Reduction in second-messenger ligand binding sites after brain ischemia--autoradiographic Bmax and Kd determinations using digital image analysis

Abstract

Changes in forskolin (FK) and phorbol 12,13-dibutyrate (PDBu) binding were evaluated in relation to local cerebral blood flow (CBF) after 6-h unilateral carotid artery occlusion in the gerbil striatum employing a quantitative autoradiographic method, which permitted these three parameters to be measured in the same brain. CBF was measured by the [14C]iodoantipyrine method at the end of the experiment. [3H]FK and [3H]PDBu were utilized as specific ligands to assess the activity of adenylate cyclase (AC) and protein kinase C (PKC), respectively. A saturation study was undertaken to measure the Kd (dissociation constant) and Bmax (maximal binding capacity) of each ligand by digital image processing of sequential autoradiograms employing pixel-by-pixel Scatchard analysis. The Bmax values of FK and PDBu were significantly decreased on the ischemic side, but the reduction in Bmax of FK was greater than that of PDBu. The K4 of each ligand remained unchanged. The FK binding underwent a progressive decline as CBF fell below 30 ml/100 g/min. The PDBu binding showed only a gradual decline in parallel with the CBF reduction. These findings suggest that a reduction in CBF below 30 ml/100 g/min for 6 h may induce a remarkable suppression of the AC system with less significant inhibition of the PKC system in the striatum.