European Journal of Pharmacology (v.388, #3)

Inhibition of cytosolic phospholipase A2 attenuates activation of mitogen-activated protein kinases in human monocytic cells by Elke Burgermeister; Ulrich Pessara; Ulrich Tibes; Andrea Küster; Peter C Heinrich; Werner V Scheuer (195-208).
Eicosanoids and platelet-activating factor generated upon activation of cytosolic phospholipase A2 enhance activity of transcription factors and synthesis of proinflammatory cytokines. Here, we show that selective inhibitors and antisense oligonucleotides against this enzyme suppressed expression of the interleukin-1β gene at the level of transcription and promoter activation in human monocytic cell lines. This inhibitory effect was due to failure of activation of mitogen-activated protein kinases (MAPK) through phosphorylation by upstream mitogen-activated protein kinase kinases (MKK). Consequently, phosphorylation and degradation of inhibitor-κBα (I-κBα) and subsequent cytoplasmic mobilization, DNA-binding and the transactivating potential of nuclear factor-κB (NF-kB), nuclear factor-interleukin-6 (NF-IL6), activation protein-1 (AP-1) and signal-transducer-and-activator-of-transcription-1 (STAT-1) were impaired. It is concluded, that lipid mediators promote activation of MAPKs, which in turn lead to phosphorylation and liberation of active transcription factors. Since inhibition of cytosolic phospholipase A2 ameliorates inflammation in vivo, this potency may reside in interference with the MAPK pathway.
Keywords: Phospholipase A2; Nuclear-factor-κB; Mitogen-activated protein kinase; Interleukin-1; Inflammation;

The potential of Ca2+ channel antagonists, particularly nifedipine, to cause apoptotic cell death has been controversial and is of considerable importance for cardiomyocytes as loss of these cells is an important component of the pathophysiology leading to heart failure. To examine the hypothesis that nifedipine induces cell death and modulates calcium-induced apoptosis, cardiomyocytes in culture from embryonic chick hearts, that readily manifest apoptosis, were studied. Apoptosis was evaluated by fluorescent activated cell sorting (FACS) analysis and by quantitative analysis of DNA fragmentation by an enzyme-linked immunosorbent assay (ELISA) specific for histone-associated DNA fragments of mono- and oligonucleosome size. Cell death was evaluated by using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Cardiomyocytes were treated with various concentrations of nifedipine over a concentration range relevant to serum concentrations in man. Nifedipine, 1 to 100 μM, did not produce cell death in cardiomyocytes. There was no evidence of apoptosis on FACS analysis of cardiomyocytes stained with fluoresceine diacetate or propidum iodide (PI). Neither was there any evidence of apoptotic nuclei on PI staining of permeabilized cardiomyocytes treated with nifedipine. In contrast, DNA fragmentation consistent with apoptosis was induced in a significant (P<0.05) concentration-dependent manner, by increases in extracellular Ca2+ concentration ([Ca2+]o). Importantly, nifedipine reduced DNA fragmentation produced by increased [Ca2+]o. Furthermore, nifedipine blocked calcium-induced cell death as high [Ca2+]o significantly (P<0.05) reduced cell viability. These data indicate that nifedipine does not induce apoptosis in cardiomyocytes rather apoptosis in cardiomyocytes is under regulatory control by Ca2+ and nifedipine can antagonize Ca2+-mediated apoptotic cell death.
Keywords: Apoptosis; Ca2+; Nifedipine; Cardiomyocyte;

The effects of magnesium ammonium phospholinoleate-palmitoleate anhydride (MAPA), a proteic aggregated polymer isolated from Aspergillus oryzae, on the growth and differentiation of granulocyte-macrophage progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM]) in normal and Ehrlich ascites tumour-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with MAPA (0.5−10 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation. No changes were observed in total and differential marrow cell counts. The dose of 5.0 mg/kg MAPA, given prior or after tumour inoculation, was the optimal biologically active dose in tumour-bearing mice and this dose schedule also stimulated myelopoiesis in normal mice. MAPA significantly enhanced survival and concurrently reduced tumour growth in the peritoneal cavity. We propose that the modulatory effect of MAPA on the myelopoietic response may be related to its antitumour activity as a possible mechanism for regulation of granulocyte-macrophage production and expression of functional activities.
Keywords: Ehrlich ascites tumour; Antitumour; Myelopoiesis; MAPA (magnesium ammonium phospholinoleate-palmitoleate anhydride); CFU-GM (colony-forming unit-granulocyte-macrophage);

cAMP-dependent potentiation of the Ca2+-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells by Makoto Sugita; Chikara Hirono; Kishio Furuya; Shunichi Yamagishi; Yoshinobu Kanno; Yoshiki Shiba (227-234).
A recent study indicates that elevation of [Ca2+]i enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca2+]i. Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca2+]i, isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca2+]i evoked by a high concentration of carbachol (10−6 M). Charybdotoxin, a K+ channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca2+-activated release and the cAMP-potentiated release may be coupled to Ca2+-activated K+ efflux, increases in both [Ca2+]i and [cAMP]i may activate the calcein conduction pathway which is not activated by an increase in [Ca2+]i alone.
Keywords: Calcium ion; cAMP; Fluid secretion; Calcein; Multidrug resistance protein;

Binding properties of [3H]gacyclidine in the rat central nervous system by Hélène Hirbec; Alain Privat; Jacques Vignon (235-239).
Gacyclidine (1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine), the racemate of (+)-and (−)-GK11, exhibits potent neuroprotective properties due to its antagonism at the NMDA receptor. In its tritiated form, gacyclidine showed a binding distribution similar to that of NMDA receptors in the rat brain. With membrane preparations, the (−)-enantiomer of gacyclidine exhibited an affinity similar to that of MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) in the low nanomolar range, while the (+)-enantiomer was about 10 times less potent. Gacyclidine affinity was lower in the cerebellum than in the forebrain or the spinal cord. In this latter region and in the cerebellum, two binding sites were evidenced, one of which was a low-affinity site insensitive to MK-801. In all regions, PRE-084 (2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate), a σ receptor ligand, had no effect on [3H]gacyclidine binding.
Keywords: NMDA receptor; Phencyclidine receptor; Gacyclidine; Gacyclidine enantiomer; Neuroprotection;

δ-Opioid receptor agonists produce antinociception and [35S]GTPγS binding in μ receptor knockout mice by Yoshiaki Hosohata; Todd W Vanderah; Thomas H Burkey; Michael H Ossipov; Carl J Kovelowski; Ichiro Sora; George R Uhl; Xiaoyan Zhang; Kenner C Rice; William R Roeske; Victor J Hruby; Henry I Yamamura; Josephine Lai; Frank Porreca (241-248).
We examined the effects of [d-Pen2,d-Pen5]enkephalin (DPDPE), [d-Ala2,Glu4]deltorphin (DELT), and (+)-4-[(αR)-α((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) on [35S]GTPγS binding in brain membranes prepared from μ-opioid receptor knockout (−/−) mice. The potency and maximal response (E max) of these agonists were unchanged compared to control mice. In contrast, while the potency of [d-Pen2,pCl-Phe4,d-Pen5]enkephalin (pCl-DPDPE) was not significantly different, the E max was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal ( DELT produced antinociceptive effects in −/− mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and DPDPE was displaced to the right by 4- and 9-fold in −/− compared to control mice, respectively. Reduced DPDPE antinociceptive potency in −/− mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from −/− mice, demonstrate that these agonists require μ-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between −/− and wild type mice, we conclude that the μ-opioid receptor is not a critical component of δ-opioid receptor function.
Keywords: δ-Opioid receptor; μ-Opioid receptor; μ-Opioid receptor-knockout mice; Antinociception; G protein; Opioid drug;

Roles of adenosine A1 and A2A receptors in the expression and development of methamphetamine-induced sensitization by Takao Shimazoe; Akiko Yoshimatsu; Atsuko Kawashimo; Shigenori Watanabe (249-254).
We studied the effects of adenosine A1 and A2A receptor agonists on the expression and development of methamphetamine-induced sensitization in rats. When animals were treated with the adenosine A1 receptor agonist, N 6-cyclohexyladenosine (CHA), along with methamphetamine every 3 days with a total of five administrations, the augmentation of hyperlocomotion by methamphetamine re-administration after 7-day withdrawal (methamphetamine challenge administration) was not inhibited. However, when the adenosine A2A receptor agonist, 2-p-(2-carboxyethyl) phenethyl-amino-5′-N-ethylcarboxy-amide adenosine (CGS21680), was administered according to the same schedule, the augmentation was significantly inhibited. On the other hand, when CHA or CGS21680 was administered 30 min before methamphetamine challenge, both drugs dose-dependently inhibited the augmentation of hyperlocomotion. These results suggested that both adenosine A1 and A2A receptors play important roles in the expression of methamphetamine-induced sensitization, and that adenosine A2A receptors do so in the development of this sensitization.
Keywords: Methamphetamine; Sensitization; Adenosine; Adenosine A1 receptor; Adenosine A2A receptor;

Effect of somatostatin on resistance and on capacitance rabbit isolated arteries by Emilio Ruiz; Marcela Del Rio; Teresa Tejerina (255-261).
The effects of somatostatin, a tetradecapeptide isolated from hypothalamus extracts, were studied on the vascular reactivity of aorta and mesenteric arteries isolated from rabbits. We also investigated whether or not Ca2+ movements were implicated in these effects. Rabbit aorta and mesenteric (fifth branch) arteries were isolated, cleaned off, and mounted in an organ bath containing Godfraind solution or physiological saline solution (PSS), respectively. Somatostatin (10−8–10−4 M) produced a concentration-dependent inhibition of the contractile responses induced by high K+ (80 mM) or noradrenaline (10−6 M in aorta or 10−4 M in mesenteric arteries) in both arteries studied. The inhibitory effect of somatostatin was greater in mesenteric resistance vessels (IC50 3.1±2.3×10−5 M, and 5.2±4.8×10−8 M with KCl and noradrenaline, respectively). Contractile responses produced by the addition of Ca2+ (1–5 mM) to Ca2+-free high K+ solution were also concentration dependently inhibited by somatostatin in aorta. Furthermore, somatostatin decreased noradrenaline-induced contraction attributed to intracellular Ca2+ release in aorta, and inhibited 45Ca2+ uptake stimulated by high K+ or by noradrenaline. However, it did not modify 45Ca2+ uptake in resting mesenteric resistance arteries. Taken together, these results suggest that somatostatin exerts an inhibitory effect on vascular contractions induced by some stimulating agents in different arteries isolated from rabbits, being more potent in mesenteric arteries.
Keywords: Somatostatin; Aorta; Mesenteric artery; Ca2+;

Effects of metoprolol and ramipril on action potentials after myocardial infarction in rats by Kay-Dietrich Wagner; Andre Kamkin; Irina Kiseleva; Heinz Theres; Holger Scholz; Joachim Günther (263-266).
The effects of chronic treatment with the β-adrenoceptor antagonist metoprolol, the angiotensin converting enzyme inhibitor ramipril, their combination, or placebo on action potential configuration 6 weeks after myocardial infarction in rats were studied. Action potentials were measured in isolated left ventricular posterior papillary muscles and compared with action potentials from a sham operated group without infarction. After infarction, the action potential amplitude was reduced and this phenomenon was partially reversed by metoprolol- and ramipril-treatment. Prolonged repolarisation after infarction compared to sham operated animals was additionally delayed after metoprolol treatment. Thus, metoprolol extends the refractory period, which may counteract tachyarrhythmia.
Keywords: Metoprolol; Ramipril; Myocardial infarction; Action potential; Papillary muscle;

Isolated epithelial layer preparations were obtained from urinary bladders of 4-week streptozotocin-diabetic rats and used for endogenous prostaglandins E2 and F determination. Tissues were incubated in modified Krebs solution under basal conditions, or in the presence of either indomethacin (5×10−7 M), ATP (10−5 and 10−3 M) or bradykinin (10−7 and 10−5 M), and samples of incubation medium were collected at 15 and 30 min. In the presence of indomethacin, the release of prostaglandins in the incubation medium was under the detection limit of the enzyme immunoassay (EIA). The epithelium from diabetic rat urinary bladders was thicker and heavier and the absolute amount of endogenous prostaglandins E2 and F was higher than for control animals, but when prostaglandin production was expressed as a fraction of tissue weight, it was reduced in diabetic epithelium. ATP and bradykinin has significantly increased the endogenous release of both prostaglandins from the epithelium when compared with the release under basal conditions. This increase was time-dependent and was higher in diabetic than in control tissues. ATP evoked a phasic and tonic contraction in bladder strips that was abolished by epithelium removal. Concentration–response curves for ATP did not differ among groups. Bradykinin evoked a long-lasting tonic contraction that was reduced significantly by epithelium removal in diabetic rat bladders only. Concentration–response curves for prostaglandin E2 and F in diabetic rat bladder differed significantly from that in controls and epithelium removal did not alter these responses. It is suggested that bradykinin receptors and P2X nucleotide receptors already found in the smooth muscle detrusor might be present in the epithelial layer of the bladder. The prostaglandin-release impairment observed in this study might be responsible, in part, for bladder abnormalities observed in pathological conditions, such as diabetes.
Keywords: Urinary bladder; Epithelium; Prostaglandin; Diabetes;

To evaluate the influence of atherosclerosis on the global production of NO, we studied the effect of a 0.3% cholesterol-enriched diet on urinary nitrate excretion in rabbits during 69 weeks. To examine whether the inducible nitric oxide synthase (iNOS) present in atherosclerotic lesions could participate in NO excretion, hypercholesterolemic rabbits were treated chronically with the selective iNOS inhibitor, N-iminoethyl-l-lysine (l-NIL; 5 mg/kg/day). Urinary nitrate excretion was higher in hypercholesterolemic than in control rabbits throughout the study period and decreased progressively with time in both groups; l-NIL had no significant effect on urinary nitrate excretion. These data illustrate that systemic NO production is enhanced in hypercholesterolemia and that iNOS, present within the plaque, might not participate in this enhanced NO production.
Keywords: (Rabbit); Nitrate; Atherosclerosis; Nitric oxide (NO) synthase; Nitric oxide (NO); l-NIL (N-iminoethyl-l-lysine);

The involvement of nitric oxide (NO) formed by the inducible isoform of NO synthase (iNOS) has been investigated in the development of rat intestinal lesions following indomethacin administration. Over a 72-h period, indomethacin (10 mg kg−1, s.c.) provoked a time-dependent increase in expression of iNOS (assessed by the conversion of radiolabelled l-arginine to citrulline) and enhancement of vascular leakage of radiolabelled human serum albumin in the jejunum which commenced 18 h after indomethacin. Similar effects were not observed in the ileum, colon or caecum. In addition, macroscopic lesions were detectable and myeloperoxidase activity (an index of neutrophil recruitment) were increased in the rat jejunum 18–24 h after indomethacin, but remained at basal levels in the ileum and colon. These findings suggest that indomethacin provokes a site-selective expression of iNOS in the rat jejunum which correlates with lesion formation and vascular leakage, whereas both the ileum and colon are spared.
Keywords: Nitric oxide (NO); Inflammation; Non-steroid anti-inflammatory drug;

Index (287-289).

Index (291-296).