Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo

The combination of microdialysis and a highly sensitive radioimmunoassay was developed in order to monitor the in vivo extracellular levels of endogenous opioid peptides from discrete regions of the rat brain. The radioimmunoassay cross-reacts 100% with peptides with alpha N-acetyl Tyr.Gly.Gly.Phe-Met or -Leu at the N terminus and thus recognizes all known endogenous opioid peptide fragments following acetylation of the sample. The assay was conducted on solid phase with antibody bound via protein A to 96-well plates and provided a limit of detection of approximately 0.2 fmol. A variety of dialysis membranes were evaluated with respect to their efficiency in recovering opioid peptides in vitro. Custom-made probes (4 mm active length) manufactured from polyacrylonitrile membranes and commercially available polycarbonate membrane probes proved most suitable with relative recoveries for [Met]- and [Leu]enkephalin in the range 6-10% at a flow rate of 2.7 microliters/min. Probes implanted in the globus pallidus/ventral pallidum of halothane/N2O anaesthetized rats recovered approximately 1.5 fmol of immunoreactive opioid material per 30-min sample in the absence of peptidase inhibitors. The majority of this immunoreactivity co-eluted with [Met]- and [Leu]enkephalin on reverse-phase high-performance liquid chromatography. A 2-min pulse of 100 mM K(+)-containing artificial cerebrospinal fluid in the perfusion medium during a 30-min sampling period increased the recovered immunoreactive material to 43.9 fmol +/- 12.4 S.E.M. A second stimulation 3 h later also resulted in elevated levels with an S2:S1 ratio of 0.64 +/- 0.03. The second stimulation was completely blocked by perfusion of a 10 mM EGTA-containing medium, basal release on average remaining unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)