European Journal of Pharmacology (v.413, #2-3)

The effects of five different protein kinase C inhibitors—calphostin C, chelerythrine, bisindolylmaleimide I, staurosporine and Gö6979—on intracellular free magnesium ([Mg2+]i) content and mobilization were investigated in primary, cultured rat aortic smooth muscle cells. All these protein kinase C inhibitors significantly and rapidly increased [Mg2+]i both in normal media (1.2 mM Mg2+) and in Mg2+ free media. These data suggest that the increments of [Mg2+]i, induced by the diverse protein kinase C inhibitors, are derived from the release of bound intracellular Mg2+ and that activation of protein kinase C isozymes are normally responsible for helping to maintain basal levels of [Mg2+]i in rat aortic smooth muscle cells.
Keywords: Mg2+; Smooth muscle; Protein kinase C inhibitor;

Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells by Katarzyna Tkaczyk-Gobis; Jolanta Tarasiuk; Olivier Seksek; Barbara Stefanska; Edward Borowski; Arlette Garnier-Suillerot (131-141).
Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux. Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells. We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1. We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells. Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2×10−17 mole cell−1 s−1, (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 μM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K m∼2 μM. In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.
Keywords: Multidrug resistance; P-glycoprotein; MRP1 (multidrug resistance-associated protein); Benzoperimidine;

Molecular and pharmacological characterization of the murine tachykinin NK3 receptor by Henry M Sarau; John A Feild; Robert S Ames; James J Foley; Parvathi Nuthulaganti; Dulcie B Schmidt; Peter T Buckley; Nabil A Elshourbagy; Mary E Brawner; Mark A Luttmann; Giuseppe A.M Giardina; Douglas W.P Hay (143-150).
Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK3 receptor from mouse brain. The murine NK3 receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK3 receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK3 receptor and human NK3 receptor, although substance P and neurokinin A were more potent stimulators of Ca2+ mobilization in murine NK3 receptor cells. NK3 receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK3 receptor compared to human NK3 receptor, and about 5- to 10-fold reduced potency in the murine NK3 receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK3 receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.
Keywords: Tachykinin NK3 receptor; Tachykinin NK3 receptor; Tachykinin; Tachykinin NK3 receptor antagonist;

The mitogen-activated protein kinase (MAPK) family consists of the p42/p44 MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. We have previously reported that the human adenosine A1 receptor stimulates p42/p44 MAPK in transfected Chinese hamster ovary cells. In this study, we have investigated whether the endogenous adenosine A1 receptor in the smooth muscle cell line, DDT1MF-2 activates p42/p44 MAPK, JNK and p38 MAPK. The adenosine A1 receptor agonist N 6-cyclopentyladenosine stimulated time and concentration-dependent increases in p42/p44 MAPK and p38 MAPK phosphorylation in DDT1MF-2 cells. No increases in JNK phosphorylation were observed following adenosine A1 receptor activation. N 6-cyclopentyladenosine-mediated increases in p42/p44 MAPK and p38 MAPK phosphorylation were blocked by the selective adenosine A1 receptor antagonist 1,3-dipropylcyclopentylxanthine and following pretreatment of cells with pertussis toxin. Furthermore, adenosine A1 receptor-mediated increases in p42/p44 MAPK were sensitive to the MAPK kinase 1 inhibitor PD 98059 (2′-amino-3′-methoxyflavone), whereas p38 MAPK responses were blocked by the p38 MAPK inhibitor SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole). The broad range protein tyrosine kinase inhibitors genistein and tyrphostin A47 (α-cyano-(3,4-dihydroxy)thiocinnamide) did not block adenosine A1 receptor stimulation of p42/p44 MAPK. For comparison, insulin-mediated increases in p42/p44 MAPK were blocked by genistein and tyrphostin A47. The Src tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and the epidermal growth factor receptor tyrosine kinase inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) also had no effect on adenosine A1 receptor stimulation of p42/p44 MAPK. Furthermore, the protein kinase C inhibitors Ro 31-8220 (3-{1-[3-(2-isothioureido) propyl]indol-3-yl}-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione), chelerythrine and GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) were without effect on adenosine A1 receptor-induced p42/p44 MAPK phosphorylation. In contrast, wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibitors of phosphatidylinositol 3-kinase, attenuated adenosine A1 receptor stimulation of p42/p44 MAPK phosphorylation. In conclusion, the adenosine A1 receptor stimulates p42/p44 MAPK through a pathway which appears to be independent of tyrosine kinase activation but involves phosphatidylinositol 3-kinase. Finally, adenosine A1 receptor stimulation in DDT1MF-2 cells also activated p38 MAPK but not JNK via a pertussis toxin-sensitive pathway.
Keywords: Adenosine A1 receptor; MAP (mitogen-activated protein) kinase; DDT1MF-2 cell;

Leukotriene D4-induced Rho-mediated actin reorganization in human bronchial smooth muscle cells by Shizue Saegusa; Hirokazu Tsubone; Masayoshi Kuwahara (163-171).
We investigated the role of cysteinyl leukotriene (CysLT) receptors on leukotriene D4-induced actin reorganization and the signaling pathways of the response in human bronchial smooth muscle cells. The effects of leukotriene D4 on actin reorganization in human bronchial smooth muscle cells were evaluated by dual-fluorescence labeling of filamentous (F) and monomeric (G) actin with fluorescein isothiocyanate (FITC)-labeled phalloidin and Texas Red-labeled DNase I, respectively. Leukotriene D4 (100 nM) induced actin reorganization in the presence and absence of extracellular Ca2+. The CysLT type 1 (CysLT1) receptor antagonist ONO 1078 (4-oxo-8(−)[p-(4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate) inhibited leukotriene D4-induced actin reorganization. Pretreatment with pertussis toxin, C3 exoenzyme, or tyrosine kinase inhibitors significantly reduced leukotriene D4-induced actin reorganization. However, phosphatidylinositol-3-kinase and protein kinase C inhibitors had little effect on these responses. These results suggest that leukotriene D4-induced actin reorganization in human bronchial smooth muscle cells is extremely dependent on the CysLT1 receptor coupled with pertussis toxin-sensitive G protein, Rho GTPases and tyrosine phosphorylation pathways.
Keywords: Actin; Smooth muscle cell; Cysteinyl leukotriene type 1 receptor; Leukotriene D4; Pertussis toxin; Protein kinase C; Tyrosine kinase;

Effects of spinorphin and tynorphin on synaptic transmission in rat hippocampal slices by Takanobu Yamazaki; Motoko Honda; Yukio Yamamoto; Tadahiko Hazato; Hideki Ono (173-178).
Spinorphin has been isolated from the bovine spinal cord as an endogenous inhibitor of enkephalin-degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase III, angiotensin-converting enzyme and enkephalinase), and tynorphin has been synthesized as a more potent inhibitor of dipeptidyl aminopeptidase III. In this study, the effects of spinorphin and tynorphin on synaptic transmission were studied in rat isolated hippocampal slices. Field potentials were recorded from the CA1 region after stimulation of Schaffer collaterals. Spinorphin (1 μM), which alone had no effect, potentiated the facilitatory effects of enkephalin on the filed potentials at a stimulation interval of 15 s. At a stimulation interval of 10–4 s, spinorphin alone frequency dependently inhibited the field potential. On the other hand, tynorphin (1 μM), which alone had no effect at any stimulus interval, tended to potentiate the facilitatory effects of enkephalin. Spinorphin blocked long-term potentiation induced by tetanic stimulation (100 Hz, 1 s), whereas tynorphin had no effect on long-term potentiation. These results suggest that, at a low stimulation frequency, spinorphin potentiates the facilitatory effects of enkephalin by preventing degradation of enkephalin, whereas at a high stimulation frequency spinorphin use dependently inhibits synaptic transmission independently of enkephalin. On the other hand, tynorphin tends to potentiate the facilitatory effects of enkephalin without use-dependent inhibition.
Keywords: Spinorphin; Tynorphin; Enkephalin-degrading enzyme; Hippocampus; Field potential;

The nature of the prejunctional inhibitory muscarinic receptor on cholinergic nerve endings in the rat urinary bladder was investigated by measuring stimulated endogenous acetylcholine release via high pressure liquid chromatography (HPLC), in the presence of various selective muscarinic antagonists. The rank order of potencies for the antagonists used was: atropine (−log concentration=7.8)>4-DAMP (4-diphenylacetoxy-N-methylpiperidine) (7.6)>tripitramine (7.3)=HHD (hexahydrodifenidol) (7.3)>pFHHSiD (p-fluoro-hexahydrosiladifenidol hydrochloride) (7.0)>himbacine (6.5)>methoctramine (5.9)≥pirenzepine (5.8)>gallamine (4.3). A comparison of the antagonist potencies obtained, with affinity constants at muscarinic M1 to M5 receptors, suggests that the prejunctional inhibitory muscarinic receptor is of the M4 receptor subtype.
Keywords: Muscarinic receptor; Cholinergic nerve; Urinary bladder;

We investigated the involvement of striatal dopamine release in electrographic and motor seizure activity evoked by kainic acid in the guinea pig. The involvement of the dopamine receptor subtypes was studied by systemic administration of the dopamine D1 receptor antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390; 0.5 mg kg−1), or the dopamine D2 antagonist, (5-aminosulphonyl)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride, 30 mg kg−1). Microdialysis and high performance liquid chromatography were used to monitor changes in extracellular levels of striatal dopamine and its metabolites, glutamate, aspartate and γ-amino-butyric acid (GABA). These data were correlated with changes in the striatal and cortical electroencephalographs and clinical signs. We found that, although neither dopamine receptor antagonist inhibited behavioural seizure activity, blockade of the dopamine D1-like receptor with SCH 23390 significantly reduced both the ‘power’ of the electrical seizure activity and the associated change in extracellular striatal concentration of glutamate, whilst increasing the extracellular striatal concentration of GABA. In contrast, blockade of the dopamine D2-like receptor with sulpiride significantly increased the extracellular, striatal content of glutamate and the dopamine metabolites. These results confirm previous evidence in other models of chemically-evoked seizures that antagonism of the dopamine D1 receptor tends to reduce motor and electrographic seizure activity as well as excitatory amino-acid transmitter activity, while antagonism of the dopamine D2 receptor has relatively less apparent effect.
Keywords: Dopamine receptor antagonist; Microdialysis; Electroencephalography; Kainic acid; Seizure; (Guinea pig);

Sodium nitroprusside-induced seizures and adenosine release in rat hippocampus by Tomohiro Kaku; Min Hai Jiang; Junichi Hada; Kazuyoshi Morimoto; Yasumasa Hayashi (199-205).
In the present study, we examined the effects of nitric oxide (NO)-related compounds, i.e. sodium nitroprusside (NO donor), diethyldithiocarbamate (NO trapper) and dithiothreitol (superoxide radical scavenger) on release of aspartate and adenosine from rat hippocampus using electrophysiological and microdialysis methods. Perfusion with 0.05 or 0.5 mM sodium nitroprusside significantly reduced high K+-evoked release of aspartate during high K+ perfusion. Perfusion with 0.5 mM sodium nitroprusside always induced seizures and significantly increased release of aspartate and adenosine during washout of sodium nitroprusside. Diethyldithiocarbamate (5 mM) reversed the effects of sodium nitroprusside. Dithiothreitol (1 mM) significantly reduced the increase in adenosine release by sodium nitroprusside. These findings indicate that adenosine release is closely related to development of seizures, which are triggered by an increase in both NO itself and in part peroxynitrite, which results in reaction with superoxide radicals.
Keywords: Seizure; Adenosine; Nitric oxide (NO); Nitric oxide (NO) donor; Sodium nitroprusside; Microdialysis; Hippocampus;

Chronic GBR 12909 administration differentially alters prodynorphin gene expression compared to cocaine by Patrizia Romualdi; Claudio D'Addario; Sergio Ferri; Brian M Cox; Sari Izenwasser (207-212).
The effect of the selective dopamine uptake inhibitor 1-[2-[bis(4-flourophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR 12909) was examined on prodynorphin gene expression. GBR 12909 or vehicle was continuously infused for 7 days via osmotic minipump, or injected daily into male rats. Both continuous infusions and daily injections of GBR 12909 produced significant decreases in prodynorphin expression in the hypothalamus (37% and 31% decreases, respectively). There were no significant changes in the caudate putamen, hippocampus or nucleus accumbens. One injection of GBR 12909 had no effects on prodynorphin expression in any of the brain regions studied, suggesting that the effect in the hypothalamus is not an acute effect. As previously reported for other treatment regimens, continuous infusion of cocaine produced a 35% significant decrease in the hypothalamus, consistent with the effects of GBR 12909. In contrast to GBR 12909, however, cocaine also produced a significant increase in prodynorphin expression in the caudate putamen. Thus, chronic inhibition of dopamine uptake can regulate prodynorphin expression in the hypothalamus. In contrast, the increase in the caudate putamen following cocaine administration may not be related to the inhibition of dopamine uptake, since it was not produced by a selective dopamine uptake inhibitor. These findings suggest that regulation of prodynorphin gene expression by cocaine in the caudate putamen may be mediated by the inhibition of norepinephrine or serotonin uptake, by a combination of effects on two or three monoamine transporters, or by a mechanism unrelated to transporter inhibition.
Keywords: Dynorphin; Dopamine uptake; Dopamine transport; Cocaine; GBR 12909;

Electroconvulsive shock increases tachykinin NK1 receptors, but not the encoding mRNA, in rat cortex by Philip W.J Burnet; Rowan Miller; Louise J Lewis; Qi Pei; Trevor Sharp; Paul J Harrison (213-219).
Recent studies have suggested that the substance P (tachykinin NK1) receptor may be a pharmacological target for the treatment of mood disorders. Here, the effects of electroconvulsive shock on tachykinin NK1 receptor gene expression in the rat brain was investigated. Rats received either a single electroconvulsive shock or five shocks on alternate days. Quantitative autoradiography with [125I]Bolton Hunter-substance P, and in situ hybridisation histochemistry, were used to measure tachykinin NK1 receptor-binding site densities and mRNA abundance, respectively. Densities of tachykinin NK1 receptor-binding sites were significantly increased in the cerebral cortex following repeated electroconvulsive shock compared to sham treated animals. Densities remained unchanged in the hippocampus, striatum and amygdala. Neither single nor repeated electroconvulsive shock altered tachykinin NK1 receptor mRNA in the brain regions examined. Hence, repeated electroconvulsive shock increases tachykinin NK1 receptors in the rat brain in a regionally specific way. Upregulation of receptor-binding sites without a change in mRNA indicates that translational or post-translational mechanisms underlie this process.
Keywords: Neurokinin; Brain; Autoradiography; Gene expression; Hybridisation;

Dehydroevodiamine attenuates β-amyloid peptide-induced amnesia in mice by Hui-Hung Wang; Cheng-Jen Chou; Jyh-Fei Liao; Chieh-Fu Chen (221-225).
Dehydroevodiamine has been reported to have anticholinesterase activity and an anti-amnesic effect. This study examined the effects of dehydroevodiamine on scopolamine- and β-amyloid peptide-(25–35)-induced amnesia in mice, using a step-through passive avoidance test. Similarly to the cholinesterase inhibitor, physostigmine (0.03–0.3 mg/kg, i.p.), dehydroevodiamine (0.75–12.0 mg/kg, i.p.) administered 30 min before the training trial, immediately after the training trial, and 30 min before the retention test significantly improved scopolamine- and β-amyloid peptide-(25–35)-induced amnesia. In β-amyloid peptide-(25–35)-induced amnesia, the rank order of anti-amnesic potency in these three administration schedules for dehydroevodiamine was different from that for physostigmine. Furthermore, dehydroevodiamine was more potent to improve β-amyloid peptide-(25–35)-induced amnesia than scopolamine-induced amnesia when administered before the training trial. These results suggested that dehydroevodiamine may have an action other than that of an anticholinesterase and may be a novel and effective ligand for improvement of β-amyloid type amnesia.
Keywords: Dehydroevodiamine; β-Amyloid peptide; β-Amyloid peptide-(25–35); Amnesia; Step-through passive avoidance; Physostigmine;

The effect of CP 55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclo-hesanol}, heroin and etonitazene on intracerebroventricular (i.c.v.) self-administration in a free-choice procedure was evaluated in rats. Animals were trained in 1-h daily sessions with a continuous reinforcement schedule to press two active levers to obtain the vehicle of each drug. Then, when a stable baseline was reached, each drug could be self-administered by pressing the lever found to be less preferred during training, while the vehicle came from the other. The number of bar pressings associated with the delivery of increasing unit doses of CP 55,940 (0.1, 0.2, 0.4, 0.8, 1.6 μg/2 μl/infusion), heroin (0.125, 0.25, 0.5, 1, 2 μg/2 μl/infusion) or etonitazene (0.1–0.2–0.5–1 μg/ 2 μl/infusion) and with the delivery of the corresponding vehicle was fitted by symmetrical parabolas. The mean drug intake was linearly related to the log of self-administered drugs. Pretreatment with SR141716A [N-piperidino-5-(4-chlorophenyl)1-(2,4-dichloro-phenyl)-4-methylpyrazole-3-carboxamide] (0.5 mg/kg) or naloxone HCl (2 mg/kg/i.p.) 15 min before each daily session reduced the self-administration of both CP 55,940 and heroin. The combination of CP 55,940 with heroin or etonitazene reduced the number of drug-associated lever pressings compared to that obtained with the maximal reinforcing unit dose of each drug alone. These findings suggest there may be a strong interaction between opioids and the cannabinoid system.
Keywords: Cannabinoid; Opiate; Self-administration; Free-choice;

The effects of intracranial injection of three β3-adrenoceptor agonists, sodium-4-[-2[-2-hydroxy-2-(-3-chloro-phenyl)ethylamino] propyl]phenoxyacetate (BRL 37344), 2-hydroxy-5(2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazole-2-yl)-phenoxy)propyl)amino)ethoxy)-benzamide monomethane sulfonate) (±)-CGP12177A) and the pro-drug RS-N-(7-carbethoxymethoxyl 1,2,3,4-tetrahydronaphth-2-yl)-2 hydroxy 2-(3-chlorophenyl)ethanamine (SR58611A), were examined on reinforcement of memory in day-old chicks. BRL37344 and CGP12177 facilitated memory, whereas SR58611A had no effect. The dose–response relationships of the β3-adrenoceptor agonists were challenged with the selective β3-adrenoceptor antagonist 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) or the β2-adrenoceptor antagonist (−)propranolol. BRL 37344 appeared to act predominantly at β3-adrenoceptors at low doses and at β2-adrenoceptors at higher doses. Facilitation of labile into long-term storage by β3-adrenoceptor agonists appears to be a class action of these drugs.
Keywords: β3-Adrenoceptor; Memory; Consolidation; Memory; Passive avoidance learning; Adrenoceptor; (Chick);

Acute administration of nicotine impairs the hypotensive responses to bradykinin in rats by Maria O Paganelli; Jose E Tanus-Santos; Juan C.Y Toledo; Joaquim Francisco do Prado; Vivian Calegari; Heitor Moreno (241-246).
Nicotine may contribute to smoking-induced endothelial dysfunction because of its ability to impair endothelium-dependent vasodilatation. We investigated whether the acute administration of nicotine changes the hypotensive responses to bradykinin in rats. The effects of pre-treatment with losartan or enalapril on the nicotine-induced changes in the responses to bradykinin were also evaluated. In study 1, anesthetized rats were cannulated via carotid artery for the measurement of mean arterial pressure. Dose–response curves to bradykinin (0.1, 0.4, 1.6, 6.4, 25 and 100 μg/kg) were generated before and 10 min after the injection of nicotine (200 μg/kg, i.v.) or saline. The individual dose–response curves were fitted to a four-parameter logistic equation using the ALLFIT program, which provided an estimate of the maximal response (E max) and of the dose of bradykinin producing the half-maximal response (ED50). In study 2, rats were pre-treated orally with losartan (10 mg/kg/day) or enalapril maleate (25 mg/kg/day) for 2 weeks. Control rats received tap water alone. After pre-treatment, the rats were anesthetized and used as described in study 1. Nicotine decreased the E max (from 73.0±7.5 to 65.7±3.3 mm Hg; P<0.05) but did not affect the ED50. In study 2, losartan or enalapril did not affect nicotine-induced decrease in responses to bradykinin; E max decreased in both groups (from 68.7±6.3 to 62.8±4.2 mm Hg, and from 53.8±13.0 to 43.1±7.1 mm Hg, respectively; P<0.05) without significantly changing the ED50. These results suggest that nicotine impairs the endothelium-dependent hypotensive responses to bradykinin. This effect is not influenced by inhibition of the angiotensin-converting enzyme or by blockade of the angiotensin AT1 receptors.
Keywords: Blood pressure; Bradykinin; Endothelial dysfunction; Enalapril; Losartan; Nicotine;

Endothelin-1-induced potentiation of adrenergic responses in the rabbit pulmonary artery: role of thromboxane A2 by José M. Vila; Pascual Medina; Gloria Segarra; Martı́n Aldasoro; Inmaculada Noguera; Salvador Lluch (247-254).
To examine whether low concentrations of endothelin-1 potentiate the vasocontrictor response to adrenergic stimulation, we recorded the isometric response of rings of rabbit pulmonary artery to electrical stimulation and noradrenaline. Endothelin-1 (10−10 M) potentiated the contractions induced by electrical stimulation and noradrenaline. The endothelin ETB receptor antagonist (2,6-dimethylpiperidinecarbonyl-γ-methyl-Leu-N in-[Methoxycarbonyl]-d-Trp-d-Nle) (BQ-788, 10−6 M), but not the endothelin ETA receptor antagonist cyclo(d-Asp-Pro-d-Val-Leu-d-TRP) (BQ-123, 10−6 M), inhibited the potentiating effects of endothelin-1. Pretreatment with the cyclooxygenase inhibitor indomethacin, the thromboxane synthase inhibitor furegrelate and the thromboxane receptor antagonist [1S-[1α,2α(Z),3α,4α]]-7-[3-[[[[(1-oxoheptyl)amino]acetyl]amino] methyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid (SQ-30741) (all at 10−5 M) prevented the potentiation induced by endothelin-1 on adrenergic stimulation. The Ca2+ channel antagonist nifedipine (10−6 M) did not affect the potentiation induced by endothelin-1. The results indicate that endothelin-1 potentiates the responses to electrical stimulation and noradrenaline by activating endothelin ETB receptors. This potentiation depends on the production of cyclooxygenase-generated factors, probably thromboxane A2.
Keywords: Endothelin-1; Electrical stimulation; Noradrenaline; Thromboxane A2; Synergism;

Experiments were undertaken, using laser-Doppler flowmetry, to determine the nature of adrenoceptors mediating sympathetic nerve evoked nasal vasoconstrictor responses in anesthetized rats. Presence of sympathetic tone was confirmed by a large (330%) increase of nasal blood flow following section of the ipsilateral preganglionic cervical sympathetic nerve. Electrical nerve stimulation produced reproducible, frequency-related nasal vasoconstrictor responses with near maximal response, observed at less than 10 Hz. Evoked nasal vasoconstrictor responses were largely blocked with intravenous treatment with the non-selective α-adrenoceptor antagonists, phentolamine (5 mg kg−1) and phenoxybenzamine (2 mg kg−1), as well as with the selective α1-adrenoceptor antagonist, prazosin (300 μg kg−1). α2-Adrenoceptor antagonism with rauwolscine (500 μg kg−1) potentiated neurally evoked nasal vasoconstriction. Neither atropine (1 mg kg−1) nor propranolol (1 mg kg−1) altered the evoked responses. Rats with intact cervical sympathetic nerves responded to rauwolscine with a modest constriction. Subsequent prazosin administration produced an increase of nasal blood flow of approximately 275%. These results suggest that the nasal vasculature of the rat is under intense sympathetic tone and that the resulting neurogenic vasoconstriction is mediated exclusively by activation of α1-adrenoceptors.
Keywords: Laser-Doppler flowmetry; α-Adrenoceptor subtype; Sympathetic nerve stimulation; Phentolamine; Phenoxybenzamine; Prazosin; Rauwolscine;

Postjunctional α2C-adrenoceptor contractility in human saphenous vein by Charles A Rizzo; Lori M Ruck; Michel R Corboz; Shelby P Umland; Yuntao Wan; Himanshu Shah; James Jakway; Lihong Cheng; Kevin McCormick; Robert W Egan; John A Hey (263-269).
The postjunctional α2-adrenoceptor-mediated contractility was characterized in human saphenous vein derived from coronary artery bypass graft surgery. Human saphenous vein contracted to α2-adrenoceptor selective agonists BHT-920 (5,6,7,8-Tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine dihydrochloride; pD 2=6.7±0.1) and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; pD 2=7.2±0.1). BHT-920-induced contractions were inhibited by the α2-adrenoceptor antagonist yohimbine (17-Hydroxy-yohimban-16-carboxylic acid methyl ester hydrochloride; pA 2=8.7±0.5), but not by the α1-adrenoceptor antagonist prazosin (1-[4-Amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine hydrochloride; 300 nM). In contrast, prazosin (pK b=7.9±0.2) potently antagonized contractions elicited by the α1-adrenoceptor agonist phenylephrine ((R)-3-Hydroxy-α-[(methylamino)methyl] benzenemethanol hydrochloride; pD 2=4.9±0.1), indicating that both α2- and α1-adrenoceptor evoke human saphenous vein contractions. Functional antagonist activity estimates (pA 2 or pK b) obtained for the α-adrenoceptor antagonists ARC 239 (2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride), WB 4101 (2-(2,6-Dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and HV 723 (α-ethyl-3,4,5-trimethoxy-α-(3-((2-(2-methoxyphenoxy) ethyl)amino)propyl)benzeneacetonitrile) against BHT-920-induced human saphenous vein contractions were 7.0±0.6, 8.3±0.6 and 7.7±0.3, respectively. The α2-adrenoceptor subtype affinities (pK i) obtained in recombinant human α2A-, α2B- and α2C-adrenoceptor competition binding assays were 8.6, 8.3 and 8.6 for yohimbine; 6.3, 8.4 and 7.0 for ARC 239; 8.4, 7.5 and 8.4 for WB 4101 and 7.5, 7.4 and 7.9 for HV 723, respectively. Taken together, the binding and functional antagonist activity estimates obtained in these investigations indicate that α2C-adrenoceptor is the predominant postjunctional α2-adrenoceptor subtype in human saphenous vein.
Keywords: Human saphenous vein; α2C-adrenoceptor;

Thromboxane A2 causes retarded clearance of aggregated protein in glomeruli of nephritic mice by Toshiyuki Nagao; Jun-ichi Koseki; Yoshio Suzuki; Tadashi Nagamatsu (271-279).
Recently, it has been demonstrated that the production of prostaglandins and thromboxane is increased in patients with chronic glomerulonephritis and lupus nephritis. We recently demonstrated that thromboxane A2 delayed the clearance of heat-aggregated bovine serum albumin deposited in glomeruli. In the present study, we investigated the effect of thromboxane A2 on the clearance of macromolecules in nephritic glomeruli. First, we attempted to clarify the conditions for the clearance of heat-aggregated bovine serum albumin in nephritic glomeruli, using glomeruli isolated from control and anti-glomerular basement membrane nephritic mice. Heat-aggregated bovine serum albumin was injected twice into each mouse. The glomeruli were then isolated and incubated in culture medium. The heat-aggregated bovine serum albumin content of control glomeruli gradually diminished with incubation time up to 24 h. The heat-aggregated bovine serum albumin content of nephritic glomeruli was 69% higher than that of control glomeruli at 24 h incubation. The production of thromboxane B2 (the stable metabolite of thromboxane A2) in nephritic glomeruli showed about a sevenfold increase compared with control. DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetrahydro-naphthalene-2-carboxylic acid hydrochloride], a thromboxane A2 synthase inhibitor, and KT2-962 [sodium 3-(4-(4-chlorophenyl-butylsulfonamido) butyl)-6-isopropylazulene-1-sulfonate], a selective thromboxane A2 receptor antagonist, significantly reduced the heat-aggregated bovine serum albumin content in nephritic glomeruli. Normal glomeruli treated with U-46619 [15S-hydroxy-11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid], a stable analogue of thromboxane A2, had significantly more heat-aggregated bovine serum albumin than control glomeruli. We next investigated whether thromboxane A2 could affect the uptake/disposal of heat-aggregated bovine serum albumin by cultured rat mesangial cells. U-46619 significantly enhanced the uptake and inhibited the disposal of heat-aggregated bovine serum albumin by mesangial cells. Finally, we performed experiments to elucidate the role of the thromboxane A2 receptor (TP receptor) in the clearance of heat-aggregated bovine serum albumin using TP-deficient mice. The glomerular heat-aggregated bovine serum albumin content of TP-receptor knockout [TP(−/−)] mice was lower than that of wild-type [WT(+/+)] mice. U-46619 dose dependently increased the uptake of heat-aggregated bovine serum albumin by mesangial cells in WT(+/+) mice, but not in the TP(−/−) mice. These findings suggest that thromboxane A2 retards the clearance of aggregated protein in nephritic glomeruli and may contribute to the pathophysiology of glomerulonephritis.
Keywords: Thromboxane A2; Clearance; Aggregated protein; Glomeruli; Nephritic mouse;

The involvement of phospholipase A2 in ethanol-induced gastric muscle contraction by Sang-Soo Sim; Jae-Chun Choi; Do Sik Min; Duck-Joo Rhie; Shin Hee Yoon; Sang June Hahn; Chang-Jong Kim; Myung-Suk Kim; Yang-Hyeok Jo (281-285).
To understand the underlying mechanism of ethanol in tonic contraction, the effect of ethanol on phospholipase A2 and phospholipase C activities and the effects of phospholipase inhibitors on ethanol-induced contraction of cat gastric smooth muscle were tested. Circular muscle strips (2.0×0.2 cm) obtained from the fundus of cat stomach were used to measure isometric contraction. Ethanol elicited tonic contraction and activated phospholipase A2 activity in a dose-dependent manner. Phospholipase A2 inhibitors, manoalide (0.1–10 μM) and oleyloxyethyl phosphorylcholine (1–10 μM), significantly inhibited ethanol-induced contraction. Furthermore, 342 mM ethanol-induced contraction was significantly inhibited by cyclooxygenase inhibitors, ibuprofen (10–100 μM) and indomethacin (10–100 μM), but not by lipoxygenase inhibitors. On the other hand, phospholipase C inhibitors had no effect on ethanol-induced contraction, indicating that phospholipase C is not involved in ethanol-induced contraction. It is suggested from the above results that ethanol-induced contraction in cat gastric smooth muscle is, in part, mediated by phospholipase A2 and cyclooxygenase pathways.
Keywords: Cyclooxygenase; Ethanol; Lipoxygenase; Phospholipase A2; Phospholipase C; Smooth muscle;

This study examined the influence of phenobarbital, an inducer of hepatic enzymes, on Na+ handling, hemodynamics and liver function (measured by the rate constant of elimination of aminopyrine in the aminopyrine breath test) after partial portal vein ligation. Rats were randomized to drink either phenobarbital+water or water only for 10 days and then underwent either sham operation or partial portal vein ligation. The aminopyrine rate constant of elimination and Na+ balance were measured daily before and after surgery; after surgery, hemodynamic measurements were obtained daily in a subset of rats. Phenobarbital raised the baseline aminopyrine rate constant of elimination. Partial portal vein ligation, but not sham operation, caused equivalent reductions in the aminopyrine rate constant of elimination in phenobarbital- and water-treated groups, such that the aminopyrine rate constant of elimination remained higher in the former. Na+ balance increased significantly in partial portal vein ligation+water, but not sham+water rats on day 1 and then decreased on days 2 and 3. In contrast, neither sham+phenobarbital nor partial portal vein ligation+phenobarbital rats had a significant increase in Na+ balance. Partial portal vein ligation resulted in vasodilation on day 3 after surgery in the water-treated rats, an effect that was prevented by treatment with phenobarbital. These results support previous suggestions that a reduction in liver function triggers renal Na+ retention in this model. Vasodilation is not necessary for the latter effect, but also appears to be dependent on a reduction in liver function.
Keywords: Portal hypertension; Na+ retention; Liver function; Aminopyrine; Phenobarbital; Hemodynamics; Vasodilation;

Author index (295-297).

Keyword index (299-304).