David B. McDougal

The distribution of choline acetyltransferase activity in vertebrate retina.

Abstract— Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90‐fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number of amacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will be found to be cholinergic.

THE EFFECTS OF ANOXIA UPON ENERGY SOURCES AND SELECTED METABOLIC INTERMEDIATES IN THE BRAINS OF FISH, FROG AND TURTLE

The levels of the main cerebral energy reserves, ATP, P‐creatine, glycogen and glucose, and of several glycolytic intermediates and lactate, were measured in the brains of fish (Carassius auratus), turtle (Pseudemys scripta elegans) and frog (Rana pipiens). The levels of glycogen in these brains were 2‐9 times higher than those reported for mammals. In frog, cerebral glycogen levels were 35 per cent higher during the winter than in spring. The P‐creatine: ATP ratios were 3 instead of the more usual (mammalian) value of 1. The levels of other intermediates were similar to those found in mammalian brain.

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN FROG SCIATIC NERVE DURING ANOXIA AND RECOVERY

Frog (Rana pipiens) sciatic nerve was incubated, with and without stimulation, in an oil bath. The correlation between changes in the magnitude of the compound action potential (α and β) and changes in metabolites, particularly energy reserves, during anoxia and recovery from anoxia was studied.

The effect of electroshock on regional CNS energy reserves in mice.

ATP, phosphocreatine, glycogen, glucose and lactate levels were measured in the cerebral cortex, thalamus, cerebellum, brain stem and spinal cord of mice following supramaximal electroshock. During the initial 17 s after the onset of a 2 s electrical stimulus high energy phosphate expenditure exceeded formation in all regions but was slower in spinal cord than in the other regions. In cerebral cortex high energy phosphate utilization continued to exceed formation for 32 s which was twice as long as in any other region studied. Altered levels of metabolites recovered most rapidly in spinal cord and least rapidly in cerebral cortex. Pretreatment with a non‐anaesthetic dose of phenobarbitone influenced the effect of electroshock. Most of the clinical seizure was prevented, and increased high energy phosphate utilization was sustained for a much shorter period. Only in cerebral cortex did high energy phosphate expenditure exceed formation for as long as 15 s after the electrical stimulus; but even in this region the excess of expenditure over formation was much less than in untreated animals.

Effect of microgravity on metabolic enzymes of individual muscle fibers

Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose‐6‐phosphate isomerase, glyccrol‐3‐phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, β‐hydroxyacyl‐CoA dehydrogenase, 3‐ketoacid CoA‐transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and TA. Group OX enzymes increased 56‐193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated for a conversion of about half of the originally predominant fast glycolytic fibers into fast oxidative glycolytic fibers. Calculation on the basis of fiber length, rather than dry weight, gave an estimate of absolute enzyme changes: hexokinase was still calculated to have increased in both soleus and TA fibers, but only by 50 and 25%, respectively. Three of the OX enzymes were, on this basis, unchanged in TA fibers, but 3‐ketoacid CoA‐transferase and thiolase had still nearly doubled, whereas TA GLY enzymes had fallen about 40%. In soleus fibers, absolute levels of OX enzymes had decreased an average of 25% and GLY enzymes were marginally decreased.—Manchester, J. K.; Chi, M. M.‐Y.; Norris, B.; Ferrier, B.; Krasnov, I.; Nemeth, P. M.; McDougal, D. B., Jr.; and Lowry, O. H. Effect of microgravity on metabolic enzymes of individual muscle fibers. FASEB J. 4: 55‐63; 1990.

AXONAL TRANSPORT OF SELECTED PARTICLE-SPECIFIC ENZYMES IN RAT SCIATIC NERVE IN VIVO AND ITS RESPONSE TO INJURY

Abstract— Orthograde and retrograde axoplasmic transport of selected axonal organelles were examined by monitoring accumulation of enzyme activities residing in various types of particles proximal and distal to a ligature placed on rat sciatic nerve as a function of time after tying.

THE EFFECT OF AUDIOGENIC SEIZURES ON REGIONAL CNS ENERGY RESERVES, GLYCOLYSIS AND CITRIC ACID CYCLE FLUX

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6‐phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α‐oxoglutarate and malate, were essentially the same in all regions studied. α‐Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased.

OXIDIZED AND REDUCED PYRIDINE NUCLEOTIDE LEVELS AND ENZYME ACTIVITIES IN BRAIN AND LIVER OF NIACIN DEFICIENT RATS

ADVANTAGE has been taken of the recent development of highly sensitive methods (LOWRY, PASSONEAU, SCHULZ and ROCK, 1961) to study the response of the individual pyridine nucleotides (NADf, NADH, NADP+, NADPHg) in the brain andliver of rats to a low tryptophan, niacin-free diet. The study of three pyridine nucleotide-dependent enzymes was included, prompted by the findings of BURCH et al. (1956,1960) that some flavin-dependent enzymes decrease in riboflavin deficiency. SINGAL, SYDENSTRICKER and LITTLEJOHN (1 948) measured the nicotinic acid levels in different tissues of rats on corn rations and found them to be subnormal in the brain, liver, and muscle of the deficient animals but normal in other tissues. BURCH et al. (1955) reported that rats fed low amounts of tryptophan and niacin had subnormal amounts of oxidized pyridine nucleotides in blood cells and liver. More recently, SPIRTES and ALPER (1 961) studied the diphosphopyridine nucleotide levels in the livers of niacin-deficient and protein-deficient mice. They found a decrease of both NAD+ and NADH in the liver of the niacin-deficient animals, with no change in the NAD+/NADH ratio.

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues☆

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.

A spectrophotometric cycling assay for reduced coenzyme A and its esters in small amounts of tissue.

Abstract Sensitive procedures for the assay of a few pmoles of CoASH and its esters in milligram amounts of tissue are described. The cycling method of Stadtman et al. , which involves the arsenolysis of acetyl- P catalyzed by CoA and phosphotransacetylase (PTA), has been used. Selective conversion of various CoA esters to free CoA, followed by oxidation of the CoA so liberated, has enabled the specific assay of CoASH, acetyl CoA, succinyl CoA, and acetoacetyl CoA, and allows partition of the remaining CoA esters into three categories: “other PTA-reactive CoA esters,” probably mostly propionyl CoA; “PTA-unreactive CoA esters plus oxidized CoA;” and long-chain (acid-insoluble) CoA esters. Two inclusive categories are “total acid-soluble CoA” and “total CoA.” Preparation of tissue extracts is described. Rapid tissue fixation is essential for the measurement of cerebral levels of succinyl CoA, which fall 50% or more with decapitation, and of acetyl CoA, which rise 25% when the head is amputated.