759
J. Physiol. (1975), 247, pp. 759-771 With 6 text-figurea Printed in Great Britain
UPTAKE OF ORTHOPHOSPHATE BY RABBIT VAGUS NERVE FIBRES
By BEATRICE ANNER, J. FERRERO, P. JIROUNEK AND R. W. STRAUB From the Departement de Pharmacologie, Ecole de Mgdecine, CH-1211 Geneve 4, Switzerland
(Received 9 October 1974) SUMMARY
1. The uptake of orthophosphate and its incorporation into ATP, ADP, and creatine phosphate (CrP) were studied in desheathed rabbit vagus nerve. 2. Using 32p labelled orthophosphate, the total amount of labelled phosphate taken up by the preparation was continuously recorded in a perfusion apparatus. For measuring the incorporation into phosphorylated compounds, phosphate esters and inorganic phosphate were extracted, separated and their total amount and radioactivity determined. 3. The total uptake of phosphate was found to be a biexponential function of time. 4. The time constant of the first process was 10-20 min and independent of the extracellular phosphate concentration, the final amount labelled by this process was relatively small and proportional to external phosphate, increasing from 0-026 m-mole/kg wet nerve at 0-04 mm phosphate to 114 m-mole/kg at 5 mm. 5. The time constant of the second process depended on the extracellular phosphate concentration varying from 4624 min at 0*04 mm to 210 min at 5 mm. The final amount labelled by this process was 5-6 m-mole/kg wet wt. and independent of the extracellular phosphate. 6. The kinetics of the slow uptake were consistent with the presence of a saturable process and a non-saturable one. 7. Extraction of ATP, ADP, and the sum of CrP and Pi, showed that the total amount of these compounds remained constant for 2 hr while their radioactivity increased slowly, approximately at same rate as the slow fraction. 8. Increasing the external phosphate from 0 04 to 5 mm increased the amount of labelled ATP. 9. A comparison with the metabolic turnover of phosphate, estimated
760 760
~BEATRICE ANNER AND OTHERS
from the oxygen consumption, shows that uptake is much slower than metabolism, so that the slow appearance of labelled nucleotides is very probably due to a limitation of the influx. 10. From the experimental data the influx can then be calculated for various phosphate concentrations. It is close to that found in squid axons. INTRODUCTION
Although the importance of phosphates for metabolism and structure of cells has been recognized for many years, relatively little is known on the way by which these ions are taken up by nerves. Studies on phosphate uptake in nerve have so far been confined to giant axons (Caldwell & Lowe, 1970) and frog nerves (Mullins, 1954; Abood & Koyama, 1963; Abood, 1968); to our knowledge (see also Hurlbut, 1970) the uptake of phosphates in mammalian nerve has not been investigated. Studies on phosphate transport are complicated by the presence of several ion species, and by the fact that phosphate, unlike the rather inert ions studied in detail, is incorporated into a number of intracellular biochemical compounds. Furthermore, labelled phosphate added to the extracellular medium seems to exchange with a phosphate fraction of the plasma membrane (Causey & Harris, 1951). In order to overcome, to some extent, these difficulties, the present experiments are based on the use of two independent methods: (i) a method that allows the continuous recording of the total uptake of radiophosphate in a perfused preparation, which has the advantage that the rate of uptake can be measured accurately under different conditions and at any time of the experiment; and (ii) methods of measuring the amounts of inorganic phosphate (Pi) and phosphorylated compounds, extracted from the preparation, and their respective radioactivities. These methods allow the study of the incorporation of radiophosphate into compounds supposedly located inside the cells, and thus give an estimation of the phosphate influx. As pointed out by Caldwell. & Lowe (1970) this technique of measuring the influx is applicable only if the rate of influx is much smaller than the rate of incorporation. This condition seems to be satisfied, as will be explained later. The experiments showed that a small amount of added radiophosphate rapidly exchanged with a fraction of loosely bound phosphate and that the bulk of the uptake was attributable to influx into the cells, transported, to a large extent, by a saturable system that will be described in more detail in a following paper. A preliminary account of some of the results has been published elsewhere (Anner, Ferrero, Jirounek & Straub, 1973a, b).
PHOSPHATE INFLUX IN C-FIBRES
761
METHODS
Preparation of nerves, apparatus for recording radioactivity. Rabbits weighing between 2-5 and 3 kg were shot and their cervical vagus nerves rapidly removed and desheathed with scissors. The preparations were then incubated for at least 1 hr in phosphate-Locke of the same phosphate concentration that was later used in the experiment. Afterwards they were mounted in the apparatus shown in Fig. 1, where they lay in a polyethylene tube that was continuously perfused. After starting with label-free phosphate-Locke, orthophosphate-labelled solutions were applied and the total radioactivity of the preparation and the surrounding medium was recorded over a length of 5 cm. The whole recording system had a time constant of approximately 0-5 sec. The solutions flowed at a rate of about 1 ml./min and could be changed by turning a tap. Their flow was maintained by a perfusion pump. Experiments in which the effect of changing the solution was recorded without nerve showed that the exchange of the solution was complete
Desheathed vagus
9
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'/ 9
I
--
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I I~~~~~
Fig. 1. Apparatus for recording total radioactivity of preparation and surrounding medium. Diagram shows desheathed cervical vagus nerve mounted in a polyethylene tube of 0-86mm inside diameter, which is continuously perfused with Locke or modified Locke; tube is placed opposite to window of beta-counter, connected to rate meter and penrecorder.
762
BEATRICE ANNER AND OTHERS
within 15 sec. The efficacy of the counting was measured by perfusing the tube with a solution of known radioactivity and without nerve. Unless otherwise stated, the temperature was 370 C. Chromatographie separation of orthophosphate, nucleotides, and creatine phosphate. For the studies on the localization of the labelled phosphate taken up by the nerves, the preparations were washed with ice-cold phosphate and label-free Locke during 5 min and then removed from the apparatus. A given length, weighing about 10 mg and corresponding to the part that lay under the counter, was cut off, and used for the analysis. In a first series of experiments the radioactive compounds extracted from the nerve were separated by thin-layer chromatography. For this, the tissue was immersed into 1 ml. trichloroacetic acid (TCA) (5 % w/v), homogenized and centrifuged at 3000 g, all these operations being done at a temperature of about 4° C. The radioactivity of 0-1 ml. of the supernatant and of the pellet was then determined. The pellet was rejected: after 40 min incubation in radioactive Locke, it contained less than 1 % of the total radioactivity. Next, the TCA of the supernatant was extracted with ether, and the remaining solution was evaporated at low pressure. The residue was dissolved in a solution of ethylenediaminetetraethylammonium (0-002M), and used for bi-dimensional thin-layer chromatography, as described by Randerath & Randerath (1964). Autoradiography showed at most 4 spots; they could be identified as ATP, ADP, guanosine 5'-triphosphate (GTP), and a spot where both orthophosphate and creatine phosphate (CrP) were found. Small amounts of these compounds were therefore added before chromatography, and the spots, revealed by ultraviolet light and autoradiography, removed, and counted in a liquid scintillation counter. The sum of all counts so obtained was compared to the number of counts found in the nerve extract and the results corrected, if the difference was not larger than 20 %. If more than 20 % of the initial radioactivity was lost during chromatography, the experiment was rejected (for details of the technique, see Anner, 1973). In other experiments, the compounds were separated by column chromatography, as described by Garrahan & Glynn (1967). This method had the advantage that the total amount of the compounds as well as their radioactivity could be determined. After removal from the apparatus the tissue was plunged into 2 ml. of boiling triethanolamine buffer at pH 8-2, rapidly cooled after 40 sec and then homogenized (see GCreengard & Straub, 1959). The homogenate was deproteinized with chloroform (1 ml.) and centrifuged for 30 min at 3000 g and 40 C. The supernatant was then passed through a Dowex 1 column, elated, and the effluent collected. Twelve fractions, allowing the separation into ATP, ADP, and the sum of CrP and Pi were taken; GTP was in the ATP fractions. The concentrations of ATP and ADP were measured spectrophotometrically with a Unicam Ultraviolet Spectrophotometer; CrP was hydrolysed and determined with the inorganic phosphate, with the method described by Anner & Moosmayer (1975). In the same fractions, the radioactivity was also counted. Weight. The nerves were weighed after desheathing, and after removal from the apparatus. All concentrations given in the text are based on the initial weight. Solutions. The composition of Locke was (in mM): NaCl, 154; KC1, 5-6; CaCl2, 0-9; MgCl4, 0-5; Tris, 10-0; glucose, 5-0. Phosphate-Locke contained in addition Na2HPO4 and NaH2PO4; the phosphate concentrations indicated in the text refer to the total concentration of orthophosphate (referred to as phosphate or Pi). The pH of all solutions was adjusted with HOl to exactly 7-40; it was stable for several days. The labelled solutions contained approximately 3 #C82P/mi. added from a radio-
763 active stock solution of [32P]Na2HPO4 and [32P]NaH2PO4 in isotonic NaCi at pH 7, which was obtained from the Eidgendssisches Institut fur Reaktorforschung, Wu~renlingen, Switzerland. The radioactivity of the stock solution had a concentration of about 3 inc/mi., and the specific activity of the 32P amounted to 2500-3500 inc/rn-mole. All other salts were analytical grade. PHOSPHATE INFLUX IN C-FIBRES76
[32P]Phosphate Locke
Phosphate Locke
1100 counts/sec
1
i
Fig. 2. Uptake of phosphate from 0-2 mm [32P]phosphate-Locke. Record of radioactivity of preparation and surrounding medium shows filling of tube and extracellular space after application of [32P]phosphate-Locke (fast upstroke), followed by uptake of phosphate by the preparation (slow increase in radioactivity). Application of inactive phosphate Locke shows loss of radioactivity from tube and extra-cellular space (fast downstroke), followed by slower loss from the preparation. 100 counts/sec correspond to uptake of 120 #tmole orthophosphate/kg nerve wet wt.; temp. 370 C. RESULTS
Total uptake When perfusion with labelled Locke containing 0-2 mm phosphate was started, the records showed a rapid increase in radioactivity during the first few minutes (Fig. 2). This initial upstroke corresponds probably to the filling of the tube and of the extracellular space. The tube fills within about 15 sec (see Methods) and the rate of filling of the extracellular space can be estimated from experiments of Keynes & Ritchie (1965), who found, with the sucrose-gap technique and similar flow conditions, that after changing from Locke to potassium sulphate-Locke, the
BEATRICE ANNER AND OTHERS 764 potassium ions filled the periaxonal space along an exponential curve with a time constant of 0x8 min at 200 C. From the diffusion coefficients of K2SO4, Na2HPO4, and NaH2PO4, and their temperature dependence (see Landolt & B6rnstein, 1969), it can be estimated that the diffusion of radiophosphate shows about the same time course. Therefore, in our experiments, the radioactive solution should have invested the extracellular space within a few minutes after its application. 10
E
0-1
0
3
6 Time (hr)
9
12
Fig. 3. Semilogarithmic plot of difference between final amount of 32p uptake and that found at any given time during experiment. Curve obtained (0) can be decomposed into two exponentials (straight lines); the corresponding final fractions are found by extrapolation to zero time. Note that extracellular phosphate was 3 mM and fast fraction relatively large in this case.
Afterwards the radioactivity increased much more slowly and reached nearly saturation after about 24 hr. By plotting the difference between the final radioactivity and that found at any given time, two approximately exponential processes could be distinguished (see Fig. 3). The first, relatively fast process had a time constant of 10 min and reached an equilibrium corresponding to the filling of a space containing 0-06 mmole/kg wet wt., at 0-2 mm extracellular phosphate. The second process was much slower and had a time constant of 1530 min, and the corresponding space contained 5-2 m-mole/kg wet wt. When experiments like that shown in Fig. 3 were repeated with different
765 PHOSPHATE INFLUX IN C-FIBRES phosphate concentrations, it was found that the time constant of the first fraction was nearly independent of the extracellular Pi (Table 1), but that the amount of phosphate that could be labelled increased with increasing extracellular phosphate concentrations. This fraction therefore shows characteristics of a non-saturable compartment, that is being filled by a diffusion process. It may tentatively be termed 'fast' fraction. TABLE 1. Final amounts exchanged in fast and slow fractions and corresponding time constants of exchange at different extracellular phosphate concentrations
Slow fraction
Fast fraction Final amount exchanged
External Pi concn. (mm) 0 04 0.1 0*2 0*3 04 0.6 1.0 2*0 3.0
5*0 Mean
Time
Final amount exchanged
(m-mole/kg wet wt.) 0X026 (3)
constant
(min) 16*2 (2)
(m-mole/kg wet wt.) 5.5 (1)
0.063 (6) 0*049 (1) 0'078 (1) 0*142 (3)
10.2 (6)
5.2 (1)
0.55 (3) 0.73 (4) 1*14 (1)
16.9 (1) 17.6 (2)
5.8 (2)
13.3
5*6
5.5 (1)
13.5 (1)
Time constant
(min) 4624 (3) 3080 (1) 1530 (7) 1380 (1) 1220 (1) 672 (3) 490 (2) 470 (2) 341 (5) 210 (2)
Values of Table were obtained from plots like that shown in Fig. 3. In the experiments where the total final amount was not measured, the time constants and the amount in the fast fraction were obtained by using the mean final amount of the slow fraction and the observation that both processes are exponential. In parentheses, number of experiments.
In the following, the second, slow process will be studied in more detail. Experiments with different extracellular phosphate concentrations showed that the time constant of the filling process depends on the extracellular Pi (Table 1), while the amount that is finally labelled is remarkably constant. At first sight, this behaviour is compatible with the filling of a constant space through a saturable process, and it was suggested in a preliminary communication (Anner et al. 1973a) that the uptake process could be described by simple saturation kinetics of the Michaelis-Menten type with an apparent Km of about 1 mm. Studying the relation between the rate of the slow uptake and the extracellular phosphate concentration over a wider range of concentrations (Fig. 4), it is now evident that the kinetics of the uptake are more complicated than initially thought.
766 766
~BEATRICE ANNER AND OTHERS
20 E
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0
0
Extracellular phosphate concentration (mm)
Fig. 4. Rate of phosphate influx at different extracellular phosphate concentrations. Rate of influx was calculated from slow uptake of Table 1; curve relating it to extracellular phosphate concentration does not show simple saturation kinetics. Bars indicate s.E.
Labelling of nudleotide8 In extracts of nerves exposed to labelled phosphate and then briefly washed, labelled ATP, ADP, GTP, CrP and orthophosphate were found. Table 2 and Fig. 5 give results of experiments in which, after incubation in 0-2 mm phosphate Locke for various times, the total amount of these compounds were measured and their activity counted. The experiments showed that the total amounts did not appreciably change during incubation. The specific activities of the extracted compounds increased slowly: only 10% of the extracted ADP, CrP and Pi had exchanged with extracellular phosphate after 2 hr, and 20 % of the ATP, counting 1 label per molecule. This behaviour could be due either to a slow influx or to a slow metabolic turnover of the nucleotides. The rate of turnover can be estimated from the oxygen consumption (see Discussion), and it is then evident that the slow appearance of labelled compounds is due to a limitation in the uptake process. The time course of the increase in specific activity parallels the slow uptake of phosphate, and since the synthesis of ATP is almost certainly intracellular, it can be assumed that the slow uptake corresponds to the influx of phosphate into the axons and the Schwann cells.
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769 PH~r-OSPHATE INFLUX IN C-FIBRES76 In another series of experiments the phosphate concentration was varied and the nerves extracted after 40 min incubation. The results, illustrated in Fig. 6, show that the amount of labelled nucleotides found in the nerve parallels the total amount of labelled Pi taken up by the slow process at different extracellular concentrations (cf. Fig. 4). This finding is consistent with the hypothesis that the slow uptake is responsible for the rate of labelling of ATP. DISCUSSION
Faet uptake Our experiments showed that radiophosphate rapidly exchanges with the phosphate of a 'superficial' fraction, and that at equilibrium, the amount of phosphate exchanged is nearly proportional to the extracellular phosphate concentration. This suggests that the phosphate of this fraction is not strongly bound and that a large number of free sites is present at low phosphate concentration. It is not clear where the fraction is located. Binding to myelin of the rather numerous myelinated fibres of the cervical vagus can almost certainly be excluded, since the fraction is also found in the thoracical vagus (unpublished observations), which in the rabbit contains only a few myelinated fibres (Evans & Murray, 1954). The fast fraction resembles a phosphate fraction observed by Causey & Harris (195 1) in the membrane of frog striated muscle. They suggested that it may be due to exchange with phosphoproteins. Slow uptake Turnover and influx. It is interesting to compare the uptake of phosphate with its metabolic turnover, which can be estimated from the oxygen consumption. The experiments of Ritchie (1967) in rabbit vagus suggest a metabolic rate of at least 0-36 in-mole Pi/kg wet wt. per minute at 370 C. In our experiments, the Pi extracted :from the nerve amounts to 9-2 in-mole/kg wet wt. (Table 2), s0 that the mean time constant of metabolic turnover becomes 9-5 min, or, if only 1 label per molecule is counted, 5-5 min. Other phosphorylated compounds than those found in our experiments are also present, but their amount is so small (Greengard & Straub, 1959) that they can be neglected. Metabolic turnover is thus not very different from the fast uptake, but much faster than the slow uptake. While the rate of fast uptake could be explained by the rate of metabolic turnover, the quantities that are taken up by the fast process are much too small to explain the amount of labelled compounds that appear in the nerve. For instance, at 0-2 mm extracellular phosphate the fast fraction does not exceed 0-06 in-mole/kg wet wt., while the Pi of
BEATRICE ANNER AND OTHERS 770 ATP exchanged after 60 min when the 'fast' uptake is complete, amounts already to 0-145 m-mole/kg wet wt. The rapid metabolic turnover and the finding that the rate of labelling is slow (Fig. 5) and parallels the slow uptake, suggest that labelling is limited by uptake. Further, since the main production of ATP is almost certainly due to reactions inside the cells, the slow uptake appears to correspond to the influx of phosphate. The situation in rabbit vagus is thus similar to that in squid axons, where the influx of phosphate is also much slower than the metabolic turnover (cf. Tasaki, Teorell & Spyropoulos, 1961; Caldwell, Hodgkin, Keynes & Shaw, 1964; Caldwell & Lowe, 1970). Influx. The influx of phosphate can then be calculated from the rate constant of slow uptake and the quantity that finally exchanges with radiophosphate (Table 1). At 0-2 mm extracellular phosphate the influx becomes 3-4 /imole/kg wet wt. per minute, and with an axon surface of 0-6 x104 cm2/g wet wt. (Keynes & Ritchie, 1965) the influx into the axons would amount to 9-4 f-mole/cm2 per second. In our experiments, phosphate may also be taken up by Schwann cells. Their surface membrane is approximately twice as large as the surface of the axons, so that the mean transmembranal flux may be around 3 f-mole/cm2 per second. At any rate, it is not very different from the flux in squid giant axons, where at 0.1 mm external phosphate, Caldwell & Lowe (1970) measured an influx of 20-9 f-mole/cm2 per second between 17 and 210C. Mechanism of influx. The experiments showed that the influx contains a large saturable component, but that it cannot be described by simple kinetics of the Michaelis-Menten type. A more detailed study of this process in various conditions will be given in a following paper. Total amount of exchangeable phosphate. At equilibrium, the quantity of phosphate in the slow fraction that exchanged with radiophosphate was almost independent of the extracellular phosphate concentration. This behaviour resembles that found in striated muscle (Causey & Harris, 1951). It is understandable if the intracellular concentration of free orthophosphate is small compared with that of the phosphorylated compounds, the total quantity of which may be limited by the availability of creatine and adenosine. Similarly, when the utilization of ATP is decreased by ouabain, the concentration of ATP is almost unchanged (Chmouliovsky & Straub, 1974). The total phosphate extracted after short incubations was at 0-2 mm external phosphate 9-2 m-mole/kg wet wt. (Table 2), which is definitely larger than the total amount of phosphate that exchanges during long term experiments, and which amounts, for the sum of both fractions to 6 m-mole/kg. Loss of compounds, able to retain phosphate, may occur during these long perfusions.
PHOSPHATE INFLUX IN C-FIBRES
771
We are grateful to Dr P. Kalix for advice in the use of the method of thin-layer chromatography, to Mrs M. Moosmayer for technical assistance throughout the experiments and to the SNSF for a grant, Number 3.0890.73. REFERENCES
Ai3OOD, L. G. (1968). Interrelationships between phosphates and calcium in bioelectric phenomena. Int. Rev. Neurobiol. 9, 223-261. ABOOD, L. G. & KOYAMA, I. (1963). Phosphate incorporation in desheathed nerves: effects of potassium and calcium ions. Science, N.Y. 141, 1277-1278. ANNER, B. (1973). Localisation, repartition et transport de l'ion phosphate dans un tissu nerveux. These No. 1609, Faculte des Sciences, Universite de Geneve. ANNER, B., FERRERO, J., JIROUNEK, P. & STRAUB, R. W. (1973a). Inhibition of intracellular orthophosphate uptake in rabbit vagus nerve by Na withdrawal and low temperature. J. Physiol. 232, 47-48P. ANNER, B., FERRERO, J., JIROUNEK, P. & STRAUB, R. W. (1973b). Na-dependent phosphate influx into mammalian nerve fibres. Experientia 29, 740. ANNER, B. & MOOSMAYER, M. (1975). Rapid determination of inorganic phosphate in biological systems by a highly sensitive photometric method. Analyt. Biochem. (in the Press). CALDWELL, P. C., HODGKIN, A. L., KEYNES, R. D. & SHAW, T. I. (1964). The rate of formation and turnover of phosphorus compounds in squid giant axons. J. Physiol. 171, 119-131. CALDWELL, P. C. & LowE, A. G. (1970). The influx of orthophosphate into squid giant axons. J. Phy8iol. 207, 271-280. CAUSEY, G. & HARRRIs, E. J. (1951). The uptake and loss of phosphate by frog muscle. Biochem. J. 49, 176-183. CHMOULIOVSKY, M. & STRAUB, R. W. (1974). Increase in ATP by reversal of the Na-K-pump in mammalian non-myelinated nerve fibres. Pfluiger8 Arch. gem. Physiol. 350, 309-320. EVANS, D. H. & MURRAY, J. G. (1954). Histological and functional studies on the fibre composition of the vagus nerve of the rabbit. J. Anat. 88, 320-337. GARRAHAN, P. J. & GLYNN, J. M. (1967). The incorporation of inorganic phosphate into adenosine triphosphate by reversal of the sodium pump. J. Physiol. 192, 237-256. GREENGARD, P. & STRAUB, R. W. (1959). Effect of frequency of electrical stimulation on the concentration of intermediary metabolites in mammalian non-myelinated nerve fibres. J. Physiol. 148, 353-361. HURLBUT, W. P. (1970). Ion movements in nerve. In Membranes and Ion Transport, ed. BITTAR, E. E., pp. 95-143. London: Wiley-Interscience. KEYNES, R. D. & RITCHIE, J. M. (1965). The movements of labelled ions in mammalian non-myelinated nerve fibres. J. Physiol. 179, 333-367. LANDOLT, H. & B6RNSTEIN, R. (1969). Landolt-Bdrnstein Zahlenwerte und Funktionen aus Physik - Chemie - Astronomie - Geophysik und Technik, 6th edn., ed. BORCHERS, H., HAUSEN, H., HELLWEGE, K. H., SCHAFFER, K. & SCHMIDT, E., Band 2, Pt. 5a, pp. 619, 623. Berlin: Springer. MULINs, L. J. (1954). Phosphate exchange in nerve. J. cell. comp. Physiol. 44, 77-86. RANDERATH, E. & RANDERATH, H. (1964). Resolution of complex nucleotide mixture by two dimensional anion exchange thin-layer chromatography. J. Chromat. 16, 126-129. RITCHIE, J. M. (1967). The oxygen consumption of mammalian non-myelinated nerve fibres at rest and during activity. J. Physiol. 188, 309-329. TASAKI, I., TEORELL, T. & SPYRopouLos, C. S. (1961). Movement of radioactive tracers across squid axon membrane. Am. J. Physiol. 200, 11-22.
J. Physiol. (1975), 247, pp. 759-771 With 6 text-figurea Printed in Great Britain
UPTAKE OF ORTHOPHOSPHATE BY RABBIT VAGUS NERVE FIBRES
By BEATRICE ANNER, J. FERRERO, P. JIROUNEK AND R. W. STRAUB From the Departement de Pharmacologie, Ecole de Mgdecine, CH-1211 Geneve 4, Switzerland
(Received 9 October 1974) SUMMARY
1. The uptake of orthophosphate and its incorporation into ATP, ADP, and creatine phosphate (CrP) were studied in desheathed rabbit vagus nerve. 2. Using 32p labelled orthophosphate, the total amount of labelled phosphate taken up by the preparation was continuously recorded in a perfusion apparatus. For measuring the incorporation into phosphorylated compounds, phosphate esters and inorganic phosphate were extracted, separated and their total amount and radioactivity determined. 3. The total uptake of phosphate was found to be a biexponential function of time. 4. The time constant of the first process was 10-20 min and independent of the extracellular phosphate concentration, the final amount labelled by this process was relatively small and proportional to external phosphate, increasing from 0-026 m-mole/kg wet nerve at 0-04 mm phosphate to 114 m-mole/kg at 5 mm. 5. The time constant of the second process depended on the extracellular phosphate concentration varying from 4624 min at 0*04 mm to 210 min at 5 mm. The final amount labelled by this process was 5-6 m-mole/kg wet wt. and independent of the extracellular phosphate. 6. The kinetics of the slow uptake were consistent with the presence of a saturable process and a non-saturable one. 7. Extraction of ATP, ADP, and the sum of CrP and Pi, showed that the total amount of these compounds remained constant for 2 hr while their radioactivity increased slowly, approximately at same rate as the slow fraction. 8. Increasing the external phosphate from 0 04 to 5 mm increased the amount of labelled ATP. 9. A comparison with the metabolic turnover of phosphate, estimated
760 760
~BEATRICE ANNER AND OTHERS
from the oxygen consumption, shows that uptake is much slower than metabolism, so that the slow appearance of labelled nucleotides is very probably due to a limitation of the influx. 10. From the experimental data the influx can then be calculated for various phosphate concentrations. It is close to that found in squid axons. INTRODUCTION
Although the importance of phosphates for metabolism and structure of cells has been recognized for many years, relatively little is known on the way by which these ions are taken up by nerves. Studies on phosphate uptake in nerve have so far been confined to giant axons (Caldwell & Lowe, 1970) and frog nerves (Mullins, 1954; Abood & Koyama, 1963; Abood, 1968); to our knowledge (see also Hurlbut, 1970) the uptake of phosphates in mammalian nerve has not been investigated. Studies on phosphate transport are complicated by the presence of several ion species, and by the fact that phosphate, unlike the rather inert ions studied in detail, is incorporated into a number of intracellular biochemical compounds. Furthermore, labelled phosphate added to the extracellular medium seems to exchange with a phosphate fraction of the plasma membrane (Causey & Harris, 1951). In order to overcome, to some extent, these difficulties, the present experiments are based on the use of two independent methods: (i) a method that allows the continuous recording of the total uptake of radiophosphate in a perfused preparation, which has the advantage that the rate of uptake can be measured accurately under different conditions and at any time of the experiment; and (ii) methods of measuring the amounts of inorganic phosphate (Pi) and phosphorylated compounds, extracted from the preparation, and their respective radioactivities. These methods allow the study of the incorporation of radiophosphate into compounds supposedly located inside the cells, and thus give an estimation of the phosphate influx. As pointed out by Caldwell. & Lowe (1970) this technique of measuring the influx is applicable only if the rate of influx is much smaller than the rate of incorporation. This condition seems to be satisfied, as will be explained later. The experiments showed that a small amount of added radiophosphate rapidly exchanged with a fraction of loosely bound phosphate and that the bulk of the uptake was attributable to influx into the cells, transported, to a large extent, by a saturable system that will be described in more detail in a following paper. A preliminary account of some of the results has been published elsewhere (Anner, Ferrero, Jirounek & Straub, 1973a, b).
PHOSPHATE INFLUX IN C-FIBRES
761
METHODS
Preparation of nerves, apparatus for recording radioactivity. Rabbits weighing between 2-5 and 3 kg were shot and their cervical vagus nerves rapidly removed and desheathed with scissors. The preparations were then incubated for at least 1 hr in phosphate-Locke of the same phosphate concentration that was later used in the experiment. Afterwards they were mounted in the apparatus shown in Fig. 1, where they lay in a polyethylene tube that was continuously perfused. After starting with label-free phosphate-Locke, orthophosphate-labelled solutions were applied and the total radioactivity of the preparation and the surrounding medium was recorded over a length of 5 cm. The whole recording system had a time constant of approximately 0-5 sec. The solutions flowed at a rate of about 1 ml./min and could be changed by turning a tap. Their flow was maintained by a perfusion pump. Experiments in which the effect of changing the solution was recorded without nerve showed that the exchange of the solution was complete
Desheathed vagus
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Fig. 1. Apparatus for recording total radioactivity of preparation and surrounding medium. Diagram shows desheathed cervical vagus nerve mounted in a polyethylene tube of 0-86mm inside diameter, which is continuously perfused with Locke or modified Locke; tube is placed opposite to window of beta-counter, connected to rate meter and penrecorder.
762
BEATRICE ANNER AND OTHERS
within 15 sec. The efficacy of the counting was measured by perfusing the tube with a solution of known radioactivity and without nerve. Unless otherwise stated, the temperature was 370 C. Chromatographie separation of orthophosphate, nucleotides, and creatine phosphate. For the studies on the localization of the labelled phosphate taken up by the nerves, the preparations were washed with ice-cold phosphate and label-free Locke during 5 min and then removed from the apparatus. A given length, weighing about 10 mg and corresponding to the part that lay under the counter, was cut off, and used for the analysis. In a first series of experiments the radioactive compounds extracted from the nerve were separated by thin-layer chromatography. For this, the tissue was immersed into 1 ml. trichloroacetic acid (TCA) (5 % w/v), homogenized and centrifuged at 3000 g, all these operations being done at a temperature of about 4° C. The radioactivity of 0-1 ml. of the supernatant and of the pellet was then determined. The pellet was rejected: after 40 min incubation in radioactive Locke, it contained less than 1 % of the total radioactivity. Next, the TCA of the supernatant was extracted with ether, and the remaining solution was evaporated at low pressure. The residue was dissolved in a solution of ethylenediaminetetraethylammonium (0-002M), and used for bi-dimensional thin-layer chromatography, as described by Randerath & Randerath (1964). Autoradiography showed at most 4 spots; they could be identified as ATP, ADP, guanosine 5'-triphosphate (GTP), and a spot where both orthophosphate and creatine phosphate (CrP) were found. Small amounts of these compounds were therefore added before chromatography, and the spots, revealed by ultraviolet light and autoradiography, removed, and counted in a liquid scintillation counter. The sum of all counts so obtained was compared to the number of counts found in the nerve extract and the results corrected, if the difference was not larger than 20 %. If more than 20 % of the initial radioactivity was lost during chromatography, the experiment was rejected (for details of the technique, see Anner, 1973). In other experiments, the compounds were separated by column chromatography, as described by Garrahan & Glynn (1967). This method had the advantage that the total amount of the compounds as well as their radioactivity could be determined. After removal from the apparatus the tissue was plunged into 2 ml. of boiling triethanolamine buffer at pH 8-2, rapidly cooled after 40 sec and then homogenized (see GCreengard & Straub, 1959). The homogenate was deproteinized with chloroform (1 ml.) and centrifuged for 30 min at 3000 g and 40 C. The supernatant was then passed through a Dowex 1 column, elated, and the effluent collected. Twelve fractions, allowing the separation into ATP, ADP, and the sum of CrP and Pi were taken; GTP was in the ATP fractions. The concentrations of ATP and ADP were measured spectrophotometrically with a Unicam Ultraviolet Spectrophotometer; CrP was hydrolysed and determined with the inorganic phosphate, with the method described by Anner & Moosmayer (1975). In the same fractions, the radioactivity was also counted. Weight. The nerves were weighed after desheathing, and after removal from the apparatus. All concentrations given in the text are based on the initial weight. Solutions. The composition of Locke was (in mM): NaCl, 154; KC1, 5-6; CaCl2, 0-9; MgCl4, 0-5; Tris, 10-0; glucose, 5-0. Phosphate-Locke contained in addition Na2HPO4 and NaH2PO4; the phosphate concentrations indicated in the text refer to the total concentration of orthophosphate (referred to as phosphate or Pi). The pH of all solutions was adjusted with HOl to exactly 7-40; it was stable for several days. The labelled solutions contained approximately 3 #C82P/mi. added from a radio-
763 active stock solution of [32P]Na2HPO4 and [32P]NaH2PO4 in isotonic NaCi at pH 7, which was obtained from the Eidgendssisches Institut fur Reaktorforschung, Wu~renlingen, Switzerland. The radioactivity of the stock solution had a concentration of about 3 inc/mi., and the specific activity of the 32P amounted to 2500-3500 inc/rn-mole. All other salts were analytical grade. PHOSPHATE INFLUX IN C-FIBRES76
[32P]Phosphate Locke
Phosphate Locke
1100 counts/sec
1
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Fig. 2. Uptake of phosphate from 0-2 mm [32P]phosphate-Locke. Record of radioactivity of preparation and surrounding medium shows filling of tube and extracellular space after application of [32P]phosphate-Locke (fast upstroke), followed by uptake of phosphate by the preparation (slow increase in radioactivity). Application of inactive phosphate Locke shows loss of radioactivity from tube and extra-cellular space (fast downstroke), followed by slower loss from the preparation. 100 counts/sec correspond to uptake of 120 #tmole orthophosphate/kg nerve wet wt.; temp. 370 C. RESULTS
Total uptake When perfusion with labelled Locke containing 0-2 mm phosphate was started, the records showed a rapid increase in radioactivity during the first few minutes (Fig. 2). This initial upstroke corresponds probably to the filling of the tube and of the extracellular space. The tube fills within about 15 sec (see Methods) and the rate of filling of the extracellular space can be estimated from experiments of Keynes & Ritchie (1965), who found, with the sucrose-gap technique and similar flow conditions, that after changing from Locke to potassium sulphate-Locke, the
BEATRICE ANNER AND OTHERS 764 potassium ions filled the periaxonal space along an exponential curve with a time constant of 0x8 min at 200 C. From the diffusion coefficients of K2SO4, Na2HPO4, and NaH2PO4, and their temperature dependence (see Landolt & B6rnstein, 1969), it can be estimated that the diffusion of radiophosphate shows about the same time course. Therefore, in our experiments, the radioactive solution should have invested the extracellular space within a few minutes after its application. 10
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9
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Fig. 3. Semilogarithmic plot of difference between final amount of 32p uptake and that found at any given time during experiment. Curve obtained (0) can be decomposed into two exponentials (straight lines); the corresponding final fractions are found by extrapolation to zero time. Note that extracellular phosphate was 3 mM and fast fraction relatively large in this case.
Afterwards the radioactivity increased much more slowly and reached nearly saturation after about 24 hr. By plotting the difference between the final radioactivity and that found at any given time, two approximately exponential processes could be distinguished (see Fig. 3). The first, relatively fast process had a time constant of 10 min and reached an equilibrium corresponding to the filling of a space containing 0-06 mmole/kg wet wt., at 0-2 mm extracellular phosphate. The second process was much slower and had a time constant of 1530 min, and the corresponding space contained 5-2 m-mole/kg wet wt. When experiments like that shown in Fig. 3 were repeated with different
765 PHOSPHATE INFLUX IN C-FIBRES phosphate concentrations, it was found that the time constant of the first fraction was nearly independent of the extracellular Pi (Table 1), but that the amount of phosphate that could be labelled increased with increasing extracellular phosphate concentrations. This fraction therefore shows characteristics of a non-saturable compartment, that is being filled by a diffusion process. It may tentatively be termed 'fast' fraction. TABLE 1. Final amounts exchanged in fast and slow fractions and corresponding time constants of exchange at different extracellular phosphate concentrations
Slow fraction
Fast fraction Final amount exchanged
External Pi concn. (mm) 0 04 0.1 0*2 0*3 04 0.6 1.0 2*0 3.0
5*0 Mean
Time
Final amount exchanged
(m-mole/kg wet wt.) 0X026 (3)
constant
(min) 16*2 (2)
(m-mole/kg wet wt.) 5.5 (1)
0.063 (6) 0*049 (1) 0'078 (1) 0*142 (3)
10.2 (6)
5.2 (1)
0.55 (3) 0.73 (4) 1*14 (1)
16.9 (1) 17.6 (2)
5.8 (2)
13.3
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13.5 (1)
Time constant
(min) 4624 (3) 3080 (1) 1530 (7) 1380 (1) 1220 (1) 672 (3) 490 (2) 470 (2) 341 (5) 210 (2)
Values of Table were obtained from plots like that shown in Fig. 3. In the experiments where the total final amount was not measured, the time constants and the amount in the fast fraction were obtained by using the mean final amount of the slow fraction and the observation that both processes are exponential. In parentheses, number of experiments.
In the following, the second, slow process will be studied in more detail. Experiments with different extracellular phosphate concentrations showed that the time constant of the filling process depends on the extracellular Pi (Table 1), while the amount that is finally labelled is remarkably constant. At first sight, this behaviour is compatible with the filling of a constant space through a saturable process, and it was suggested in a preliminary communication (Anner et al. 1973a) that the uptake process could be described by simple saturation kinetics of the Michaelis-Menten type with an apparent Km of about 1 mm. Studying the relation between the rate of the slow uptake and the extracellular phosphate concentration over a wider range of concentrations (Fig. 4), it is now evident that the kinetics of the uptake are more complicated than initially thought.
766 766
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Fig. 4. Rate of phosphate influx at different extracellular phosphate concentrations. Rate of influx was calculated from slow uptake of Table 1; curve relating it to extracellular phosphate concentration does not show simple saturation kinetics. Bars indicate s.E.
Labelling of nudleotide8 In extracts of nerves exposed to labelled phosphate and then briefly washed, labelled ATP, ADP, GTP, CrP and orthophosphate were found. Table 2 and Fig. 5 give results of experiments in which, after incubation in 0-2 mm phosphate Locke for various times, the total amount of these compounds were measured and their activity counted. The experiments showed that the total amounts did not appreciably change during incubation. The specific activities of the extracted compounds increased slowly: only 10% of the extracted ADP, CrP and Pi had exchanged with extracellular phosphate after 2 hr, and 20 % of the ATP, counting 1 label per molecule. This behaviour could be due either to a slow influx or to a slow metabolic turnover of the nucleotides. The rate of turnover can be estimated from the oxygen consumption (see Discussion), and it is then evident that the slow appearance of labelled compounds is due to a limitation in the uptake process. The time course of the increase in specific activity parallels the slow uptake of phosphate, and since the synthesis of ATP is almost certainly intracellular, it can be assumed that the slow uptake corresponds to the influx of phosphate into the axons and the Schwann cells.
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Fig. 6. Uptake of phosphate and incorporation into nucleotides. Preparations exposed to Locke with different phosphate concentrations during 40 min, and extracted after a brief wash ip phosphate and label-free Locke. Temp. during exposure to phosphate Locke 370 C. CrP + Pi ( x ); ATP (A); ADP (AL); GTP (E).
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769 PH~r-OSPHATE INFLUX IN C-FIBRES76 In another series of experiments the phosphate concentration was varied and the nerves extracted after 40 min incubation. The results, illustrated in Fig. 6, show that the amount of labelled nucleotides found in the nerve parallels the total amount of labelled Pi taken up by the slow process at different extracellular concentrations (cf. Fig. 4). This finding is consistent with the hypothesis that the slow uptake is responsible for the rate of labelling of ATP. DISCUSSION
Faet uptake Our experiments showed that radiophosphate rapidly exchanges with the phosphate of a 'superficial' fraction, and that at equilibrium, the amount of phosphate exchanged is nearly proportional to the extracellular phosphate concentration. This suggests that the phosphate of this fraction is not strongly bound and that a large number of free sites is present at low phosphate concentration. It is not clear where the fraction is located. Binding to myelin of the rather numerous myelinated fibres of the cervical vagus can almost certainly be excluded, since the fraction is also found in the thoracical vagus (unpublished observations), which in the rabbit contains only a few myelinated fibres (Evans & Murray, 1954). The fast fraction resembles a phosphate fraction observed by Causey & Harris (195 1) in the membrane of frog striated muscle. They suggested that it may be due to exchange with phosphoproteins. Slow uptake Turnover and influx. It is interesting to compare the uptake of phosphate with its metabolic turnover, which can be estimated from the oxygen consumption. The experiments of Ritchie (1967) in rabbit vagus suggest a metabolic rate of at least 0-36 in-mole Pi/kg wet wt. per minute at 370 C. In our experiments, the Pi extracted :from the nerve amounts to 9-2 in-mole/kg wet wt. (Table 2), s0 that the mean time constant of metabolic turnover becomes 9-5 min, or, if only 1 label per molecule is counted, 5-5 min. Other phosphorylated compounds than those found in our experiments are also present, but their amount is so small (Greengard & Straub, 1959) that they can be neglected. Metabolic turnover is thus not very different from the fast uptake, but much faster than the slow uptake. While the rate of fast uptake could be explained by the rate of metabolic turnover, the quantities that are taken up by the fast process are much too small to explain the amount of labelled compounds that appear in the nerve. For instance, at 0-2 mm extracellular phosphate the fast fraction does not exceed 0-06 in-mole/kg wet wt., while the Pi of
BEATRICE ANNER AND OTHERS 770 ATP exchanged after 60 min when the 'fast' uptake is complete, amounts already to 0-145 m-mole/kg wet wt. The rapid metabolic turnover and the finding that the rate of labelling is slow (Fig. 5) and parallels the slow uptake, suggest that labelling is limited by uptake. Further, since the main production of ATP is almost certainly due to reactions inside the cells, the slow uptake appears to correspond to the influx of phosphate. The situation in rabbit vagus is thus similar to that in squid axons, where the influx of phosphate is also much slower than the metabolic turnover (cf. Tasaki, Teorell & Spyropoulos, 1961; Caldwell, Hodgkin, Keynes & Shaw, 1964; Caldwell & Lowe, 1970). Influx. The influx of phosphate can then be calculated from the rate constant of slow uptake and the quantity that finally exchanges with radiophosphate (Table 1). At 0-2 mm extracellular phosphate the influx becomes 3-4 /imole/kg wet wt. per minute, and with an axon surface of 0-6 x104 cm2/g wet wt. (Keynes & Ritchie, 1965) the influx into the axons would amount to 9-4 f-mole/cm2 per second. In our experiments, phosphate may also be taken up by Schwann cells. Their surface membrane is approximately twice as large as the surface of the axons, so that the mean transmembranal flux may be around 3 f-mole/cm2 per second. At any rate, it is not very different from the flux in squid giant axons, where at 0.1 mm external phosphate, Caldwell & Lowe (1970) measured an influx of 20-9 f-mole/cm2 per second between 17 and 210C. Mechanism of influx. The experiments showed that the influx contains a large saturable component, but that it cannot be described by simple kinetics of the Michaelis-Menten type. A more detailed study of this process in various conditions will be given in a following paper. Total amount of exchangeable phosphate. At equilibrium, the quantity of phosphate in the slow fraction that exchanged with radiophosphate was almost independent of the extracellular phosphate concentration. This behaviour resembles that found in striated muscle (Causey & Harris, 1951). It is understandable if the intracellular concentration of free orthophosphate is small compared with that of the phosphorylated compounds, the total quantity of which may be limited by the availability of creatine and adenosine. Similarly, when the utilization of ATP is decreased by ouabain, the concentration of ATP is almost unchanged (Chmouliovsky & Straub, 1974). The total phosphate extracted after short incubations was at 0-2 mm external phosphate 9-2 m-mole/kg wet wt. (Table 2), which is definitely larger than the total amount of phosphate that exchanges during long term experiments, and which amounts, for the sum of both fractions to 6 m-mole/kg. Loss of compounds, able to retain phosphate, may occur during these long perfusions.
PHOSPHATE INFLUX IN C-FIBRES
771
We are grateful to Dr P. Kalix for advice in the use of the method of thin-layer chromatography, to Mrs M. Moosmayer for technical assistance throughout the experiments and to the SNSF for a grant, Number 3.0890.73. REFERENCES
Ai3OOD, L. G. (1968). Interrelationships between phosphates and calcium in bioelectric phenomena. Int. Rev. Neurobiol. 9, 223-261. ABOOD, L. G. & KOYAMA, I. (1963). Phosphate incorporation in desheathed nerves: effects of potassium and calcium ions. Science, N.Y. 141, 1277-1278. ANNER, B. (1973). Localisation, repartition et transport de l'ion phosphate dans un tissu nerveux. These No. 1609, Faculte des Sciences, Universite de Geneve. ANNER, B., FERRERO, J., JIROUNEK, P. & STRAUB, R. W. (1973a). Inhibition of intracellular orthophosphate uptake in rabbit vagus nerve by Na withdrawal and low temperature. J. Physiol. 232, 47-48P. ANNER, B., FERRERO, J., JIROUNEK, P. & STRAUB, R. W. (1973b). Na-dependent phosphate influx into mammalian nerve fibres. Experientia 29, 740. ANNER, B. & MOOSMAYER, M. (1975). Rapid determination of inorganic phosphate in biological systems by a highly sensitive photometric method. Analyt. Biochem. (in the Press). CALDWELL, P. C., HODGKIN, A. L., KEYNES, R. D. & SHAW, T. I. (1964). The rate of formation and turnover of phosphorus compounds in squid giant axons. J. Physiol. 171, 119-131. CALDWELL, P. C. & LowE, A. G. (1970). The influx of orthophosphate into squid giant axons. J. Phy8iol. 207, 271-280. CAUSEY, G. & HARRRIs, E. J. (1951). The uptake and loss of phosphate by frog muscle. Biochem. J. 49, 176-183. CHMOULIOVSKY, M. & STRAUB, R. W. (1974). Increase in ATP by reversal of the Na-K-pump in mammalian non-myelinated nerve fibres. Pfluiger8 Arch. gem. Physiol. 350, 309-320. EVANS, D. H. & MURRAY, J. G. (1954). Histological and functional studies on the fibre composition of the vagus nerve of the rabbit. J. Anat. 88, 320-337. GARRAHAN, P. J. & GLYNN, J. M. (1967). The incorporation of inorganic phosphate into adenosine triphosphate by reversal of the sodium pump. J. Physiol. 192, 237-256. GREENGARD, P. & STRAUB, R. W. (1959). Effect of frequency of electrical stimulation on the concentration of intermediary metabolites in mammalian non-myelinated nerve fibres. J. Physiol. 148, 353-361. HURLBUT, W. P. (1970). Ion movements in nerve. In Membranes and Ion Transport, ed. BITTAR, E. E., pp. 95-143. London: Wiley-Interscience. KEYNES, R. D. & RITCHIE, J. M. (1965). The movements of labelled ions in mammalian non-myelinated nerve fibres. J. Physiol. 179, 333-367. LANDOLT, H. & B6RNSTEIN, R. (1969). Landolt-Bdrnstein Zahlenwerte und Funktionen aus Physik - Chemie - Astronomie - Geophysik und Technik, 6th edn., ed. BORCHERS, H., HAUSEN, H., HELLWEGE, K. H., SCHAFFER, K. & SCHMIDT, E., Band 2, Pt. 5a, pp. 619, 623. Berlin: Springer. MULINs, L. J. (1954). Phosphate exchange in nerve. J. cell. comp. Physiol. 44, 77-86. RANDERATH, E. & RANDERATH, H. (1964). Resolution of complex nucleotide mixture by two dimensional anion exchange thin-layer chromatography. J. Chromat. 16, 126-129. RITCHIE, J. M. (1967). The oxygen consumption of mammalian non-myelinated nerve fibres at rest and during activity. J. Physiol. 188, 309-329. TASAKI, I., TEORELL, T. & SPYRopouLos, C. S. (1961). Movement of radioactive tracers across squid axon membrane. Am. J. Physiol. 200, 11-22.
