Cyclooxygenase-2-Derived Prostaglandins Mediate Cerebral Microcirculation in a Juvenile Ischemic Rat Model.

International audienceBACKGROUND AND PURPOSE:We previously showed that the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) increases cerebral microcirculation in a juvenile ischemic rat model. We address the roles of cyclooxygenase (COX)-elaborated prostaglandins in collateral recruitment and blood supply.METHODS:Fourteen-day-old rats were subjected to ischemia-reperfusion and treated with either PBS or 7-NI (25 mg/kg) at the reperfusion onset. Six-keto-prostaglandin F1α was measured using ELISA. COX-1 and COX-2 and prostaglandin terminal synthesizing enzymes were evaluated using reverse-transcriptase polymerase chain reaction and immunofluorescence. Microvascular blood flow indexes (artery diameter and capillaries number) were measured using sidestream dark-field videomicroscopy in PBS- and 7-NI-treated ischemic rats in the absence or presence of the COX-2 inhibitor NS-398 (5 mg/kg). Cell death was measured with the TUNEL (terminal transferase dUTP nick end labeling) assay and cleaved-caspase-3 immunostaining.RESULTS:Six-keto-prostaglandin F1α and COX-2, associated with a prostaglandin E synthase, were significantly increased in PBS- and 7-NI-treated animals 15 minutes and 1 hour after ischemia-reperfusion, respectively. In contrast and as compared with PBS, 7-NI significantly decreased prostacyclin synthase and cytosolic prostaglandins E synthase mRNA. Selective COX-2 inhibition significantly decreased blood flow indexes and significantly reversed the effects of 7-NI, including the number of TUNEL+- and cleaved-caspase-3+-nuclei.CONCLUSIONS:These results show that the juvenile rat brains mostly respond to ischemia by a COX-2-dependent prostaglandins production and suggest that the transcriptional responses observed under 7-NI facilitate and reorient COX-2-dependent prostaglandins production

Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT(1B) receptors in C6-glial cells: similarities with the human 5-HT(1B) receptor

1. The guinea-pig recombinant 5-hydroxytryptamine(1B) (gp 5-HT(1B)) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [(35)S]-GTPγS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT(1B) (h 5-HT(1B)) receptor under similar experimental conditions. 2. Membrane preparations of C6-glial/gp 5-HT(1B) cells exhibited [(3)H]-5-carboxamidotryptamine (5-CT) and [(3)H] - N- [4-methoxy-3,4 - methylpiperazin-1-yl) phenyl] -3 - methyl - 4-(4 - pyridinyl)benzamide (GR 125743) binding sites with a pK(d) of 9.62 to 9.85 and a B(max) between 2.1 to 6.4 fmol mg(−1) protein. The binding affinities of a series of 5-HT receptor ligands determined with [(3)H]-5-CT and [(3)H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r(2): 0.74, P<0.001) with those determined at the recombinant h 5-HT(1B) receptor. 3. [(35)S]-GTPγS binding to membrane preparations of C6-glial/gp 5-HT(1B) cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethylsulphonamide (CP122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r(2): 0.72, P=0.015) with their potency at the recombinant h 5-HT(1B) receptor. 1-naphthylpiperazine, (±)-cyanopindolol and (2′-methyl-4′-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (E(max): 0.32 to 0.63). 4. The ligands 1′-methyl-5-(2′-methyl-4′-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro [furo[2,3-f]indole-3-spiro-4′-piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [(35)S]-GTPγS binding at concentrations relevant to their binding affinity for the gp 5-HT(1B) receptor. Methiothepin and SB224289 behaved as competitive antagonists at gp 5-HT(1B) receptors; pA(2) values were 9.74 and 8.73, respectively when 5-HT was used as an agonist. These estimates accorded with the potencies measured in antagonism of zolmitriptan-mediated inhibition of forskolin-stimulated cyclic AMP formation. Ketanserin acted as a weak antagonist (pK(B): 5.87) at gp 5-HT(1B) receptors. 5. In conclusion, the recombinant gp 5-HT(1B) receptor shares important pharmacological similarities with the recombinant h 5-HT(1B) receptor. The finding that negative activity occurs at these receptors further suggests that SB224289, methiothepin and ritanserin are likely to be inverse agonists