Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 297, 213 - 217 (1977)

Archivesof Pharmacology 9 by Springer-Verlag 1977

Studies on Sodium Ion Retention and Antidiuretic Effects after Administration of L-Tryptophan to Rats E. REUTER, H.-J. WEBER, and H. H E R K E N PharmakoIogisches Institut der Freien Universitfit Berlin, Thielallee 69/73, D-1000 Berlin 33

Summary. Under certain conditions the i.p. injection of L-tryptophan leads to a reduction of elimination of Na +. At the same time, the fractional Na+-reab sorption increases. No increase in the absolute tubular sodium transport rate was observed since the significantly reduced plasma-sodium concentration leads to a decreased sodium load. The most probable cause of a decreased plasma-sodium concentration seems to be a retention of sodium-free water under the chosen conditions of infusion. The water retention is compatible with the antidiuretic effect of 5-hydroxytryptamine.

Key words."L-Tryptophan - 5-Hydroxytryptamine Electrolyte and water balance - Water intoxication Antidiuresis.

INTRODUCTION Decreased elimination of sodium ions in unanaesthetized rats can be produced by L-tryptophan injection (Herken and Weber, 1971). The authors deduced from their results that the sodium retention is based on an increased reabsorption. However, apart from an increased rate of transport other factors can also lead to a decreased elimination of sodium. In the present study the alterations of water and electrolyte balances after an injection of L-tryptophan have been analysed in more detail by the use of clearance experiments.

an equilibration period of at least 4 h, L-tryptophan (50 rag/100 g body weight), L-tryptophanethyl ester HC1 (60 or 30 mg/100 g body weight), L-phenylalanine (50 mg/100 g body weight), D-tryptophan (50 mg/100g body weight) or 6-aminonicotinamide (5 mg/100 g body weight) with L4ryptophan (50 mg/100 g body weight) were injected intraperitoneally. In I experiment rats were adrenalectomized 4 days prior to the assay. For clearance experiments a carotid catheter was introduced into rats (about 220 g body weight) under hexobarbital anaesthesia 3 days before the assay. The rats were kept in individual cages with normal food (Altromin | and tap water. The evening before the day of the assay, a catheter was inserted into the bladder and an infusion was made as described above. Eight hours before injection, an initial dose of 10 mg inulin/100 g body weight was given and the infusion rate was calibrated to 83 gl/min, The solution contained 1 ~ inulin. L-Tryptophanethyl ester HC1 (30 mg/100 g body weight) was injected in neutral solution. For the determination of osmolality, inulin and electrolytes in the plasma, 150 gl blood samptes were taken from carotid artery every hour at the beginning and at the end of each clearance period. Urine was collected over 1 h. The respective analyses were carried out with a semi-micro osmometer (Knauer) and with an atomic absorption spectrometer (Type 1200, Varian Techtron). Inulin was determined according to the method of Ffihr et al. (1955). All experiments included the appropriate controls, the rats receiving identical volumes of solution without the test substance. Analysis of the parameters in the time course of controls and experimental groups by variance analysis revealed significant changes within the experimental groups at a level of P = 0.01, but not in the controls. At identical times, differences between experimental group and controls were analysed by Student's t-test. Given are the mean _+ S.E.M. P-Values of less than 0.01 are indicated by asterisks in the figures.

RESULTS

Excretion Experiments METHODS Male Wistar rats ( 1 4 0 - 170 g body weight) were used in excretion experiments according to the method described by Herken et al. (1964). The rats received a solution containing 34 mM NaC1 and 265 mM D-glucose at a rate of 85 gl/min through the tail vein. After Send o fJprint requests to." H. Herken at the above address

When the fluid balance of rats is followed over a period of 8 h, volume retention is seen immediately after L-tryptophan injection (Fig. 1). In the following 3 - 5 h the fall in sodium excretion rate is a typical phenomenon. The sodium retention calculated over a 7-h period amounted to 409 ~Eq in the experimental group compared with - 7 0 gEq in the controls.

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The differences in s o d i u m excretion rate, however, were small in contrast to the differences observed when the water-soluble derivative L-tryptophanethyl ester HC1 was injected (Fig.2). As the conditions for rea b s o r p t i o n seem to be m o r e favourable, a very strong effect was obtained with a dose equivalent to 46 m g L-tryptophan/100 g b o d y weight. U n d e r this condition sodium retention was 742 g E q over 7 h which a m o u n t s to 63 ~o o f the sodium intake. Even m o r e

Fig. 2 Time course of volume and sodium excretion rates after injection of 60 m g L-tryptophanethyl ester HC1/I00 g body weight. Controls n = 4, experimental group n = 4. Symbols as in Figure 1

than with L-tryptophan, the injection with this derivative caused a stronger fall in volume excretion rate. Since these p h e n o m e n a could be based on several mechanisms, clearance experiments with unanaesthetized rats were carried out for m o r e detailed information. A typical experiment with the highly effective dose o f 30 m g L-tryptophanethyl ester HC1//00 g b o d y weight is shown in Figure 3. The glomerular filtration rate d r o p p e d in t h e / s t h. T h e significant fall in urine

E. Reuter et al. : Tryptophan Induced Antidiuresis

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Fig.3. Clearance data of water and sodium in controls (n = 4) and 4 rats treated with 30 mg L-tryptophanethyl ester HCI/100 g body weight i.p. R = cumulative retention of sodium-free solution (see Results). Symbols as in Figure 1

formation lasted 2 h and paralleled the negative free water clearance during this time. Since only 1/5 o f the infusion solution was isotonic NaC1, the greater part of the retained volume was consequently sodium-free glucose solution. Cumulative calculation R = ( V I - INs" VUPN=)- ( V - UN, V/PNa)t demonstrates that sodium-free solution up to 4 ml/100 g body weight could be retained. Plasma sodium concentration decreased due to this retention which resulted in a lower sodium filtration rate in the 5th h compared with the controls. In the 1st h after injection the sodium filtration rate was obviously influenced by the decrease of G F R . Although the fractional sodium reabsorption increased in the experimental group, the absolute amounts of sodium transport rates did not. The drastic decrease o f sodium elimination therefore correlated with the decreased sodium filtration rate 9

[//" = infusion rate, Its== sodium concentration in infusion solution 1

due to the diminished plasma sodium concentration. Theoretically the decreased plasma sodium concentration may be due to : 1. a migration of sodium from the plasma into other compartments, 2. the dilution of the plasma with sodium-free isotonic fluid. The retention of sodium-free solution during the infusion with low-sodium glucose solution favours the latter possibility while not excluding the former. The data compiled in Table 1 show that a close relationship exists between the volume retention under the chosen conditions of infusion and a consecutive sodium retention. Both in normal and in adrenalectomized rats the volume excretion was reduced in the I st h following injection of L-tryptophan or its ester derivative. A decrease of the sodium excretion rate followed which was evident in the 3rd h. In contrast, L-phenylalanine and o - t r y p t o p h a n failed to produce either of these two effects. The antimetabolite 6-aminonicotinamide antagonized the effect of g-tryptophan in so far as the decreased sodium

216

Naunyn-Schmiedeberg's Arch. Pharmacol. 297 (1977)

Table 1. Excretion volume and sodium excretion 1 and 3 h after injection respectively. L- and D-tryptophan, L-tryptophanethyl ester HC1 and phenylalanine were given during infusion with 83 gl/min of an isoosmolar solution containing glucose and 34 mEq/l sodium V l h p. inj. (~tl/min) contr,

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(n = 5) h-Tryptophan i.p. (50 rag/100 g b.wt.) (n = 4)

107 • 8 P < 0.01

65 • 4

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L-Tryptophanethyl ester HC1 i.p. (30 rag/100 g b.wt.) (n = 4)

80 • 4 P < 0.005

38 _+ 9

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L-Tryptophan i.p. (70 rag/100 g b.wt.), infusion of 0.9 ~ NaC1 (n = 5)

97 _+ 8 62 _+ 8 0.05 > P > 0,025

13.7 + 0.8 n.s.

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excretion was masked by a relative sodium diuresis, the ant• effect was not influenced. If L-tryptophan was injected into rats which were infused with isotonic saline solution, a slight volume retention occurred in the 1st h. As no dilution of the plasma sodium was possible, the sodium load and the sodium excretion remained unchanged.

DISCUSSION In the liver, 1 ~o of L-tryptophan is catabolised via the tryptophan-pyrrolase (EC 1.13.1.12) to metabolites, part of which are known to be inhibitors of gluconeogenesis (Sourkes, 1971). Quinolinic acid which specifically inhibits the phosphoenolpyruvate carboxykinase (PEPCK) (EC 4.1.1.32) (Foster et al., 1966) is a potent inhibitor of the renal gluconeogenesis (Endou et al., 1975). At the time when, after application of L-tryptophan the activity of the renal PEPCK is markedly inhibited (not shown here) and the changes in the carbohydrate metabolism of the kidney are most pronounced (Weber et al., 1973), a decrease in sodium excretion ,could be identified in excretion experiments. The sod• retention could be confirmed in the present clearance experiments if a 0.2 ~ NaC1 solution was used for infusion. It was supposed that the change in sodium excretion was due to an influence on the tubule

n.s.

cell metabolism, caused by an inhibitor of gluconeogenesis derived from tryptophan catabolism (Herken and Weber, 1971). In experiments with quinolinic acid, however, we could not produce a sodium retention, only a potassium diuresis, In experiments on isolated per• rat kidney no influence of tryptophan or quinolinic acid on the sodium excretion could be shown (Ross, personal communication, 1976). Also, no sodium retention could be demonstrated in vivo, when isotonic saline solution was used. These findings comply with the observation that the absolute quantity of transported sodium does not change, thus no additional energy supply is necessary. Friedriehs and Schoner (1974) showed that an inhibition of the renal sodium transport can probably influence the tubular gluconeogenesis in vitro. On the other hand it does not seem probable that the renal gluconeogenesis, which only needs about 3 ~ of the total renal consumption of ATP (Cohen and BaracNieto, 1973), can exert a decisive influence on the regulation of the tubular sodium transport. The diminished sodium excretion can however, easily be understood when the water balance is also taken into account. If about 4 ~o of the body weight of sodium-free fluid is added to the total body fluid, the total sodium is diluted by 5.7 ~ i.e. a decrease of the plasma sodium concentration by about 8 mEq/1. With constant G F R this leads to a diminution of the

E. Reuter et al. : Tryptophan Induced Antidiuresis

filtrated sodium by 5.__7~. If, under these conditions, the kidneys reabsorb the same quantity of sodium as do control animals, the percentage reabsorption rate must be increased and the sodium excretion must be decreased without additional work having been performed. These relationships could be shown in the present clearance experiments. The question remains as to what causes the volume retention and the limitation of the clearance of free water directly following the injection of >-tryptophan. 5-Hydroxytryptamine is said to be able to affect the kidney in three ways: it exerts an antidiuretic effect either directly or via a stimulation of the neurohypophysis (review see Garattini and Valzelli, 1965); it causes a vasoconstriction of the glomerular vascular bed (Erspamer and Ottolenghi, 1953); furthermore, it is said to cause a sodium retention by release of aldosterone from the adrenal cortex in the dog (Park et al., 1968). The diminished GFR found particularly after administration of t-tryptophan ethylester as well as the diminution of the free-water clearance correlate with the effects found with 5-hydroxytryptamine. The mechanism involving aldosterone seems unlikely in this condition since decreased sodium excretion is also evident in adrenalectomized rats. The stimulation of aldosterone release or antidiuresis with 5-hydroxytryptamine is possibly dependent on the species since in dogs, either only a slight volume retention (Park et al., 1968) or no antidiuresis (Bhargava et al., 1972) was found. Studies by Koe and Weissman (1966) showed that a maximal brain level of 5-hydroxytryptamineis attained within I h after injection of t-tryptophan, thus the antidiuresis can most probably be attributed to the effect of the metabolite of >-tryptophan. Antidiuresis is not followed by a diminution in sodium excretion during infusion of isotonic saline. The decrease in sodium excretion only becomes evident during infusion of 0.2 ~ NaC1. So we can conclude that this effect is based on a dilution hyponatremia.

217

REFERENCES Bhargava, K.B., Kulshrestha, V.K., Srivastava, Y.P.: Central cholinergic and adrenergic mechanisms in the release of antidiuretic hormone. Brit. J. PharmacoI. 44, 617-627 (1972) Cohen, J. J., Barac-Nieto, M. : Renal metabolism of substrates in relation to renal function. In: Handbook of physiology, Vol. 8, Renal physiology (J. Orloff, R. W. Berliner, and S. R. Geiger, eds.), pp. 909-927. Washington, D.C.: Amer. Physiological Society 1973 Endou, H., Reuter, E., Weber, H. J. : Inhibition of gluconeogenesis in rat renal cortex slices by metabolites of L-tryptophan in vitro. Naunyn-Schmiedeberg's Arch. Pharmacol. 287, 297 - 308 (1975) Erspamer, V., Ottolenghi, A.: Pharmacological studies on enteramine, action of enteramine on diuresis and renal circulation of rat, Arch. int. Pharmacodyn. 93, 177-201 (1953) Foster, D. O., Ray, P. D., Lardy, H. A.: A paradoxical in vivo effect of L-tryptophan on the phosphoenolpyruvate carboxykinase of rat liver. Biochemistry 5, 563-569 (1966) Friedrichs, D., Schoner, W. : Stimulation of renal gluconeogenesis by inhibition of the sodium pump. Biochem. biophys. Acta (Amst.) 304, 142-160 (1973) Ffihr, J., Kaczmarczyk, J., Krfittgen, C. D.: Eine einfache colorimetrische Methode zur Inulinbestimmung ffir Nieren-Clearance-Untersuchungen bei Stoffwechselgesunden und Diabetikern. Klin. Wschr. 33, 729-730 (1955) Garattini, S., Valzelli, L.: Serotonin, pp. 137-147. Amsterdam: Elsevier Publ. Co. 1965 Herken, H., Weber, H. J. : L-Tryptophan-induced increase of renal sodium reabsorption. Naunyn-Schmiedebergs Arch. Pharmak. 271, 206-210 (1971) Herken, H., Senti, G., Zemisch, B. : Die Einschr~inkung des tubuIfiren Natrium- und Kaliumtransportes durch Biosynthese 6-Aminonicotinsgureamid enthaltender Nucleotide. NaunynSchmiedebergs Arch. exp. Path. Pharmak. 249, 5 4 - 7 0 (1964) Koe, B. K., Weissman, A. : p-Chlorophenylalanine : A specific depletor of brain serotonin. J. Pharmacol. exp. Ther. 154, 499 - 516 (1966) Park, C. S., Chu, C. S., Park, Y. S., Hong, S. K.: Effect of 5-hydroxytryptamine on renal function of the anaesthetized dog. Amer. J. Physiol. 214, 384-388 (1968) Sourkes, T. L. : Effects of amino acid derivatives and drugs on the metabolism of tryptophan. Amer. J. clin. Nutr. 24, 815-820 (1971) Weber, H.-J., Reuter, E., Endou, H. : Inhibition of gluconeogenesis in the rat kidney after application of sodium retaining doses of L-tryptophan. Naunyn-Schmiedeberg's Arch. Pharmacol. 279, R 9 (1973)

Acknowledgment. We thank Miss Ursula Brandt for excellent technical assistance.

Received November 1, 1976/Accepted January 3, 1977