Studies on hypoxia. 8. Ultrastructural and biochemical effects of prolonged exposure on rat parotid glands

The effect of chronic hypoxia upon the rat parotid gland is studied by electron microscopy and biochemistry. Male Sprague-Dawley rats are subjected to 88% N₂ and 12% O₂ at less than 2 psi pressure for 7 days. For electron microscopy, the tissues are perfusion-fixed, embedded and sectioned in a routine manner. In the biochemical studies, rats are injected with ³H-phenylalanine (2 μCi/g; s.i. ～ 5 Ci/mM) 60 min before sacrifice. Fresh glands are then prepared for analysis of amylase (Cibachrome-amylase substrate method), DNA (diphenylamine reaction), and total protein (Lowry method). The radioactivity in the acid precipitable and soluble fractions is determined by liquid scintillation spectrometry. The control animals are pair-fed and handled identically except that they are maintained in an ambient atmosphere. The ultrastructure of the hypoxic cells is altered in several areas. The Golgi apparatus demonstrates a decrease in organization and contains fewer transport vesicles. The rough-surfaced endoplasmic reticulum is broken and presents many vesicular and concentric profiles. The mitochondria have undergone several changes including swelling, clumping of intramitochondrial matrix, and fragmentation of cristase. The nucleus demonstrates a fibrillar pattern and contains a reduced amount of heterochromatin. The biochemical results indicate that, compared with controls, the hypoxic cells contain only 55% of amylase and 84% of the DNA, and thus suggest a drastic reduction of exportable protein production and an increase in cellular size, respectively. On the contrary, the incorporation of ³H-phenylalanine demonstrates an increase in the amount of radioactivity in the acid precipitable fraction of the hypoxic cells. These results lead to the conclusion that hypoxic stress causes a suppression of exportable protein synthesis, but may induce other cytoplasmic protein production, reflecting the biochemical and morphologic adjustment of the cell to a recovery phase.