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Potassium pyroantimonate histochemistry, coupled with ethyleneglycoltetraacetic acid (EGTA)-chelation and Xray microprobe analysis, was employed to localize intracellular calcium binding sites in the normal and degenerating flight musculature in queens ofSolenopsis, the fire ant. In normal animals, calcium distribution was light to moderate within myofibrils and mitochondria. In the early contracture stages of the insemination-induced degeneration, both myofilament and mitochondrial calcium loading was markedly increased. In the terminal stages of myofibril breakdown, only Z-lines

(5,6). The two decarboxylative steps in the tricarboxylic acid cycle (isocitrate dehydrogenase and a-ketoglutarate dehydrogenase) can be bypassed and two-carbon units (acetyl-CoA) can be converted to four-carbon acids (succinate) instead of being oxidized to CO2 (Fig. 1).In previous studies of aldosterone action in the toad urinary bladder, we demonstrated that the hormone-induced increase in sodium transport is preceded by stimulation of endogenous phospholipase activity, increased phospholipid fatty acid synthesis, and the recycling of [14C]acetyl-CoA derived from several 1-14C-labeled fatty acids. This [14C]acetyl-CoA did not appear to be derived from mitochondrial /3-oxidation because aldosterone stimulated a generalized increase in phospholipid fatty acid specific activities without altering fatty acid oxidation, measured by [14C]CO2 production, during the first 30 min of exposure to hormone (7,8). Consequently, we have investigated the possibility that nonmitochondrial fatty acid metabolism might be altered by aldosterone in the toad urinary bladder.Our results indicate that the toad urinary bladder is capable of cyanide-insensitive fatty acid oxidation. More importantly, the tissue possesses the two enzymatic activities unique to the glyoxylate cycle. These two enzymes have been reported previously only in unicellular organisms, certain nematodes, and higher plants (9-13). In toad urinary bladder, fatty acid can serve as a gluconeogenic substrate; in the presence of aldosteThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1521 rone, glycogen levels are higher. These data indicate that the toad urinary bladder is unique among higher animal tissues in that it has the enzymes necessary for the synthesis of carbohydrate from fatty acid. MATERIALS AND METHODSLarge female toads (Bufo marinus) were obtained from National Reagents (Bridgeport, CT). Hemibladders were removed from doubly pithed animals and incubated in an amphibian Ringer's solution (14). For enzyme assays, epithelial cells were scraped from 10-12 hemibladders and washed twice in a calcium-free amphibian Ringer's solution. To prepare a cell extract the cell pellet was frozen and thawed five times in calcium-free buffer and a 33% (wt/vol) homogenate was prepared in a Potter-Elvehjem homogenizer. The homogenate was then centrifuged at 3000 X g for 20 min at 4°C and the supernatant was used as the cell extract.Palmitoyl-CoA oxidation was assayed by the spectrophotometric reduction of NAD at 340 nm (15 from acetyl-CoA to be converted to four-carbon acids, bypassing the decarboxylative steps in the citric acid cycle.

Information regarding the presence of the glyoxylate cycle in chick liver was sought. This metabolic pathway has long been thought to be absent from vertebrate tissues. Previous studies in other tissues have shown that, when present, this pathway is sensitive to vitamin-D. Thus, the effect of long-term vitamin-D deficiency and subsequent vitamin-D replacement on liver structure was studied by light microscopy. In addition, specific biochemical assays for the presence of glyoxylate cycle enzymes were performed. Light microscopy of lipid extracted tissues, light microscopic histochemistry, and quantitative histochemistry showed that the hepatocytes from vitamin-D-deficient animals contained primarily lipid. Hepatocytes from normal and vitamin-D-replete livers contained primarily carbohydrate as judged by their staining with periodic acid-Schiff (PAS). Also, malate synthase positive peroxisomes were seen in hepatocytes from normal and vitamin-D-treated chicks. Structures positive for this glyoxylate cycle enzyme were rarely seen in the hepatocytes from vitamin-D-deficient animals. Biochemical analyses showed the presence of the two unique glyoxylate cycle enzymes, isocitrate lyase and malate synthase, in chick hepatocytes. The activity of these enzymes was markedly increased in the vitamin-D-replete livers. In addition, chick hepatocytes demonstrated the capacity to oxidize fatty acid in the presence of cyanide. This activity, which is characteristic of peroxisomal B-oxidation rather than mitochondrial, was stimulated by vitamin-D treatment. Lastly, incubation of chick liver in the presence of a fatty acid substrate (palmitate) led to higher tissue glycogen content. The latter was further increased in liver from vitamin-D-replete animals. These data show the presence of glyoxylate cycle enzymes in a higher vertebrate and indicate that this tissue is endowed with the capacity to convert lipid to carbohydrate.