Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity.
In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.
Louin
Gaëlle
G
Laboratoire de Pharmacologie (UPRES EA 2510), Université René Descartes, 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France.
Besson
Valérie C
VC
Royo
Nicolas C
NC
Bonnefont-Rousselot
Dominique
D
Marchand-Verrecchia
Catherine
C
Plotkine
Michel
M
Jafarian-Tehrani
Mehrnaz
M
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001930:Brain Injuries / Q000139:chemically induced / Q000201:enzymology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002531:Cerebellum / Q000187:drug effects / Q000201:enzymology; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004660:Encephalitis / Q000139:chemically induced / Q000201:enzymology; D005786:Gene Expression Regulation / Q000187:drug effects; D020128:Inhibitory Concentration 50; D008070:Lipopolysaccharides; D008297:Male; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010474:Percussion / Q000379:methods; D011014:Pneumonia / Q000139:chemically induced / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley
D002614:Chelating Agents; D008070:Lipopolysaccharides; D004492:Edetic Acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D002118:Calcium