morphine. 2 The twitch contractions of the tissue (0.1 Hz, 0.5 ms, supramaximal voltage) were inhibited by all the opioid peptides and morphine in a concentration-dependent manner. The order of potency was dynorphin-(1-13) greater than alpha-neo-endorphin greater than beta-endorphin greater than [D-Ala2]-methionine-enkephalin much greater than alpha-endorphin, methionine-enkephalin, leucine-enkephalin and morphine. 3 The inhibitory actions of dynorphin-(1-13) (20 nM), alpha-neo-endorphin (100 nM) and beta-endorphin (3 microM) were completely reversed either by naloxone (1 microM) or by morphine (100 microM). The Ke values of naloxone against dynorphin-(1-13) and alpha-neo-endorphin were 30 and 25 nM, respectively. 4 Increasing the concentration of calcium from 1.8 to 3.6 mM in Tyrode solution decreased the sensitivity of the tissue to dynorphin-(1-13) 7.4 times and to alpha-neo-endorphin 462 times. 5 The inhibitory actions of dynorphin-(1-13) (100 nM) and alpha-neo-endorphin (300 nM) were inversely related to stimulus frequency, being most active at low frequencies (0.1-1 Hz), and least active at high (30 Hz). 6 Exogenously applied acetylcholine produced concentration-dependent contractions of the isolated muscularis mucosae, with an EC50 of 72.6 +/- 4.5 nM. The contractile response of the oesophagus to acetylcholine was not affected by the pretreatment of the tissue with dynorphin-(1-13) (100 nM), alpha-neo-endorphin (300 nM) or beta-endorphin (3 microM). 7 It is concluded that the submucous plexus-longitudinal muscularis mucosae of the guinea-pig oesophagus is inhibited by opioid peptides acting at prejunctional opioid receptors, probably of the kappa-subtype.

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae.

1. In the oesophageal muscularis mucosae, we examined the effects of endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ-123 and RES-701-1 as endothelin receptor antagonists. 2. All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration-dependent manner. The order of potency (-log EC50) was ET-1 (8.61)=SX6c (8.65)>ET-2 (8.40)>ET-3 (8.18). 3. FR139317 (1-3 microM) and BQ-123 (1 microM) caused parallel rightward shifts of the concentration-response curve to ET-1, but at higher concentrations caused no further shift. RES-701-1 (3 microM) caused a rightward shift of the concentration-response curve to ET-1, while RES-701-1 (10 microM) had no additional effect. RES-701-1 (0.1-1 microM) concentration-dependently caused a rightward shift of the concentration-response curve to SX6c. The contraction to ET-1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 microM). 4. Modulation of the Ca2+ concentration in the Krebs solution caused the concentration-response curve to ET-1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 microM) abolished rhythmic motility induced by ET-1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET-1- or SX6c-induced tonic contraction. SK&F 96365 (60 microM) completely inhibited ET-1-induced contractions. 5. We conclude that there are two types of ET-receptors, excitatory ET(A)- and ET(B)-receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor-operated Ca2+ channels (ROCs) and partly by opening T-type Ca2+ channels, and mediate rhythmic motility by opening L-type Ca2+ channels.

Inhibitory actions of catecholamines on electrically induced contractions of the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus.

1 The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of catecholamines on the twitch responses to electrical stimulation.2 When the preparation was stimulated coaxially (0.1 Hz, 0.5 ms, supramaximal voltage), stable twitch-like contractions were obtained. These were abolished by tetrodotoxin (0.1 muM) and atropine (0.1 muM), potentiated by physostigmine (0.1 muM), and were mediated presumably by stimulation of intramural cholinergic nerves.3 The twitch contractions of the muscularis mucosae were inhibited by catecholamines, in a concentration-dependent manner. The order of potency was isoprenaline > adrenaline > noradrenaline > dopamine.4 The inhibitory actions of noradrenaline (1 muM) and adrenaline (1 muM) were partly reversed by phentolamine (1 muM) or by propranolol (1 muM), and completely abolished by both antagonists together. The inhibitory effect of dopamine (300 muM) was largely reversed by phentolamine (1 muM), but not by propranolol (1 muM), while the inhibitory action of isoprenaline was competitively antagonized only by propranolol (pA(2) of 7.6).5 The contraction of the muscularis mucosae to exogenously applied acetylcholine (ACh, 20 nM) which was comparable in magnitude with that to electrical stimulation was also inhibited by isoprenaline (0.1 muM), adrenaline (1 muM) and noradrenaline (1 muM), but not by dopamine (300 muM). In the presence of propranolol (1 muM), noradrenaline, adrenaline and dopamine potentiated the ACh-induced contraction, while the effect of isoprenaline was mainly antagonized. The potentiating effects were antagonized by further treatment with phentolamine (1 muM).6 Adrenaline, noradrenaline and dopamine but not isoprenaline, produced a weak contraction of the longitudinal muscularis mucosae in the presence of propranolol (3 muM). The contractile responses were completely inhibited by phentolamine (3 muM). Tone in the muscularis mucosae induced by carbachol (3 muM) in the presence of phentolamine (10 muM) was inhibited by catecholamines, in a concentration-dependent manner, an effect that was competitively antagonized by propranolol.7 In the submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus there are three types of adrenoceptor, inhibitory prejunctional alpha-adrenoceptors, excitatory postjunctional alpha-adrenoceptors and inhibitory postjunctional beta-adrenoceptors, and cholinergic neurotransmission is inhibited by catecholamines acting at both prejunctional alpha- and postjunctional beta-adrenoceptors.

Pharmacological characterization of the histamine receptor in the isolated muscularis mucosae of the guinea-pig oesophagus.

To characterize the histamine receptors in the muscularis mucosae, the isotonic responsiveness of the isolated muscularis mucosae of the guinea-pig oesophagus to histamine receptor agonists and antagonists was examined in vitro. Histamine (0.1-100 microM) produced a concentration-dependent contraction of the muscularis mucosae (EC50 = 1.6 +/- 0.2 microM). The contractions were rapid in onset, sustained, reversible by washing and the preparation did not show tachyphylaxis. 2-Methylhistamine (2-MH), 2-pyridylethylamine (PEA) and 4-methylhistamine (4-MH) produced similar sustained contractions of the muscularis mucosae. The order of sensitivity was histamine greater than 2-MH greater than PEA greater than 4-MH. Impromidine (10-300 microM) and dimaprit (10-300 microM) caused no response in this tissue. The contractile responses to histamine, 2-MH, and PEA were competitively antagonized by diphenhydramine, and the pA2 values were almost the same (approximately 8.1). Cimetidine (100 microM) could not modify the contractile response to these agonists. The contractile response to histamine was slightly inhibited by tetrodotoxin (0.3 microM), atropine (1 microM), indomethacin (0.1-3 microM) or aspirin (30-300 microM), and the EC50 value was increased about 2-6 times by these drugs. When the preparation was incubated in Tyrode solution containing various calcium concentrations (0, 0.45, 0.9 and 1.8 mM), the concentration-response curve to histamine was shifted to the right and downward; the effect was inversely dependent on the calcium concentration, and in a calcium-free medium the response to histamine was abolished. Verapamil (1-10 microM) partially inhibited the contractile response to histamine. 7 The present results indicate that the contraction of the guinea-pig oesophageal muscularis mucosae to histamine is mediated mainly by a direct action on the smooth muscle and partly by indirect actions via the stimulation of either endogenous prostaglandin biosynthesis or intramural cholinergic nerves. The histamine receptors responsible for contractions of this tissue are probably mainly of the H,- subtype with H2-receptors having a negligible role.

Comparison of the effect of zopiclone and brotizolam on sleep EEG by quantitative evaluation in healthy young women.

We studied the effect of brotizolam (BRO) (0.25 mg) and zopiclone (ZPC) (7.5 mg) on the sleep electroencephalograms (EEG) of seven healthy young women (ages 20-21 years). In addition to conventional sleep scoring, we performed spectral analysis of EEG wave forms using the Fast Fourier Transformation (FFT) method. Four weeks following two consecutive polysomnographic recordings with placebo administration (for baseline data), polysomnography with BRO or ZPC administration was performed on the subjects who were then crossed over to polysomnography with ZPC or BRO, respectively, 1 week later. Total rapid eye movement (REM) time was decreased on the ZPC and BRO nights and REM latency was prolonged on the ZPC night compared with baseline (BL) night. Other parameters, however, were not significantly different between the drug nights and the BL night. There were also no differences in standard scoring results between both drug nights. FFT analysis revealed significant differences in total power density percentage and mean power density percentage in both nonrapid eye movement (NREM) and REM sleep between ZPC and BRO nights and between both drug nights compared to the BL night. Total power density and mean power density per epoch showed significant decreases in the theta band (6.0-8.0 Hz) and sigma band (12.0-14.0 Hz) on the ZPC night versus the BRO night or BL night during specific NREM periods. The results of this study corroborate previous reports that suggest computer-aided quantitative electroencephalography aids in differentiating specific effects of hypnotics and other central-nervous-system-acting agents on the sleep EEG.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine selectively inhibits noncholinergic transmission in guinea pig bronchi.

The neuromodulatory action of adenosine and ATP was investigated in isolated guinea pig bronchial strip chain preparations contracted with electrical field stimulation. The tissues were placed in organ baths containing physiological salt solution and stimulated at 8-Hz frequency, 0.5-ms pulse duration, and 30 V (approximately 100 mA) for 5 s. Electrical field stimulation evoked a biphasic contraction of bronchial muscle, consisting of an initial contraction followed by a sustained contraction, which was mediated by intramural cholinergic and noncholinergic nerve stimulations, respectively. Adenosine, at concentrations greater than M, caused a concentration-dependent inhibition in the height of the noncholinergically mediated contraction, accompanied by a very weak inhibition on the cholinergically mediated response. ATP (10(-5) to 3 x 10(-3) M) also produced a similar inhibitory effect on the noncholinergically mediated contraction, but the inhibitory potency was less than that of adenosine. The inhibitory response to adenosine was enhanced by the pretreatment with dipyridamole (2 x 10(-6) M) but antagonized with aminophylline (10(-5) M). Contractions of bronchial muscle evoked by exogenous acetylcholine (2 x 10(-6) M) or substance P (2 x 10(-7) M) were significantly inhibited by the adenosine (3 x 10(-4) M) pretreatment. These data suggest that in isolated guinea pig bronchi adenosine selectively inhibits noncholinergic neurotransmission through prejunctional P1-purinoceptors.

Different spasmolytic effects of smooth muscle relaxants on the guinea-pig esophageal muscularis mucosae contracted by carbachol or high potassium in vitro.

The responsiveness of the guinea-pig esophageal muscularis mucosae to smooth muscle relaxants was examined in vitro during the contractile state induced by carbachol (3 microM) or high potassium (60 mM). In the presence of phentolamine (3 microM), all catecholamines tested (10 nM-30 microM) relaxed the carbachol-induced tone more effectively than the high potassium-induced tone, and the maximum relaxations reached about 90-95% for carbachol but only about 40% for high potassium. Verapamil produced a concentration-dependent relaxation of the muscularis mucosae precontracted with either spasmogen; the mean EC50 values (-log M) were 6.73 for high potassium and 4.65 for carbachol. Methylene blue (1-300 microM) relaxed the carbachol-contracted muscularis mucosae in a concentration-dependent manner but relaxed the high potassium-contracted one less potently. Forskolin (1-300 microM), papaverine (1-100 microM), aminophylline (10-300 microM), trifluoperazine (1-300 microM), 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (10-300 microM), quinacrine (1-300 microM) and dibutyryl cyclic AMP (100 microM-3 mM) produced relaxation almost equipotently in both contractile states whereas adenosine (10 microM-1 mM), sodium nitroprusside (10-300 microM) and dibutyryl cyclic GMP (100 microM-3 mM) were virtually ineffective. The present results indicate that a variety of smooth muscle relaxants have different spasmolytic effects on the guinea-pig isolated esophageal muscularis mucosae which was precontracted with carbachol or high potassium and that catecholamines and methylene blue may produce relaxation independent of the changes in intracellular cyclic nucleotides, calmodulin or phosphoinositides.

Ba2+ selectively inhibits receptor-mediated contraction of the esophageal muscularis mucosae.

The aim of the present study was to examine the effect of Ba2+ on acetylcholine- and KCl-induced contractions of the guinea-pig esophageal muscularis mucosae. When the muscularis mucosae was pretreated with nicardipine (1 microM), Ba2+ (0.1-30 mM) markedly inhibited the acetylcholine (3 microM)-induced tone, and at 10-30 mM the tone returned to its basal level. In contrast, Ba2+ (0.1-30 mM) slightly increased the KCl (60 mM)-induced tone. Moreover, the Ba2+ (30 mM)-increased KCl tone was completely inhibited by treatment with nicardipine (0.3-1 microM). In conclusion, Ba2+ both selectively inhibits receptor-mediated contraction of the muscularis mucosae and itself permeates through voltage-dependent Ca2+ channels.

Some possibilities for prostaglandin mediation in the contractile response to ATP of the guinea-pig digestive tract.

Several contractile responses of longitudinal muscles of the guinea-pig digestive tract to exogenously applied ATP (10-300 micrometer), including "rebound" contractions, were inhibited by indomethacin (3-20 micrometer) or polyphloretin phosphate (10-100 microgram/ml). Relaxations to ATP in stomach and large intestinal muscles were increased by these drugs. Prostaglandin release might therefore contribute to the contractile responses of the guinea-pig digestive tract to ATP.

Contractile responses of the guinea-pig esophageal muscularis mucosae in vitro to arachidonic acid and its metabolites.

The responsiveness of the guinea-pig esophageal muscularis mucosae to arachidonic acid (AA) and its cyclooxygenase and lipoxygenase metabolites was examined in vitro. AA (0.1-30 microM) produced a concentration-dependent contraction of the muscularis mucosae (mean EC50 +/- S.E.M. = 5.1 +/- 1.0 microM). The contractions in response to low concentrations of AA (0.1-3 microM) were prevented by pretreatment of the tissue with indomethacin (1-10 microM), while those in response to high concentrations (10-100 micron) were prevented by BW755C (10-100 microM). The contractile response to AA was antagonized by polyphloretin phosphate (PPP, 1-10 micrograms/ml) and by FPL 55712 (1-10 microM). All cyclooxygenase and lipoxygenase metabolites of AA tested also produced a sustained contraction of the muscularis mucosae with the following order of sensitivity; leukotriene (LT) D4 greater than LTC4 greater than prostaglandin (PG) E2 greater than PGF2 alpha greater than PGI2 greater than thromboxane B2. The responses to LTC4 and LTD4 were antagonized by FPL 55712 (0.1-1 microM), while those to PGE2 and PGF2 alpha were antagonized by PPP (3-100 micrograms/ml). The present results indicate that exogenously applied AA contracts the isolated muscularis mucosae of the guinea-pig esophagus by an indirect action via its metabolism to both PGs and LTs. The putative PG and LT receptors located in this tissue are probably similar to those in the ileal longitudinal muscle, but differ from those in the airway smooth muscle.

Diminished purinergic modulation of the vascular adrenergic neurotransmission in spontaneously hypertensive rats.

The inhibitory effects of adenosine and ATP on the pressor response of the perfused mesenteric vascular bed to perivascular adrenergic nerve stimulation were compared between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR). Both adenosine and ATP inhibited the neurogenic vasoconstriction in a dose-dependent manner in WKR, in which the inhibitory effect of adenosine was approximately eight times greater than that of ATP on the basis of 50% inhibitory doses (ID50). In the same preparation isolated from SHR, the inhibitory effects of both adenosine and ATP were significatly smaller than that in WKR. The ID50 values in the SHR for both agonists were approximately six times those found in WKR. These compounds also inhibited the pressor response to norepinephrine (0.3 microgram/ml) infusion, but the degrees of inhibition were markedly less than those with the neurogenic pressor response, and were not significantly different between WKR and SHR. These results suggest that presynaptic inhibition of vascular adrenergic neurotransmission by purine compounds is reduced in SHR.

Assessment of apoptosis in oesophageal carcinoma preoperatively treated by chemotherapy and radiotherapy.

Apoptosis, programmed cell death, was immunohistochemically determined in 55 samples of oesophageal squamous cell carcinoma using the BM1 Mab. Sections from patients not treated (group 1, n = 12) or preoperatively treated by chemotherapy (group 2, n = 11), radiation (group 3, n = 13) or both (group 4, n = 8), and 11 additional cases of high-grade dysplasia or early cancer were examined. Most of the apoptotic cells were BM1-positive and checked by TUNEL proved to be nick end positive. They accounted for 7 (11%), 19 (29%), 21 (32%) and 26 (38%) cells per field in those 4 groups respectively. Chemotherapy and/or radiation significantly increased the number of apoptotic cells as compared to controls (p = 0.029 and p = 0.029, respectively). To assess the implications of the oncogene expression in the apoptotic pathway, additional section stained with bcl2 and p53 were negative for bcl2 and were positive for p53 in 16 samples (37%). Overall, positive cases for p53 mutation showed a significantly decreased incidence of apoptotic cells (p = 0.03). These results suggest that in situ assessment of apoptotic response better correlates to the apoptosis induced by radiation than that by chemotherapy, that abnormalities of the p53 protein decrease the apoptotic response in oesophageal carcinoma, and that immunohistochemical analysis of p53 protein helps to determine the sensitivity to these anticancer agents.

Inhibitory effects of sympathomimetic drugs on cholinergically mediated contractions of guinea-pig isolated tracheal muscle.

The experiments examine the actions of sympathomimetic drugs on the responses evoked by electrical field stimulation or by acetylcholine in guinea-pig tracheal strip chains. Electrical field stimulation evoked contractions which were cholinergically mediated, in the presence of guanethidine (10 microM) and indomethacin (2 microM). All the sympathomimetic drugs tested caused a concentration-dependent reduction in the height of these contractions. Inhibitory effects of isoprenaline and terbutaline were largely prevented by propranolol (2 microM) alone, whereas those of clonidine, oxymetazoline, lidamidine and WHR1370 were prevented by yohimbine alone (2 microM). Treatments with both propranolol and yohimbine were required to prevent the inhibitory effects of noradrenaline, adrenaline and dopamine. Contractions evoked by exogenous acetylcholine (0.1-3 microM) were also inhibited by all catecholamines and terbutaline, but not by clonidine, oxymetazoline, lidamidine and WHR1370. The inhibitory effects were antagonized by propranolol (2 microM) alone. The results suggest that in guinea-pig isolated tracheal muscle, sympathomimetic drugs can inhibit cholinergic neurotransmission not only by postjunctional beta 2-adrenoceptors but also by prejunctional alpha 2-adrenoceptors.