Carnitine transport by rat kidney cortex slices: Stimulation by dibutyryl cyclic AMP

Life Sciences, Vol . 23, pp . 715-722 Printed in the U.S .A .

Pergamoa Press

CARNITINE TRANSPORT BY RAT KIDNEY CORTE% SLICES : STIMULATION BY DIBUTYRYL CYCLIC AMP + Peter J . Huth, James H. Thomaea and Austin L . Shug* Metabolic Research Laboratory, William S . Middleton Memorial Veterans Administration Hospital 2500 Overlook Terrace, Madison, Wisconsin 53705 (Received in final form June 19, 1978) Summary The transport of caraitine by rat kidney cortex slices against a concentration gradient has been demonstrated . Similarities to other transport systems included a linear period of uptake, as well as indications of saturability of the system with increasing concentrations of substrate . The transport of caraitine was inhibited by anoxia, and carbonyl cyaaide~chloropheaylhydroxazone (CCC1P), an uncoupler of ozidative phosphorylation . Caraitine uptake was stimulated approximately 50X when kidney alicea were treated with dibutyryl cAt~ . A transport system specific for the cellular accumulation of caraitine (3-hydroay-4-N-trimethylaminobutyrate) has been auggeated by previous studies (1,2) . Recently, caraitine transport has been demonstrated in both isolated liver cells and human heart cells in culture (3,4) . In liver cells, caraitine transport appears to be an active process (3) . IIrinary excretion and reabsorptioa mechanisms for caraitine have not been studied in detail, although creatinine clearance and the glomerular filtration rates have been shown to exceed caraitine clearance in rats and dogs (1,5) . Furthermore, it can be calculated from the daily loss of caraitine in urine (80-175 uméles/24 hr) that more than 95x of the ultrafiltrated caraitine is normally reabsorbed (6) . It has also been suggested that caraitine might be reabsorbed across the renal tubule to about the same extent as amino acids. Since normal serum caraitine levels are maintained at 40-50 umol/1 in humans (7), the observation of large reductions is serum caraitine is chronic hemodialysie patients after dialysis further auggeata caraitine reabaorption by the kidney (7,8) . In this paper we present evidence for mediated and possible active transport of caraitine into rat kidney cortes slices . In addition, data are presented which show stimulated cellular accumulation of caraitine by dibutyryl cAMP, and suggest that caraitine transport is rat kidney may be under hormonal control . Address correspondence to this author .

0300-9653/78/0821-0715$02 .00/0 Copyright (c) 1978 Pergamon Preen

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Materials and Methods Male rate of the Holtzman strain weighing 200-250 g were used in all experiments . They were given water and commercial rat chow (Wayne Lablox) _ad libitum and housed in individual stainless steel cages with wire mesh bottoms in a room with approximately a 14- and 10-hour natural light-dark cycle. The animals were routinely sacrificed in the afternoon . The radioactive compounds used were D,Ir[ 3H-CHg]l~arnitine, specific activity 2 .1 Ci/umole (Ameraham Corp .) and [carboxyl- C] inulin, specific activity 1 .95 uCi/g (New England Nuclear) . Other chemicals were obtained as follows : dibutyryl cAMP, Sigma Chemical Corp ; and Carbonyl cyanide-mchlorophenylhydrozone, Calbiochem . Preparation and Incubation of Slices The animals were killed by decapitation after which the kidneys were quickly removed and bisected transversely . The capsule was stripped off and cortical slices (about 0 .4 mm) were made with a Stadie-Riggs microtome (9) . Groups of three slices (100-150 mg) were preincubated in 25 ml Erlenmeyer flasks containing 3 ml of carnitine-free Kreba-Ringer bicarbonate buffer pH 7 .4 for 15 min in a shaking water bath at 37°C . Slices were then transferred and incubated in an identical aliquot containing 5.5 mM glucose, 80 umol/R-carnitine and 0.05 uCi D, L[ HCH~]-carnitine (9) . The incubation medium was continually aerated with 0 .CO2 (95 :5X) . At the end of the incubation period, slices were removed aâd dapped twice in isotonic saline to rinse off surface radioactivity, blotted, weighed and placed in test tubes containing 2 ml water for 5 min at 100° C. Radioactivity Measurements Radioactivity was measured by counting 0.2 ml portions of the tissue supernatant or the remaining incubation medium in a Packard Liqu~d Scintillation Spectrometer with a counting efficiency of 32-34X for H in this system . Calculation and Expression of Results Disant ./min per ml of intracellular fluid were calculated from measurements of radioactivity in incubation medium and tissue, total tissue water and extracellular space (9) . Total tissue water was deter mined as the difference in weight before and after drying tissue slices at 80 °C to l~ constant weight . Extracellular space was estimated using [carboxyl- C] inulin (10) . The transport of carnitine was expressed as the distribution ratio (D .R .) : D.R . a DPM per ml intracellular fluid DPM per ml medium In some cases the uptake of carnitine was expressed as nmoles per ml intracellular fluid :

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Caraitiae Transport is Ridaey Slices

71 7

nmoles carnitine per ml intracellular fluid ~ DPM per ml intracellular fluid DPM per nmole carnitine All results are expressed as + S .D . unless otherwise stated . Chromatogr~c Procedures Thin-layer chromatography was used to characterize the radioactive compound present is kidney slice extracts after incubation with D,Ir[ HCH ]-carnitine for 30 min. After kidney'slices were incubated and The slices were then rinsed they were immediately frozen in liquid N2 . eztracted 3 tines with 1.0 ml of 5X trichloroacetic acid and the ineoluable residue was removed by centrifugation . By this method no radioactivity was observed in the final wash . The acid was roved from the supernatant fraction by. three extractions in 2 .5 volumes of diethylether . The aqueous fraction was evaporated to dryness under vacuum and rediesolved is 60 pl of ethanol and water (3 :1v/v) . Aliquots of the concentrated extract containing 20,000-40,000 D.P .M . were spotted on silica gel-G thin-layer plates (Brialmtan Instruments, Inc .) along with nonradioactive L-carnitine and acetylcarnitine as carriers . Ascending chromatography was performed on the plates using a solvent system consisting of : methanol, chlroform, water, concentrated-ammonia and concentrated-formic acid, 55 :50:10:7 .5 :2 .5/(v/v/v/v/v) (11) . The lanes to which radioactivity was applied were cut into 0.5 cm sections . Each section was placed in a scintillation vial and eluted with 0.5 ml of water, covered with 12 ml of Brays solution and counted . In every case, the radioactive extract migrated se a single major peak . The remaining portion of the plate containing nonradioactive carnitine and acetylcarnitine standards was exposed to iodine vapors and the relative mobilities were calculated from the visualized spots . Long-chain acylcarnitines were measured as the butanol extractible radioactivity from the remaining tissue slice pellet previously washed with 5% TCA. Results and Discussion Sffect of Time It is generally believed that a distribution ratio of (DPM per ml ICF/DPM per ml ECF) greater than 1.0 represents uptake is eacees of simple diffusion (9) . Fig . lA shows the uptake of carnitine by kidney slices as a function of incubation time, with a distribution ratio of 5 .50 +0 .88 observed after 60 min incubation . These results sugeest accumulation of caraitine a¢ainat a concentration eradient . Additional exveriments were undertaken to rule out other possibilities for these observations including : non-specific binding and carnitine exterification . Various tissue treatments were examined for non-specific carnitine binding. The results indicated that the radioactivity retained by acid-treated (2 min, lOX TCA), heat-treated ( 2 min 100°C), or freeze-thawed kidney slices was as in significant portion of the total radioactivity retained by nontreated kidney slices . This indicated that nonspecifically bound radioactive carnitine was not a significant factor for the caraitine accumulation observed in control slices .

718

Carnitine Transport in Ridaey Slices

0 0 ss

Vol . 23, No . 7, 1978

A

a G Q' KF 2 W É ~4 z

z I

ar

~ 3~-

V

2

V

JI J I

dd

TIME (MIN)

100

ô ~ B00 a w 600 u a

F Z

É 400

w 200

F 2 Q U

200 400 600 900 CARNITINE (nmales/ml EXTRACELLULAR FLUID)

1000

FIG . 1 Uptake of D,Ir[ 3H-CH ]-carnitine by rat kidney cortex slices . (A) Uptake as a fuac~ion of lima showing (upper) the concentration gradient achieved at 37°C and (lower) at 4°C. Incubation conditions same as described is teat . Bare represent _+ S .E .M . (B) Uptake as a function of extracellular Ir~carnitine concentration . Five minute incubations at 37°C . Incubation conditions same as described in text . Each data point (A and B) represents 3-11 experiments . No correction was made for esterification of carnitine .

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Caraitine does not appear to be metabolized under normal conditions (12) . In addition, free carnitiae is found in greater amounts is the kidney than Intracellular aeterification of carnitiae acyl carnitiae eaters (13) . is accomplished by a family of enzymes known ae carnitiae acyltraasferaees (14) . These enzymes, which display activity toward short-, medium- and long-chain acyl CoA's, are found is the membranes of mitochondria, nicrosomes, and peroaisomea (15) . To determine whether the incorporated carnitiae was terified, we examined the chromatographic properties of the acid soluble -co~pouad present in kidney slices incubated for 30 min in the presence of D,L-[ H-CH ]-carnitiae while the acid insoluble long-chain carnitiae astern were determined se the butanol eatractible radioactivity from the previously acid washed tissue slice pellet . Table I summarizes the results obtained from thin-layer chromatographic ~rofiles of the acid-soluble radioactive kidney extract, comeercial D,L-[ H-CH3 ]-carnitiae, and nonradioactive carnitiae and acetylcarnitine standards. The radioactive extract migrated ae a single major peak and had the same relative mobility se the radioactive and nonradioactive free carnitiae standards . In addition, approximately 2 .4x + 0.3 ; n~3, of the total tissue radioactivity was found as acid insoluble long-chain carnitiae esters . These results suggest that only a minor portion of the total carnitiae accumulated during the incubation period was estarified . TABLE I Characterization of radioactive compound preseat~ in kidney cortex slice acid eatracta after incubation with D,L-[ H-CH3 ]-carnitiae. Compound Applied

Rf values

Radioactive Kidney extract

0.25

Commercial D,L-[ 3H-CH3]-carnitiae

C.25

Carnitine

0 .26

Acetylcarnitina

0.36

All values are the mean of three experiments . Tissue slice incubation conditions, extraction procedures and chromatographic system were as described in Materials and Methods .

Sffect of Initial Substrate

Concentretton

Kidney slices were incubated for 5 min with varying concentrations of Lcarnitiae (Fig . 1B) . Tha uptake of carnitiae appeared to be a saturable protean, since a linear increase in the coacentratioa of carnitiae in the intracellular fluid was not maintained with increasing amounts of substrate . This is consistent with the hypothesis that carnitiae accumulation involves a catalytic process. Similar indications of saturability have been found is numerous other mediated transport systems in vitro . A Liaeweaver-Burk plot was prepared from the data, and the R for the transport of carnitiae was calculated to ba 0.3 mM. This value is considered only an approximation, because it was obtained in a tissur slice preparation and because the specificity of the transport system for carnitiae has not been established.

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Carnitine Transport in Kidney Slices

yol,

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TABLE II Treatment

Distribution Ratio

Control

5 .58 ± 0 .67

Anoxia

0 .89 ± 0 .08*

CcciP (1 umol/k)

3 .51 ± 0 .03*

CCC1P (2 umol/R)

1 .10 ± 0 .13*

* Significantly different from control, p < 0 .001 . All values are expressed as mean ± S.D . ; n = 3-9 . Incubation medium and conditions as in Fig. 1 . No correction was made for esterification of carnitine. 8ffect of Anoxia, CCC1P and Temperature To determine the possible energy requirements for carnitine accumulation, kidney slices were incubated under anoxic conditions (N2 :C02, 95 :55) for 60 min . Table II shows that accumulation of carnitine against a gradient ceases and approaches values compatible with simple diffusion (D .R . 0 .89 t 0 .08) . A similar degree of inhibition was observed with carbonyl cyanide-m-chlorophenylhydrazone (CCC1P), an uncoupler of oxidative phoaporylation (2 x 10-6 M), while partial inhibition was observed at a weaker concentration (1 x 10'6 M) . The Fig. effect of temperature reduction on carnitine uptake was also tested . lA shows that reduction of the incubation temperature from 37 ° C to 4°C is accompanied by a marked reduction in D,L-[3H-CHg]-carnitine uptake and the final distribution ratio . These data suggest the existence of an active transport system for carnitine accumulation by rat kidney slices . 8ffect of Dibvtyryl cA1~ Hormonal stimulation of membrane transport systems involving dibutyryl cht~ as a second messenger has been observed in liver and kidney cortex slices after treatment for prolonged periods (16,17) . When rat kidney cortex slices were treated similarly with 0 .1 mM dibutyryl CAMP for a total of 165 min ., a significant increase in the cellular accumulation of carnitine was observed (D .R . 10 .72 ± 0.77) (p < 0 .001) . In addition, the apparent stimulation of carnitine uptake by dibutyryl cAi~ follows a characteristic dose response curve (Fig . 2) . Preliminary experiments with glucagon and epinephrine have failed to show that they act as primary mediators of the effect observed with These results are in contrast to those in which glucagon has dibutyryl cAMP . been shown to increase carnitine uptake in isolated liver cells (18) .

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Carnitine Transport is Kidney Slices

72 1

0 d: Q W U Q F2 É w W Z H 2 Q u i u

u G~

O

FIG. 2 Concentration gradients is kidney slices incubated with graded levels of dibutyryl cAMP . Slices were preincubated for 105 min in a carnitine-free medium followed by 60 min incubation with 80 umol/B-caraitine plus the radioactive label ; all preparations included dibutyryl cAMP . Asterisks indicate a significant difference from control, p < 0.001 ; n = 3-9.

Conclusion The present study has demonstrated the accumulation of carnitine by rat kidâey cortex slices against a concentration gradient of free carnitine . Substrate saturation by carnitine and dependence upon oxidative phoaphorylation and temperature have also been shown . These observations fulfill the major criteria for the existence of a mediated transport system sad support earlier is vivo studies 1, 13) which suggest active transport of carnitiae by the kidney . The physiological significance of increased carnitine uptake in kidney slices by dibutyryl cAMP is unclear, but suggests the possibility of hormonal induction. These results indicate that the transport of L-carnitine by the kidney may be crucial for both carnitine conservation by the animal and the maintenance of normal kidney cell levels .

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Ref erences 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .

K .T . YDE and I .B . FRITZ, Amen . J . Physiol . _202 122-128 (1962) . W .R . MARRSBERRY, M .P . MCQUILLEN, P .G . PROCOPIS, A .R . AARRISON, and A .G . ENGEL, Arch . Néurol . 31 320-324 (1974) . R .Z . C~ISTIANSEN and J . BREMER, Bioclüm . Biophys . Acta 448 562-577 (1976) . T . BOAR, K . EIKLID, and J . JONSEN, Biochim . Bio_phys . Acta , 462 627-633 (1977) . C . CEDERBLAD and S . LINDSTEDT, Arch . Biochem . Bioptiys . _173 173-180 (1976) . C . CEDERBLAD and S . LINDSTEDT, Clin . Chin . Acta . _33 117(1971) . T . BOHMER, A . RYDNING, and A .E . SOLBERG, Clin . Chin . Acta 57 55-61 (1974) . T . BOHMER, A . BERGREIS, and R . EIKLID, Lencet 1 126-128 (1978) . L .E . ROSENBERG, A . BLAIR, and S . SEGAL, Biochim . Biophys . Acta _54 479-499 (1961) . L .E . ROSENBERG, S .J . DOWNING, and S . SEGAL, Amer . J . Physiol . 202 800-804 (1962) . S .A . GDMPEN, and R .R . NORUM, Biochim . Biophys . Acta 316 48-55 (1973) . D .E . BROOKS, and J .E . MCINTOSH, Biochem . J . _148 439-445 (1975) . T . BOHMER, K .R . NORDM, and J . BRBMFR, Biochim . Biophys . Acta 125 244-251 (1966) . .A. E NEWSAOLMS and C . START, Regulation in Metabolism, pp . 203, 298-300, John Wiley and Soas, New York, 1974 . M .A. MARRWELL, N .E . TOLBERT, and L .L . BIEBER, Arch . Biochem . Biophys . 176 479-488 (1976) . J . TSWS, N .A . WOODCOCK, and A .E . HARPER, J . Biol . Chem . 245 .R 3026-3032 (1970) . I .W . WEISS, R . MORGAN, and J .M . PRANG, J . Biol . Chem . 247 760-764 (1972) . R . CARISTIANSEN, Biothun. Bio phys_Açta 488 249-262 (1977) .

+ A preliminary report of this work was presented in Fed . Proc . 36 3 :692 (1977) .