Martin G. Larrabee

EFFECTS OF TEMPERATURE, CALCIUM AND ACTIVITY ON PHOSPHOLTPID METABOLISM IN A SYMPATHETIC GANGLION*

THE MAJOR purpose of these experiments was to investigate the changes in phospholipid metabolism which occur when nerve cells are activated by naturally conducted impulses. For this purpose, impulses were initiated under experimental control at a point effectively remote from that at which the metabolic effects of conduction and synaptic transmission were measured (Fig. 1). Observations such as those of

Lactate Metabolism and Its Effects on Glucose Metabolism in an Excised Neural Tissue

Abstract: Chains of lumbar sympathetic ganglia, excised from 15‐day‐old chicken embryos, were incubated for 4 h at 36°C in a bicarbonate‐buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO2–95% O2. [U‐14C]Glucose and [U‐14C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO2, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50–70% of the glucose carbon otherwise used for CO2 production and provided about three times as much carbon for CO2 as did glucose. The lactate addition increased the total carbon incorporated into CO2 and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U‐14C]‐glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10‐fold relative to that of the tissue, the average output of CO2 from [U‐14C]glucose during a 4‐h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate. Consumable substrates other than lactate, such as glutamine and certain amino acids, also affected glucose metabolism.

CHEMICAL EXCITATION OF NERVE

One of the noteworthy characteristics of neurones is their sensitivity to changes in the chemical environment. Even within the relatively protected interior of :he body, the properties of nerves are subject t o modification by variations in the composition of the body fluids. Indeed, the alterations of irritability and the trains of nerve impulses, which are the result of changes in the chemical environment, are among the most important factors involved in the regulation of the activity of the organism. This is one of t,he significant reasons for studying the chemical activation of nerve. A second reason derives from the current interest in the role of chemical agents in the mechanism of synaptic transmission. Furthermore, the investigation of the effects of various chemical agents is one of the most fruitful sources of information regarding the role of the several chemical components of the nerve structure and of the chemical processes involved in nervous action. This last consideration suggests that the most significant chemical agents for use in the study of the processes of activation are those which have an important part in the normal structure of nerve. Calcium is such an element. Potassium is another; it modifies the action of calcium, to which it is closely related in the regulation of nerve action, and i t has a marked influence on the electric potential difference across the interfaces a t which the nerve comcs in contact with its environment. Finally, the effects of acetylcholine on the initiation and conduction of the nerve impulse make an important and timely subject for investigation in such a study as this. It is with the effects of these agents that we shall be primarily concerned. There are others of significance for a general study of this probIem, but from these three we can derive many of the basic phenomena involved in chemical excitation. I

Effects of a nerve-growth factor, embryo age and metabolic inhibitors on growth of fibres and on synthesis of ribonucleic acid and protein in embryonic sympathetic ganglia.

Incorporation of labelled precursors into RNA and protein was measured in lumbar sympathetic ganglia from chicken embryos (usually 13‐14 days old) in the presence . or absence of nerve‐growth factor. The ganglia were incubated with labelled precursors while embedded in plasma clots, so that the outgrowth of nerve fibres could be measured in the same ganglia as the incorporation. Fibre outgrowth was estimated quantitatively by the use of a newly‐devised objective measure of mean halo width.

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15‐day‐old chicken embryos were incubated for 4 h at 36°C in a bicarbonate‐buffered salt solution equilibrated with 5% CO2‐95% O2. Glucose (1–10 mM), lactate (1–10 mM), [U‐14C]glucose, [1‐14C]glucose, [6‐14C]glucose, and [U‐14C]lactate were added as needed. 14CO2 output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO2 from [U‐14C]glucose, and glucose reduced that from [U‐14C]lactate. When using uniformly labeled substrates in the presence of 5.5 mM glucose, the output of CO2 from lactate exceeded that from glucose when the lactate concentration was >2 mM. The combined outputs at each concentration tested were greater than those from either substrate alone. The 14CO2 output from [1‐14C]glucose always exceeded that from [6‐14C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood‐brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.

Oxygen consumption of excised sympathetic ganglia at rest and in activity.

SYMPATHETIC ganglia of mammals have recently been found suitable for a variety of metabolic studies. By the use of these ganglia metabolism can be measured in a preparation which contains nerve cell bodies and synapses, and which survives for long periods after excision when kept in appropriate solutions at or below normal body temperature. Thus well-controlled measurements can be made under reproducible conditions, simultaneously with electrical tests of axonal conduction and synaptic transmission, and with nerve cells at rest or in repetitive action as desired by the experimenter. The superior cervical ganglion of the rat has been chosen for a number of studies in this laboratory, some of which have already been published (LARRABEE and BRONK, 1952; EDWARDS and LARRALEE, 1955). It is the purpose of this paper, first to describe an improved method for determining the rate of oxygen uptake of these preparations, secondly to report measurements of oxygen uptake at rest and during various frequencies of stimulation, and finally to compare certain of the results with those previously reported by others for frog nerve. These data will subsequently be compared with observations on ganglionic glucose uptake and lactate production (HOROWICZ and LARRABEE, 1957).

PHOSPHOINOSITIDES AND OTHER PHOSPHOLIPIDS IN SYMPATHETIC GANGLIA AND NERVE TRUNKS OF RATS

Abstract— Paired vagus nerves, phrenic nerves or superior cervical sympathetic ganglia from adult white rats were incubated for 4 h at 37°C in a bicarbonate‐buffered physiological solution containing glucose and 32P1. At the end of incubation triphosphoinositide (TPI) contained more 32P than any other lipid in the vagus nerves and was second only to phosphatidylcholine (PC) in the phrenic nerves. In the sympathetic ganglia phosphatidylinositol (PI) contained more 32P than did TPI, but both had less than PC. Conducted nerve impulses, initiated by electrical stimulation during the final 3 h of incubation, caused a highly significant increase in the [32P]‐labelling of PI in ganglia (as previously reported) probably decreased the labelling of TPI in the vagus nerves, and decreased the labelling of phosphatidylethanolamine (PE) in the ganglia. Addition to the incubation medium of §‐ or γ‐hexachlorocyclohexane (analogs of inositol) reversibly blocked transmission through the sympathetic ganglia at concentrations less than 0·1 mM. The §‐isomer also blocked conduction along axons at similar concentrations; only the γ‐isomer (lindane) exerted a selective effect on synaptic transmission. In the ganglia, the §‐isomer increased the [32P]‐labelling of PI and diphosphoinositide (DPI) relative to that of PC. The γ‐isomer did not affect the relative labelling of PI in the ganglia, whereas it decreased that of TPI, but only at relatively high concentrations. Thus, various affects of the hexachlorocyclohexanes were not explicable by assuming that they acted as analog inhibitors of inositol metabolism. In the ganglia, the hexachlorocyclohexanes reduced the effect of neuronal activity on the labelling of PI in proportion to the extent by which they blocked transmission. This metabolic effect was therefore presumed to be secondary to a ganglionic blocking action.

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagles minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U‐14C]lactate, [1‐14C]glucose, or [6‐14C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis‐Menten equation, with Vmax = 360 μmol/g protein/h and Km = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO2. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady‐state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO2 and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood‐brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.

TRANSYNAPTIC STIMULATION OF PHOSPHATIDYLINOSITOL METABOLISM IN SYMPATHETIC NEURONS IN SITU

Abstract— An increase in 32P labelling of phosphatidylinositol, associated with synaptic activity, has previously been reported in excised sympathetic ganglia excited by preganglionic nerve impulses. In the present experiments a similar effect occurred in naturally‐stimulated ganglia. It occurred when the preganglionic impulses were either evoked by electrical stimulation of the preganglionic nerve or were discharged spontaneously from the central nervous system. Thus increased turnover of phosphatidylinositol appears to be a normal accompaniment of synaptic transmission in these ganglia.

OXIDATION OF GLUCOSE IN A MAMMALIAN SYMPATHETIC GANGLION AT REST AND IN ACTIVITY

THE EXPERIMENTS to be reported in this paper, employing glucose labelled with radioactive carbon, confirm one conclusion concerning glucose metabolism previously reached with less direct methods, but contradict another. The conclusion confirmed is that enough glucose is oxidized to carbon dioxide by resting neurons to account for most or all of the resting oxygen uptake. The other conclusion, that some substrate in addition to glucose is oxidized during neuronal activity, is not supported by results of the present study. With the direct procedures permitted by tracer methods, it has been shown that the production of CO, derived from labelled glucose is sufficiently accelerated during activity in a sympathetic ganglion to account for the previously reported increase in oxygen consumption. This new result is at variance not only with our own previous conclusion, but with inferences concerning metabolism in various nervous tissues reached by several other investigators. The uptake of glucose by an excised sympathetic ganglion has previously been measured both at rest and during activity (EDWARDS and LARRABEE, 1955; HOROWICZ and LARRABEE, 1958; DOLIVO and LARRABEE, 1958). The present report is the first of several concerned with the compounds into which the carbon of glucose finds its way, as determined by tracer methods. In these experiments the rate of oxidation of glucose was measured in superior cervical ganglia of rats by observing the rate of production of labelled CO, when the tissue was bathed in a solution containing [14C]glucose. In subsequent papers the movement of carbon from glucose will be traced into other compounds, including lactic acid and certain materials retained by the tissue such as lipids and amino acids (HOROWICZ and LARRABEE, 1962; LARRABEE and KLINGMAN, 1962). These results, combined with the present experiments, will provide a reasonably complete accounting of the carbon released in the breakdown of glucose by a resting ganglion. Another purpose of this paper is to provide a detailed description of the methods used for the continuous measurement of labelled CO, produced when an excised tissue is immersed in a solution containing a labelled substrate.