Yuichiro Kamikawa

Decreased Expression of Cytochromes P450 1A2, 2E1, and 3A4 and Drug Transporters Na+-Taurocholate-Cotransporting Polypeptide, Organic Cation Transporter 1, and Organic Anion-Transporting Peptide-C Correlates with the Progression of Liver Fibrosis in Chronic Hepatitis C Patients

Patients with chronic hepatitis C viral infection underwent liver biopsies and laboratory studies for evaluation and to determine subsequent treatment. Changes in status of drug metabolism and disposition may vary with chronic hepatitis C stage and should be assessed. Total RNA was extracted from liver biopsy specimens (n = 63) and reverse transcribed to yield cDNA. Relative mRNA levels of drug-metabolizing enzymes, transporters, nuclear receptors, and proinflammatory cytokines were analyzed with normalization to glyceraldehyde 3-phosphate dehydrogenase expression. mRNAs encoding cytochromes P450 1A2, 2E1, and 3A4, and drug transporters, Na+-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1 showed remarkable decreases, and tumor necrosis factor-α showed an increase according to fibrosis stage progression. HepG2 cells and primary hepatocytes of two human individuals were treated with interleukin 1β, interleukin 6, or tumor necrosis factor-α. CYP1A2 and Na+-taurocholate-cotransporting polypeptide mRNA levels significantly decreased in HepG2 cells with interleukin 1β and interleukin 6 treatments. CYP2E1 and organic cation transporter 1 mRNA levels significantly decreased with tumor necrosis factor-α treatment only in HepG2. These results suggested that down-regulation of CYP1A2, 2E1, and 3A4, and drug transporters, Na+-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1, manifested in livers of patients with chronic hepatitis C viral infection, was associated, at least in part, with the elevated production of proinflammatory cytokines, including tumor necrosis factor-α.

Heterogeneity of muscarinic receptors in the guinea pig esophageal muscularis mucosae and ileal longitudinal muscle

Pharmacologic characteristics of muscarinic receptors in the muscularis mucosae of the guinea pig esophagus were examined in vitro and compared with those of the longitudinal muscle of the guinea pig ileum. All cholinomimetics tested produced a sustained contraction of the muscularis mucosae, in a concentration-dependent manner, which was associated with a small biphasic change in membrane potential, initially a hyperpolarization followed by a depolarization. The contractile response was hardly modified by verapamil, but was depressed by calcium removal from the bathing medium. Both atropine and pirenzepine antagonized the contractile response in a competitive manner, with higher pA2 values than those in the ileum. These results indicate that the muscarinic receptors of the muscularis mucosae of the guinea pig esophagus mainly link with calcium ion channels which are independent of changes in membrane potential and that their subtype populations are probably pharmacologically distinct from those in the ileal longitudinal muscle.

Pharmacological differences of non‐adrenergic inhibitory response and of ATP‐induced relaxation in guinea‐pig tracheal strip‐chains

The presence of non-adrenergic inhibitory neurons in the guinea-pig tracheal muscle has been demonstrated by Coburn & Tomita (1973) and Richardson & Bouchard (1975), using isolated tracheal strips under isometric conditions. On the other hand, Coleman & Levy (1974) also recognized these neurons using the isolated tracheal tube preparation under isotonic conditions and proposed adenosine 5’-triphosphate (ATP) as a transmitter in these neurons. Previously (Kamikawa & Shimo, 1976), we reported that the inhibitory response to ATP of guinea-pig tracheal strip-chains is antagonized by indomethacin, aspirin and polyphloretin phosphate (PPP) so that this response may be an indirect one mediated by prostaglandin (PG) &. The present report thus describes the influences of these drugs on the nonadrenergic inhibitory response to field stimulation of tracheal strip-chains of guinea-pigs under isometric conditions. Male guinea-pigs, 300 to 6OOg, were used. The tracheal strip-chain was prepared according to the method described in our previous paper and suspended in a 30ml organ bath with modified Krebs-Ringer solution (composition mM: NaCI, 120; KCl, 4.7; CaClp, 2.5; MgCI2, 1-2; NaHCO,, 25; KH2POo, 1.2; glucose, 14; pH 7.4) aerated with 5 % C 0 2 in oxygen at 37 ’. The preparation was mounted under 0.5g of initial tension and contracted by an application of histamine 10 p~ before every stimulation. The tracheal response was recorded isometrically using a forcedisplacement transducer coupled to a Recticorder (Nihon Kohden). The field stimulation was with rectangular pulses of varied frequencies from 1 to 50Hz, pulse duration of 0.3 ms and supramaximal voltage, through bipolar platinum electrodes which were 10 mm apart and connected to a Nihon Kohden stimulator, model SEN 1101. The total number of stimulating pulses was kept constant at 60. For the elimination of parasympathetic components in responses to field stimulation, the Krebs-Ringer solution contained 1 p~ atropine sulphate. Drugs used were: adenosine 5’-triphosphate disodium salt (Sigma), histamine dihydrochloride, atropine sulphate (Wako Pure Chem.), guanethidine sulphate (Ciba-Geigy), propranolol hydrochloride, tetrodotoxin, indomethacin (Sankyo), and PPP (AB Leo). Indomethacin was dissolved in 50% ethanol, and all other drugs were dissolved in physiological saline. Field stimulation of the tracheal strip-chains with the stimulus parameters described above elicited only relaxation which depended on the stimulus frequency.

Contractile response to a cannabimimetic eicosanoid, 2-arachidonoylglycerol, of longitudinal smooth muscle from the guinea-pig distal colon in vitro

The effect of 2-arachidonoylglycerol, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), 2-arachidonoylglycerol (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.2 microM), and partially by hexamethonium (100 microM) pretreatment. The response to 2-arachidonoylglycerol was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(2) receptor agonist, R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(2) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for 2-arachidonoylglycerol to the right. The contractile action of 2-arachidonoylglycerol was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that 2-arachidonoylglycerol produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(2) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of 2-arachidonoylglycerol.

Inhibitory effects of catecholamines on cholinergically and non‐cholinergically mediated contractions of guinea‐pig isolated bronchial muscle

Abstract— The actions of catecholamines on the responses evoked by electrical field stimulation or by acetylcholine and substance P in guinea‐pig bronchial strip chain have been examined. Electrical field stimulation evoked a biphasic contraction, consisting of a cholinergically‐mediated fast contraction followed by a non‐cholinergically‐mediated slow contraction. All catecholamines tested caused a concentration‐dependent reduction in the height of the biphasic contraction, where non‐cholinergic contractions were more potently inhibited. The inhibitory effect of isoprenaline was largely prevented by propranolol (2 μM) alone, whereas those of noradrenaline and adrenaline were prevented by treatment with both propranolol (2 μM) and yohimbine (2 μM). The inhibitory effect of dopamine was unaffected either by propranolol (2 μM), yohimbine (2 μM) or haloperidol (10 μM). Submaximal contractions of bronchial muscle evoked by exogenous acetylcholine (2 μM) or substance P (0.2 μM) were also inhibited by catecholamines, except dopamine, but the effects were antagonized by propranolol (2 μM) alone. The results suggest that in guinea‐pig isolated bronchial muscle, catecholamines can inhibit both cholinergic and non‐cholinergic excitatory neurotransmissions not only by postjunctional β‐adrenoceptors but also by prejunctional α2‐adrenoceptors.

Mediation of prostaglandin E2 in the triphasic response to ATP of the isolated tracheal muscle of guinea-pigs

ATP, at a dose higher than 0·1 μg ml−1, showed a biphasic action consisting of an initial increase followed by a gradual decrease of muscle tension in the isolated tracheal strip‐chains of guinea‐pigs. The pattern of this biphasic response to ATP varied with the level of basal tone of the preparation at the moment of application of ATP. A similar biphasic action was obtained by prostaglandin (PG) E2 among the various active substances studied including acetylcholine, histamine, catecholamines and various types of PG. Indomethacin (0·1 μg ml−1) and aspirin (30 μg ml−1) completely abolished the ATP‐induced inhibitory response observed in the presence of histamine (10 μm). Polyphloretin phosphate (100 μg ml−1) also significantly depressed the inhibitory response to ATP or PGE2. It is concluded that the response to ATP of the preparation is mediated by PGE2 released via the stimulation of its biosynthesis.

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) > α‐neo‐endorphin > β‐endorphin > [d‐Ala2]‐methionine‐enkephalin ≪≪ α‐endorphin, methionine‐enkephalin, leucine‐enkephalin and morphine. 3 The inhibitory actions of dynorphin‐(1–13) (20 nm), α‐neo‐endorphin (100 nm) and β‐endorphin (3 μm) were completely reversed either by naloxone (1 μm) or by morphine (100 μm). The Ke values of naloxone against dynorphin‐(1–13) and α‐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 α‐neo‐endorphin 462 times. 5 The inhibitory actions of dynorphin‐(1–13) (100 nm) and α‐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), α‐neo‐endorphin (300 nm) or β‐endorphin (3 μm). 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 κ‐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 μM) and BQ‐123 (1 μM) caused parallel rightward shifts of the concentration‐response curve to ET‐1, but at higher concentrations caused no further shift. RES‐701‐1 (3 μM) caused a rightward shift of the concentration‐response curve to ET‐1, while RES‐701‐1 (10 μM) had no additional effect. RES‐701‐1 (0.1–1 μM) 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 μM). 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 μM) 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 μM) completely inhibited ET‐1‐induced contractions. 5 We conclude that there are two types of ET‐receptors, excitatory ETA‐ and ETB‐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.

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 μm. 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 μm, 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 mucosae, the 50% effective concentration (EC50) 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 μm)‐induced contraction was completely abolished by tetrodotoxin (0.2 μm) and atropine (0.2 μm). 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 μm). 6 Among several 5‐MT antagonists examined, methysergide (1 μm), ketanserin (1 μm) and morphine (100 μm) failed to modify the 5‐HT (100 μm)‐induced contraction significantly. Cinanserin (0.1–3 μm), cyproheptadine (3–100 nm) and phenoxybenzamine (0.1–3 μm) 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 μm) or ACh (0.1 μm)‐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.

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 μm) and atropine (0.1 μm), potentiated by physostigmine (0.1 μm), 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 μm) and adrenaline (1 μm) were partly reversed by phentolamine (1 μm) or by propranolol (1