Uptake of horseradish peroxidase at isolated nerve terminals in relationship to transmitter release; Biochemical results

Abstract

The uptake of horseradish peroxidase (HRP) into isolated nerve terminals (synaptosomes) has been studied by using a spectrophotometric method to determine the enzyme activity. HRP is rapidly taken up by synaptosomes, it is not removed by multiple washes in iso-osmotic medium but is lost if the particles are ruptured by hypo-osmotic conditions. The uptake is not affected by metabolic poisons, is reduced at lower temperature and is not with any significant release of cytoplasmic lactate dehydrogenase suggesting an endocytotic mechanism. Intra-synaptosomal HRP can be released by a process that is similar to uptake and also not accompanied by any loss of synaptosomal lactate dehydrogenase exocytosis. Depolarization of synaptosomes (by high potassium concentrations) was found to release [ 14C]ACh but to have no effect on HRP uptake either simultaneouly or after recovery in an non-depolarizing medium. Absence of Ca 2+ prevented depolarization evoked release of [ 14C]ACh but had no effect on the uptake of HRP. The release of HRP was not increased by depolarization even though [ 14C]choline taken up during the same period wasreleased as [ 14C]ACh. It is concluded that the endo-exocytotic cycle that transport HRP across the synaptosomal membrane is unrelated to tranmitter release. A discrete vesicular localization of HRP reaction product was only occasionally in the EM nor could consistent differences resulting from depolarization be observed. However, nthe untrastructural localization was found to be unreliable because glutaradehyde fixation irreversiy inactivated 80–90% of the HRP even when it was sequestered within synaptosomes and the insoluble reaction product precipitated from a supersaturated solution onto membranes.