European Journal of Pharmacology (v.427, #3)

Immunosuppressant rapamycin inhibits protein kinase C α and p38 mitogen-activated protein kinase leading to the inhibition of chondrogenesis by Chun-Do Oh; Song-Ja Kim; Jung-Won Ju; Woo Keun Song; Jae-Hong Kim; Yung Joon Yoo; Jang-Soo Chun (175-185).
Immunosuppressants are now known to modulate bone metabolism, including bone formation and resorption. Because cartilage, formed by differentiated chondrocytes, serves as a template for endochondral bone formation, we examined the effects of the immunosuppressant rapamycin on the chondrogenesis of mesenchymal cells and on the cell signaling that is required for chondrogenesis, such as protein kinase C, extracellular signal-regulated kinase-1 (ERK-1), and p38 mitogen-activated protein (MAP) kinase pathways. Rapamycin inhibited the expression of type II collagen and the accumulation of sulfate glycosaminoglycan, indicating inhibition of the chondrogenesis of mesenchymal cells. Rapamycin treatment did not affect precartilage condensation, but it prevented cartilage nodule formation. Exposure of chondrifying mesenchymal cells to rapamycin blocked activation of the protein kinase C α and p38 MAP kinase, but had no discernible effect on ERK-1 signaling. Selective inhibition of PKCα or p38 MAP kinase activity, which is dramatically increased during chondrogenesis, with specific inhibitors in the absence of rapamycin blocked the chondrogenic differentiation of mesenchymal cells. Taken together, our data indicate that the immunosuppressant rapamycin inhibits the chondrogenesis of mesenchymal cells at the post-precartilage condensation stage by modulating signaling pathways including those of PKCα and p38 MAP kinase.
Keywords: Chondrogenesis; Mesenchymal cells; Micromass culture; Protein kinase C α; p38 MAP (mitogen-activated protein) kinase; Rapamycin;

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors by Michael S Lidow; Amy Roberts; Ling Zhang; Phil-Ok Koh; Nelson Lezcano; Clare Bergson (187-193).
The recently cloned protein, calcyon, potentiates crosstalk between Gs-coupled dopamine D1 receptors and heterologous Gq/11-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca2+ release, in addition to cAMP production. This crosstalk also requires the participating Gq/11-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous Gq/11-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.
Keywords: G protein; Agonist; Antagonist; Affinity; Receptor crosstalk; Receptor binding assay;

Inhibition by docosahexaenoic acid of receptor-mediated Ca2+ influx in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine by Masahiko Hirafuji; Takashi Ebihara; Fumito Kawahara; Noaya Hamaue; Toru Endo; Masaru Minami (195-201).
The effect of docosahexaenoic acid treatment on intracellular Ca2+ dynamics in rat vascular smooth muscle cells stimulated with 5-hydroxytryptamine (5-HT) has been investigated in order to elucidate one of the mechanisms for its beneficial effect on cardiovascular disorders. The treatment of cells with 30 μM docosahexaenoic acid for 2 days inhibited an increase in intracellular Ca2+ concentration induced by 5-HT (10 μM) and a depolarizing concentration of KCl (80 mM). Docosahexaenoic acid treatment significantly inhibited divalent cation influx stimulated by 5-HT and KCl, as measured by Mn2+ quenching method, whereas had no effect on 5-HT-induced Ca2+ release from the internal stores. Docosahexaenoic acid treatment also significantly inhibited 5-HT receptor-mediated Ca2+ influx through Ni2+-insensitive channels that were distinct from store-operated channels. These results suggest that the specific inhibition of intracellular Ca2+ dynamics in vascular smooth muscle cells may contribute to the beneficial properties of docosahexaenoic acid on cardiovascular disorders.
Keywords: Docosahexaenoic acid; Smooth muscle cell; Ca2+ concentration; Ca2+ channels; Cardiovascular protection;

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse by Shinobu Sakurada; Takafumi Hayashi; Masayuki Yuhki; Tohru Orito; James E Zadina; Abba J Kastin; Tsutomu Fujimura; Kimie Murayama; Chikai Sakurada; Tsukasa Sakurada; Minoru Narita; Tsutomu Suzuki; Koichi Tan-no; Leon F Tseng (203-210).
Two highly selective μ-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for μ-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the μ1-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with κ-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the δ2-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the δ1-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met5]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of μ1-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met5]enkephalin which subsequently act on κ-opioid receptors and δ2-opioid receptors to produce antinociception.
Keywords: Endomorphin; Antinociceptive effect; μ-Opioid receptor agonist; (Mouse);

A novel selective σ1 receptor ligand, (R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone l-tartrate (MS-377), inhibits phencyclidine (1-(1-phenylcyclohexyl)piperidine; PCP)-induced behaviors in animal models. In this study, we measured extracellular dopamine and serotonin levels in the rat brain after treatment with MS-377 alone, using in vivo microdialysis. We also examined the effects of MS-377 on extracellular dopamine and serotonin levels in the rat medial prefrontal cortex after treatment with PCP. MS-377 itself had no significant effects on dopamine release in the striatum (10 mg/kg, p.o.) nor on dopamine or serotonin release in the medial prefrontal cortex (1 and 10 mg/kg, p.o.). PCP (3 mg/kg, i.p.) markedly increased dopamine and serotonin release in the medial prefrontal cortex. MS-377 (1 mg/kg, p.o.), when administered 60 min prior to PCP, significantly attenuated this effect of PCP. These results suggest that the inhibitory effects of MS-377 on PCP-induced behaviors are partly mediated by inhibition of the increase in dopamine and serotonin release in the rat medial prefrontal cortex caused by PCP.
Keywords: σ1 Receptor ligand; MS-377; Phencyclidine; Microdialysis; Prefrontal cortex;

The effects of 5-HT2 receptor antagonists on antidepressant effects of imipramine were investigated in the forced swimming test. Imipramine induced anti-immobility effects in mice dose dependently. Pretreatment with the 5-HT2A/2B/2C receptor antagonist, 4-isopropyl-7-methyl-9-(2-hydroxy-1-methyl-propoxycarbonyl)-4,6A,7,8,9,10,10A-octahydro-indolo[4,3-FG]quinolone maleate (LY 53857) significantly enhanced the anti-immobility effects of imipramine. The 5-HT2C/2B receptor antagonist, N-3-pyridinyl-3,5-dihydro-5-methyl-benzo[1,2-b:4,5-b′]dipyrrole-1(2H)-carboxamide (SB 206553), also enhanced, while the 5-HT2A receptor antagonist, ketanserin, was without effect. These results suggest that blockade of the 5-HT2C/2B receptor leads to potentiation of the antidepressant effects of imipramine.
Keywords: Imipramine; 5-HT (5-hydroxytryptamine, serotonin); 5-HT2C/2B receptor; 5-HT2A receptor; Forced swimming test; Antidepressant; (Mouse);

Alcohol dependence has been correlated with decreases in heart rate variability. However, the time course of recovery of heart rate variability after cessation of alcohol consumption is unknown. We used electrocardiogram (ECG) data serially obtained from a population of detoxifying alcoholic subjects to determine the Hurst exponent of the ECG interbeat interval time series. Higher values of the Hurst exponent are associated with decreased heart rate variability when H≤0.5. We tested a series of response-surface models relating the Hurst exponent (H) thus obtained to the following independent variables: the time interval T (days since last use of alcohol), A (age in years at time of admission), and gender. The best-fit model was: H(T)=(KA+H m T+H f T)/(1+T), F=5.2, P(F)≤0.01. Model parameters were: K=0.008±0.002 (mean±SEM); asymptotic H-values for males and females: H m=0.24±0.02 and H f=0.16±0.03, respectively, significantly different at P≤0.05. Age was the strongest predictor of initial H-values in this alcoholic population sample.
Keywords: Heart rate; Electrocardiography; Alcohol-related disorders; Fractal; Toxicology;

The effects of endothelin-1 on ischaemia-induced ventricular arrhythmias in rat isolated hearts by Isam Sharif; Thomas R Crockett; Kathleen A Kane; Cherry L Wainwright (235-242).
We have shown previously that a small bolus dose of endothelin-1, given intravenously before coronary occlusion, exerts a marked antiarrhythmic effect in anaesthetised rats. The aim of the current study was to determine whether or not this is due to a direct effect of endothelin-1 on the heart by assessing the antiarrhythmic effect of endothelin-1 against occlusion-induced arrhythmias in rat isolated hearts. Rat isolated hearts were perfused in Langendorff mode (constant flow) and subjected to coronary artery occlusion for 30 min. Coronary perfusion pressure and a surface electrocardiogram (ECG) were monitored throughout the experiment. In the first series of studies, the effects of three 5-min infusions of endothelin-1 (0.1–10 nM), given prior to coronary occlusion, were assessed. A second series of hearts was given a single bolus dose of endothelin-1 (10 pmol) 5 min prior to ischaemia. A third series of experiments was performed using a modified (low K+) Krebs Henseleit solution to increase the incidence of ischaemia-induced ventricular fibrillation (VF). In these hearts, endothelin-1 (0.1 or 2 pmol) was administered as a bolus injection 5 min before ischaemia. Infusion of endothelin-1 prior to ischaemia did not modify the incidence or severity of arrhythmias at any of the concentrations used. Bolus administration of endothelin-1 (10 pmol) in hearts perfused with Kreb's Henseleit solution containing normal K+ (4.4 mM) was found to cause a small rise in coronary perfusion pressure, with no preceding depressor response. Under these conditions, endothelin-1 exerted only a very moderate reduction in arrhythmias, by reducing the arrhythmia count in the 21–30-min post-occlusion period. Furthermore, in hearts perfused with low K+ solution, bolus injection of endothelin-1, in a dose that either had no effect on coronary perfusion pressure (0.1 pmol) or produced a significant vasodilator effect with no significant pressor effect (2 pmol), had no effect on ventricular fibrillation. Thus, in concentrations that are sufficient to exert effects on the coronary vasculature, endothelin-1 fails to modify arrhythmias in an isolated heart preparation. These results suggest that the antiarrhythmic effects of endothelin-1 previously observed in vivo are not due to a direct effect on either the myocardium or the coronary blood vessels.
Keywords: Endothelin-1; Arrhythmias; Heart; (Rat); Isolated;

Diabetes abolishes the gender difference in vasopressin-mediated potentiation of sympathetic vasoconstriction by Elena Sanz; Luis Monge; Nuria Fernández; Marı́a Angeles Martı́nez; Godofredo Diéguez; Angel Luis Garcı́a-Villalón (243-250).
Electrical field stimulation (4 Hz, 0.2 ms pulse duration, at a supramaximal voltage of 70 V, for 1 s) of isolated rat tail artery segments produced contraction which was lower in female than in male rats, and was reduced by streptozotocin-induced diabetes in both genders. This contraction was potentiated by vasopressin (10−12–10−10 M) more in normoglycemic male than in normoglycemic female rats, and this effect of vasopressin was increased by the cyclooxigenase inhibitor meclofenamate (10−5 M) in female control rats, but not in diabetic female, or control and diabetic male rats, and it was not modified by the nitric oxide synthase inhibitor N ω-nitro-l-arginine methyl ester (l-NAME, 10−4 M). Endothelin-1 (10−10–3×10−9 M) also potentiated the contraction to electrical stimulation. This potentiation was similar in all experimental groups, and it was not modified by meclofenamate or l-NAME. These results suggest that the potentiating effect of vasopressin, but not that of endothelin-1, on the sympathetic vasoconstriction, is lower in females than in males, probably by an increased release of vasodilating prostanoids, and this release may be reduced by diabetes in females.
Keywords: Caudal artery; Electrical stimulation; Male; Female; Nitric oxide; Prostanoid;

Long-term inhibition of nitric oxide synthase potentiates effects of anandamide in the rat mesenteric bed by Victoria E Mendizábal; Marı́a Luz Orliac; Edda Adler-Graschinsky (251-262).
In rat isolated mesenteric beds, anandamide induced a concentration-dependent reduction (0.01–50 μM) of the contractile responses elicited by bolus administration of noradrenaline. The anandamide-induced reductions of noradrenaline responses were unmodified by the in vitro exposure to the nitric oxide synthase (NOS) inhibitor, 100 μM l-N G-nitro-l-arginine methyl ester (l-NAME), whereas they were significantly potentiated after the long-term in vivo administration of l-NAME (70 mg/kg/day during 4 weeks). Responses to anandamide were not potentiated and even reduced in mesenteric beds from rats made hypertensive by aortic coarctation. In mesenteric beds isolated from either untreated or in vivo l-NAME treated rats, concentration–response curves to anandamide were significantly attenuated by the non-selective K+ channel blocker tetraethylammonium (TEA) but were not modified by either endothelium removal, or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) or the cannabinoid receptor antagonists 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl] (4-methoxyphenyl) methanone (AM630) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281). On the other hand, the vanilloid receptor agonist (E)-N-[4-hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide (capsaicin) induced a concentration-dependent inhibition of noradrenaline-induced vasoconstriction, and the vanilloid receptor antagonist N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine) caused a significant reduction of anandamide-induced responses in mesenteric beds isolated from both control and chronic l-NAME treated rats. The non-metabolizable analogue of anandamide, methanandamide, produced higher reductions of noradrenaline responses than anandamide in mesenteric beds isolated from controls but not from the l-NAME treated rats. Moreover, in mesenteric beds from untreated but not from l-NAME treated rats, the effects of anandamide were significantly potentiated by the inhibitor of endocannabinoid degradation, 200 μM phenylmethylsulphonyl fluoride (PMSF), and by the inhibitor of anandamide uptake, 5 μM (all Z)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404). It is concluded that long-term inhibition of NOS potentiates anandamide-induced relaxations probably through changes in either endocannabinoid metabolism or uptake. A possible compensatory role for endocannabinoids in vascular function in situations in which nitric oxide (NO) synthesis is long-term impaired arises from the present results.
Keywords: Endocannabinoid; Anandamide; Nitric oxide (NO); Hypertension; l-N G-nitro-l-arginine methyl ester (l-NAME); Mesenteric bed;

Ischemic preconditioning, the most effective gastroprotective intervention: involvement of prostaglandins, nitric oxide, adenosine and sensory nerves by Robert Pajdo; Tomasz Brzozowski; Peter C Konturek; Slawomir Kwiecien; Stanislaw J Konturek; Zbigniew Śliwowski; Michal Pawlik; Agata Ptak; Danuta Drozdowicz; Eckhart G Hahn (263-276).
Various organs, including heart, kidneys, liver or brain, respond to brief exposures to ischemia with an increased resistance to severe ischemia/reperfusion and this phenomenon is called “preconditioning”. No study so far has been undertaken to check whether such short, repeated gastric ischemic episodes protect gastric mucosa against severe damage caused by subsequent prolonged ischemia/reperfusion and, if so, what could be the mechanism of this phenomenon. The ischemic preconditioning was induced by short episodes of gastric ischemia (occlusion of celiac artery from one to five times, for 5 min each) applied 30 min before prolonged (30 min) ischemia followed by 3 h of reperfusion or 30 min before topical application of strong mucosal irritants, such as 100% ethanol, 25% NaCl or 80 mM taurocholate. Exposure to regular 30-min ischemia, followed by 3-h reperfusion, produced numerous severe gastric lesions and significant fall in the gastric blood flow and prostaglandin E2 generation. Short (5-min) ischemic episodes (1–5 times) by itself failed to cause any gastric lesions, but significantly attenuated those produced by ischemia/reperfusion. This protection was accompanied by a reversal of the fall in the gastric blood flow and prostaglandin E2 generation and resembled that induced by classic gastric mild irritants. These protective and hyperemic effects of standard preconditioning were significantly attenuated by pretreatment with cyclooxygenase-2 and cyclooxygenase-1 inhibitors, such as indomethacin, Vioxx, resveratrol and nitric oxide (NO)-synthase inhibitor, N G-nitro-l-arginine (l-NNA). The protective and hyperemic effects of standard preconditioning were restored by addition of 16,16 dm prostaglandin E2 or l-arginine, a substrate for NO synthase, respectively. Gastroprotective and hyperemic actions of standard ischemic preconditioning were abolished by pretreatment with capsaicin-inactivating sensory nerves, but restored by the administration of exogenous CGRP to capsaicin-treated animals. Gene and protein expression of cyclooxygenase-1, but not cyclooxygenase-2, were detected in intact gastric mucosa and in that exposed to ischemia/reperfusion with or without ischemic preconditioning, whereas cyclooxygenase-2 was overexpressed only in preconditioned mucosa. We conclude that: (1) gastric ischemic preconditioning represents one of the most powerful protective interventions against the mucosal damage induced by severe ischemia/reperfusion as well as by topical mucosal irritants in the stomach; (2) gastric ischemic preconditioning resembles the protective effect of “mild irritants” against the damage by necrotizing substances in the stomach acting via “adaptive cytoprotection” and involves several mediators, such as prostaglandin derived from cyclooxygenase-1 and cyclooxygenase-2, NO originating from NO synthase and sensory nerves that appear to play a key mechanism of gastric ischemic preconditioning.
Keywords: Gastric preconditioning; Ischemia/reperfusion; Gastric blood flow; Prostaglandin; Cyclooxygenase; Nitric oxide (NO); Adenosine; CGRP (Calcitonin gene-related peptide); Adaptive cytoprotection;

Tumor necrosis factor-α (TNF-α) stimulates the expression CXC chemokines, i.e. macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), and neutrophil extravasation. However, the individual role of MIP-2 and KC in the recruitment process of neutrophils in vivo remains elusive. By use of intravital microscopy in the mouse cremaster muscle, we analyzed the effect of specific inhibition of CXC chemokines, alone and together, on TNF-α-induced leukocyte rolling, firm adhesion and recruitment. After stimulation with TNF-α, the mRNA levels of both MIP-2 and KC were increased. Notably, separate administration of antibodies directed against MIP-2 and KC had no effect on TNF-α-induced neutrophil extravasation. In contrast, combined injection of anti-MIP-2 and anti-KC antibodies markedly inhibited extravascular migration of neutrophils. Moreover, MIP-2 and KC dose-dependently increased neutrophil recruitment, however, no synergistic effect of combined stimulation with MIP-2 and KC on the neutrophil response was found. Taken together, these data suggest that MIP-2 and KC are functionally redundant in TNF-α-induced neutrophil accumulation and that neutralization of both MIP-2 and KC may be necessary in order to reduce accumulation of neutrophils in cytokine-activated tissues.
Keywords: Chemokine; Inflammation; Intravital microscopy; Leukocyte; Microcirculation;

High fat fed hamster, a unique animal model for treatment of diabetic dyslipidemia with peroxisome proliferator activated receptor alpha selective agonists by Pei-Ran Wang; Qiu Guo; Marc Ippolito; Margaret Wu; Denise Milot; John Ventre; Tom Doebber; Samuel D. Wright; Yu-Sheng Chao (285-293).
Dyslipidemia, a major risk factor for cardiovascular disease, may be directly linked to diabetic hyperglycemia and insulin resistance. An appropriate dyslipidemic animal model that has diabetes would provide an important tool for research on the treatment of diabetic dyslipidemia. Ten days of high fat feeding in golden Syrian hamsters resulted in a significant increase in insulin resistance and baseline serum lipid levels accompanied by a pronounced dyslipidemia. Thirteen days of treatment with fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) selective agonist, produced a dose-dependent decrease in serum lipid levels. The pattern observed was characterized by lowered very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) and raised high-density lipoprotein (HDL) cholesterol in a fashion similar to that seen in man. Diabetic conditions were also significantly improved by fenofibrate with a normalization of impaired glucose tolerance and an improvement of insulin sensitivity during an oral glucose tolerance test. These data suggest that fenofibrate may correct not only the dyslipidemia but also the insulin resistance caused by a high fat diet, and the high fat fed hamster may be a good animal model for research on the treatment of diabetic dyslipidemia with PPARα selective agonists.
Keywords: Fenofibrate; Diabetic dyslipidemia; Insulin sensitivity; Animal model;

Author index (295-297).

Keyword index (299-304).