Pathways of hydrogen utilization from NADPH generated by glucose-6-phosphate dehydrogenase in circumventricular organs and the hypothalamo-neurohypophysial system: A cytochemical study

Cytochemistry was used to examine the distribution of two pathways of utilization of hydrogen (Type I and Type II H) generated by glucose-6-phosphate dehydrogenase (G6PD) in circumventricular organs (CVOs) and the hypothalamo-neurohypophysial system in cryostat sections of rat brain. Type I H is defined as that portion of the total reducing equivalents (Total H) that is passed, in the intact cells, along the cytochrome chain (NADPH-diaphorase system). In the liver, energy from Type I H is used for cytochrome P-450-dependent oxidation of steroids, as well as xenobiotics. We proposed that mixed function oxidation, and therefore Type I H, would be preferentially localized in brain regions lacking a blood-brain barrier, such as CVOs and magnocellular cells with terminals in such brain regions. Type I H was identified in tissue sections using neotetrazolium. This reagent, when reduced, precipitates as formazan granules that can be quantified. The large difference in redox potential between NADPH and neotetrazolium ensures that only hydrogen (Type I H) passed in the intact cell along the cytochrome chain, can reduce the tetrazole. Total NADPH generation (Total H) from glucose-6-phosphate, was identified using medium containing phenazine methosulphate, a hydrogen acceptor that transfers all reducing equivalents from NADPH to the tetrazole. Type II H, the difference between Total and Type I H, is presumed to be used for NADPH-dependent biosynthetic functions such as lipid synthesis, or reduction of glutathione. In CVOs formazan granules indicative of Type I H were selectively concentrated and localized within cells throughout the SFO, organum vasculosum of the lamina terminals, pineal gland and in the apical cytoplasm of columnar ependymocytes in the subcommissural organ. Formazan granules attributable to Type I H were also prominent throughout the hypothalamo-neurohypophysial system. Reaction product was present in the cytoplasm of some magnocellular neurons in both the supraoptic and paraventricular nuclei, in the median eminence, including the zona interna, and in and between cells in the neurohypophysis. The distribution of NADPH-diaphorase in sections incubated with NADPH instead of glucose-6-phosphate was similar to that of type I H. These findings are consistent with the hypothesis that mixed function oxidation involving NADPH and the cytochrome chain occur in these brain regions. Formazan granules attributable to Total H were both more ubiquitously distributed than Type I H throughout the brain, and heavily concentrated in CVOs, particularly in the SFO, and in or between cells in the supraoptic and paraventricular nuclei. Reaction product attributable to Total and Type I H in magnocellular neurons was quantified by microspectrophotometry. Absorbance (mean integrated extinction × 100) for Total H was approximately 5-6× greater than for Type I H in magnocellular neurons in both the supraoptic and paraventricular nuclei. Relatively more Type II than Type I H was generated in magnocellular neurons and ependymal cells from glucose-6-phosphate (ratio Type I/Type II = 0.13 ± 0.01 to 0.29 ± 0.08). No reaction product was formed in the absence of substrate (glucose-6-phosphate) or NADPH.