Journal of Neurochemistry 34(1):213-215, January. Raven Press, New York © 1980 International Society for Neurochemistry 0022-3042/80/0101-0213/$02.00/0 Short Communication Activity-dependent Energy Metabolism in Rat Posterior Pituitary Primarily Reflects Sodium Pump Activity Marina Mata, David J. Fink, and Harold Gainer; Carolyn B. Smith, Leslie Davidsen, Helen Savaki, William J. Schwartz, and Louis Sokoloff Recent studies suggest (Schwartz et al., 1979) that local cerebral glucose utilization, measured with the deoxyglu- cose technique (Sokoloff et al., 1977), correlates most closely with electrical activity in the neuropil in general and synaptic terminals in particular. Presumably, in- creased glucose utilization associated with increased im- pulse activity in nervous tissue is, as is oxygen consump- tion (Ritchie, 1967; Greengard and Ritchie, 1971; De- Weer, 1975), principally due to enhanced activity of the sodium pump. If the increased energy metabolism during impulse activity is used mainly for reconstitution of elec- trochemical gradients, then it is to be expected that cel- lular components with larger surface-to-volume ratios will have larger energy demands (Ritchie, 1967; Greengard and Ritchie, 1971; DeWeer, 1975) and, thus, greater rates of glucose utilization. It would be of:value for the in- terpretation of studies that employ the autoradiographic deoxyglucose method to identify the cellular elements in which neural activity and energy metabolism are most closely linked. We have, therefore, studied an in vitro preparation of rat posterior pituitary, which represents a relatively enriched population of axon terminals (Nordmann, 1977) and may serve as a model for synaptic endings in the brain. Because the pituitary is a neurose- cretory organ, we have also studied the influence of the secretory process in this system on energy metabolism. As an index of glucose utilization, we have measured the rate at which ['*C]deoxyglucose is phosphorylated by hexokinase and trapped in the tissue incubated in vitro. This is the in vitro equivalent of the deoxyglucose method in which trapped ['*C]deoxyglucose-6-phosphate is visu- alized and measured autoradiographically (Sokoloff et al., 1977). MATERIALS AND METHODS Male Sprague-Dawley rats (180~250 g) were decapi- tated, and the pituitary glands were removed rapidly and placed in balanced salt solution (BSS) consisting of 10 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (adjusted to pH 7.4 with NaOH), 130 mm-NaCl, 4.5 mM-KCl, 1.8 mm-CaCl,, 0.9 mm-MgSO,, and 11.1 mm- glucose. The final osmolarity of the solution was 290 mosM. One hundred percent oxygen was continuously bubbled through the BSS. The posterior pituitaries were gently teased from the anterior lobes. Posterior pituitaries from three to five rats (approximately 3—5 mg wet weight of tissue) were combined in a small sack made of 200-4m mesh Nytex. Following a 15-min equilibration at 37°C in the BSS, the tissue was transferred to a vial containing 13.6 «Ci/ml of 2-deoxy-p-[ 1-'*C]glucose (54.4 mCi/mmol; New England Nuclear Corp., Boston, Massachusetts) in BSS. Pharmacological agents were included in some ex- periments as described below. In experiments carried out in the absence of calcium, the preincubation medium was also calcium-free. In experiments in which the tissue was stimulated electrically, the Nytex bag was placed be- tween two platinum grid electrodes. After 5 min in {'*C]deoxyglucose-containing medium, the tissue was stimulated with 1-V rectangular pulses at 10 Hz for 10 min; the pulses were 0.4 msec in duration. In all of the experiments the tissue was incubated for a total of 15 min in the medium containing the ['*C]deoxyglucose. Experi- ments were terminated by washing the tissue for 50 min in five successive changes of BSS in order to remove any free deoxyglucose. The tissue was digested in | M-NaOH and portions were taken for protein determination (Lowry et al., 1951) and for measurement of '4C by liquid scintillation count- ing. The results are expressed as c.p.m. of ['*C]deoxy- glucose/100 xg protein/15 min. In a separate experiment, the identity of the labeled product was determined by thin-layer chromatography. Eighty-eight percent of the radioactivity was in the form of ['*C]deoxyglucose-6- phosphate. The results have been analyzed with Dunnett’s statisti- cal test for multiple comparisons with a single control group (Dunnett, 1955; 1964). From the Section on Functional Neurochemistry, Behavioral Biology Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20014; and the Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, Maryland 20014. Address reprint requests to Dr. C. B. Smith, Laboratory of Cerebral Metabolism, Building 36, Room 213 1A-27, National Institute of Mental Health, 9000 Rockville Pike, Bethesda, Maryland 20005. Received May 22, 1979; accepted July 2, 1979. Abbreviations used: BSS, Balanced salt solution; mosM, Mil- liosmolar; Hz, Cycles per second. 214 M. MATA ET AL. TABLE 1. Effects of repetitive electrical stimulation on [!4C] deoxyglucose uptake in posterior pituitary ['*C]Deoxyglucose uptake Statistical Condition (c.p.m./100 yg protein/15 min) significance Controls (4) 988 + 19 Stimulated at 10 Hz (4) 1272 + 57 p < 0.01 Stimulated at 10 Hz + ouabain (4) 1018 + 51 n.s. The values represent the means + S.E.M. of the results obtained in the number of experiments, indicated in parentheses. RESULTS AND DISCUSSION Electrical stimulation of the posterior pituitary in vitro at 10 Hz produced a 29% increase in ['*C]deoxyglucose- 6-phosphate accumulation in the tissue above control val- ues (Table 1). In the presence of 10,.M-ouabain (an inhib- itor of the Nat—K+-—ATPase and sodium pump), how- ever, electrical stimulation of the tissue produced no sig- nificant change in ['tC]deoxyglucose-6-phosphate ac- cumulation from control values (Table 1). Inasmuch as ouabain does not inhibit either spike activity or hormone release in the neurohypophysis (Dicker, 1966), these data strongly indicate that activation of the ouabain-sensitive sodium pump is the critical event coupling energy metabolism to electrical activity. Additional evidence was obtained in experiments with veratridine, an alkaloid that activates the sodium con- ductance mechanism in the membrane in association with spike activity. In the posterior pituitary, veratridine de- polarizes the nerve terminals, produces an influx of sodium ions, and, consequently, causes a large release of hormones (Dyball and Nordmann, 1977). The results in Table 2 (A) show that in the presence of 60 um- veratridine, deoxyglucose-6-phosphate formation was significantly increased over control values by an average of 37%. The addition of 6 4.M-tetrodotoxin, a neurotoxin which blocks the activation of sodium channels, pre- vented the veratridine effect on deoxyglucose phosphor- ylation. The effect of veratridine appeared, therefore, to be mediated by its effects on the sodium permeability of cell membranes. Furthermore, as in the experiments with electrical stimulation, inhibition of the sodium pump by ouabain also prevented the veratridine stimulation of deoxyglucose phosphorylation [Table 2 (A)}. A major function of the terminals in the posterior pituitary is neurosecretion. In order to examine the con- tribution of the process of neurosecretion to energy con- sumption we measured the effect of veratridine on deoxyglucose phosphorylation in posterior pituitaries in- cubated in the absence of calcium and a raised magnesium concentration [Table 2 (B)]. Under these conditions neurosecretion is inhibited, but electrical activity is not (Dyball and Nordmann, 1977). In spite of the absence of neurosecretion, veratridine continued to cause a signifi- cant, 47% increase in deoxyglucose phosphorylation above the calcium-free control values. The contribution of neurosecretion to the energy utilization of these termi- nals appears, therefore, to be negligible in comparison with that of sodium-pump activity. The results of the present studies suggest that the major energy-consuming function of activated nervous tissue is ion pumping by the sodium pump. Glucose utilization traced by deoxyglucose phosphorylation was increased in the posterior pituitary by electrical stimulation and by TABLE 2. Influence of sodium-pump activity and neurosecretion on ['4C]deoxyglucose uptake in posterior pituitary ['*C]Deoxyglucose uptake Statistical Condition (c.p.m./100 wg protein/15 min) significance A. Dependence on activation of sodium-pump activity Controls (14) 1381 + 50 Veratridine (14) 1891 + 85 p < 0.001 Tetrodotoxin (9) 1209 + 84 ns. Tetrodotoxin + veratridine (8) 1551+ 72 n.s. Ouabain (4) 1318 + 57 n.s. Ouabain , + veratridine (4) 1218 + 120 ns. B. Independence from activation of neurosecretion Controls Ca?+-free medium (6) 1142 + 38 Veratridine in Ca**-free medium 1681 + 78 p < 0.001¢ The values represent the means + s.E.M. of the results obtained in the number of experiments, indicated in parentheses. @ The statistical significance of the difference between these two groups was calculated by Student’s t-test. J. Neurochem., Vol. 34, No. 1, 1980 ENERGY METABOLISM AND THE SODIUM PUMP 215 veratridine, both of which produce an activation of the sodium conductance mechanism, with a consequent in- flux of sodium. With both modes of stimulation the in- crease in glucose utilization was prevented by ouabain, which blocks the sodium pump. Both electrical stimula- tion and veratridine should have activated neurosecre- tion, the primary function of the terminals in the posterior pituitary. If the secretory process, however, made a sub- stantial contribution to the energy consumption of an ac- tivated nerve terminal, then the ouabain should not have blocked the effects of electrical stimulation and vera- tridine on glucose utilization. Furthermore, the veratridine stimulation of glucose utilization was unimpaired by in- cubation conditions that blocked neurosecretion. There- fore, we can find no evidence for a significant contribu- tion of neurosecretion to the consumption of energy in nerve terminals. Our results also show that the rate of glucose con- sumption by the nerve terminals of the posterior pituitary at rest is not affected by ouabain in the medium. Thus, relatively little energy metabolism at rest appears to be used for the maintenance of ionic gradients by the ouabain-sensitive sodium pump. The resting energy metabolism is probably devoted to other cellular pro- cesses, e.g., cell maintenance and repair, axoplasmic transport, and—in neuronal perikarya—protein synthe- sis. The proportions of total resting energy metabolism associated with each of these processes remain to be de- termined. These results obtained with deoxyglucose uptake con- firm and extend the results of other studies in which oxy- gen consumption was measured (Ritchie, 1967; Green- gard and Ritchie, 1971; DeWeer, 1975). Increases in energy metabolism with stimulation are a function of the increased activity of the sodium pump which is activated by the influx of sodium ions and efflux of potassium ions accompanying a depolarizing stimulus. The increment in energy utilization accompanying membrane depolariza- tion is, in turn, a function of the surface-to-volume ratio of the tissue (Ritchie, 1967; Greengard and Ritchie, 1971; DeWeer, 1975). Because the highest surface-to-volume ratios are found in nerve endings and dendrites, one would expect brain areas rich in these structures to have the greatest relative increase in glucose consumption ac- companying stimulation. Recognition of this will be of importance in interpreting future studies using the au- toradiographic deoxyglucose method. ACKNOWLEDGMENT We thank K. D. Pettigrew for help with the statistical analysis of the data. REFERENCES DeWeer P. (1975) Aspects of the recovery processes in nerve, in Physiology (Hunt C. 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