Metabolism and Excretion of Exogenous Adenosine 3’S ... · significant effects on the handling of cyclic AMP by the kidney. Thus, inulin was routinely employed as a measure of the

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 251, No. 16. Issue of Agust 25, pp. 4958-4967, 1976

Printed in U.S.A

Metabolism and Excretion of Exogenous Adenosine 3’S’-Monophosphate and Guanosine 3’S’-Monophosphate

STUDIES IN THE ISOLATED PERFUSED RAT KIDNEY AND IN THE INTACT RAT*

(Received for publication, December 26, 1975, and in revised form, April 8, 1976)

RICHARD COULSON

From the Department of Pharmacology and Veterans Administration Hospital, Upstate Medical Center, Syracuse, New York 13210

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3’:5’-monophosphate (cyclic AMP) or guanosine 3’:5’-monophosphate (cyclic GMP) at an initial concentration of 0.1 IIIM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate.

The metabolism of the cyclic nucleotides was studied by including ‘C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [l’C]AMP was metabolized to renal [“Cladenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [“C]uric acid. Similarly, 20% of cyclic[“C]GMP was metabolized to renal [14C ]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [“C]uric acid. Urine contained principally unchanged “C-labeled cyclic nucleotide.

Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold.

The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 pmol of either ‘C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for “C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [l*C]AMP and cyclic [l’C]GMP lost from the plasma, respectively. The “C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [“C lallantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged “C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [“Cluric acid was not a significant metabolite of the “C-labeled cyclic nucleotides in these in uiuo experiments.

It has long been considered that the cell membrane is poorly permeable to exogenous cyclic nucleotides (2). Recent evidence from human in uiuo experiments suggests that the cyclic nucleotides, adenosine 3’:5’-monophosphate and guanosine 3’:5’-monophosphate are very permeant in some tissues (3). Studies in the intact rat and dog have demonstrated that the kidney and, to a slightly lesser extent, the liver are the principal tissues involved in the removal of plasma cyclic AMP’ (4, 5). Studies with isolated perfused rat tissues have shown that the kidney (6) and the liver (7) are rapidly penetrated by exogenous cyclic AMP. In the perfused rat kidney it was concluded that the majority of the cyclic AMP removed from the perfusate was metabolized, since no signifi-

* This work was supported by United States Public Health Service Grant ZROl-AM 14401-04 and by a grant-in-aid from the Heart Association of Upstate New York. A part of these studies has been reported in an abstract (1).

‘The abbreviations used are: cyclic AMP or CAMP, adenosine 3’:5’-monophosphate; cyclic GMP or cGMP, guanosine 3’:5’-monophosphate.

cant intracellular accumulation of cyclic AMP was apparent and the urinary excretion of cyclic AMP accounted for only a small fraction of its total renal clearance (6). A similar conclusion could be made for the perfused rat liver (7). Cyclic GMP appears to share the ability of cyclic AMP to readily penetrate renal and hepatic tissues (5, 7) and is presumably also metabolized once it gains access to the cell.

In order to interpret the effects obtained with exogenous cyclic AMP and cyclic GMP in renal tissue, it is essential to understand the nature and distribution of the compounds produced from the metabolism of these cyclic nucleotides. Currently the metabolic fate of cyclic AMP and cyclic GMP in renal tissue is not known.

This communication describes and compares the handling and metabolic products of exogenous cyclic AMP and cyclic GMP in the isolated perfused rat kidney and the intact rat.

EXPERIMENTAL PROCEDURES

Materials-Unless otherwise indicated all nucleotides, nucleosides, purine bases, and enzymes were purchased from Boehringer Mann-

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Renal Metabolism of Exogenous Cyclic Nucleotides 4959

h&n. Adenine, guanine, hypoxanthine, xanthine, xanthosine, and XMP were purchased from Sigma Chemical Co. Uricase was from Worthington Biochemical Corp. Cyclic [8-“C]AMP (49.2 mCi/mmol), 3H,0 (5 mCi/ml), succinyl cyclic AMP [‘zLI]iodotyrosine methyl ester and its antibody were obtained from Schwarz/Mann. Cyclic [8-“C]GMP (60 mCi/mmol), [“Clurea (54.4 mCi/mmol), and Phase Combining System were supplied by Amersham/Searle. [“C]Toluene (0.2 &i/g) was a product of Packard Instrument Co. Inc. Bovine serum albumin Fraction V was from Armour Pharmaceutical Co. Sephadex G-10, DEAE-Sephadex A-25, and blue dextran 2000 were from Pharmacia Fine Chemicals Inc. [“ClAllantoin was prepared by treating [2-“Cluric acid (56.7 mCi/mmol, Amersham/Searle) with uricase (8).

Kidney Perfusion-Nonfasted male Charles River rats (350 to 450 g body weight) were used throughout these studies. Perfusion medium was prepared and kidneys were perfused by techniques previously described (9, 10). Unless indicated otherwise, kidneys were recirculated with 50 ml of perfusion medium consisting of Krebs-Henseleit bicarbonate buffer and 5 g of Fraction V bovine serum albumin per 100 ml. The antibiotics penicillin G and streptomycin, routinely employed in the perfusion medium in this laboratory, were omitted from these studies to prevent possible interference with nucleotide transport and metabolism. Other chemicals, when used, were present in the perfusion medium from the beginning of the perfusion and included inulin (400 &ml), cyclic AMP (0.1 mM) and cyclic GMP (0.1 mM). In some experiments additional cyclic AMP or cyclic GMP (2 or 5 mM, respectively) was infused directly into the perfusate oxygenation chamber of the perfusion apparatus at a rate sufficient to maintain the perfusate concentration at about 0.1 mM. Cyclic nucleotides labeled with “C were used to study metabolite formation. Previous experience’ with SH-labeled cyclic AMP injected intravenously into rats revealed a significant production of aH,O, making this a poor choice of label for studying metabolite formation. Cyclic [8-‘C]AMP and cyclic [8-“C]GMP were purified before use on columns (15 x 0.6 cm) of Dowex 5OW-X8 eluted with 0.1 M HCl. Kidneys were perfused with 0.1 mM (i.e. 5 pmol and 2 to 3 pCi) cyclic nucleotide. Urine samples were collected directly into preweighed tubes containing 1 ml of 1.7 M perchloric acid. Perfusate samples, taken at the beginning and end of all perfusions and at various times throughout the perfusion, were immediately acidified with an equal volume of I.7 M perchloric acid. At the end of the experiment, the kidneys, while still being perfused, were frozen between aluminum blocks cooled in Dry Ice (11).

Intact Rat St&&-Rats were anesthetized with pentobarbitol, the right carotid artery and left jugular vein were cannulated with PE 50 tubing (Clay Adams) and the bladder was catheterized with PE 90 tubing (Clay Adams). After an intravenous priming dose of 3 ml of 280 mM mannitol, 3.3 rmol and 2 to 4 FCi of “C-labeled cyclic nucleotide in 0.9 ml of 150 mM saline (0.9% NaCl solution) were rapidly injected into the vein and washed in with a further 1 ml of the saline. The amount of cyclic nucleotide was chosen to approximate 0.1 to 0.2 mM in the plasma assuming complete mixing. The mannitol solution was infused at 0.2 ml/min for the duration (60 min) of the experiment. Mannitol diuresis was maintained since normal urine flow was too low for an accurate estimate of urinary contents. Two consecutive 30-min urine collections were made into preweighed tubes containing 1 ml of 1.7 M perchloric acid. Likewise, approximately 1 ml of arterial blood was taken at 30 and 60 min after the isotope injection and added to similar preweighed tubes. Blood samples (0.2 ml) were also taken at intervals and centrifuged in heparin-treated tubes to obtain plasma. At the end of the experiment, 60 min after the isotope injection, one kidney and one liver lobe were rapidly excised and frozen in acetone/ Dry Ice for the subsequent analysis of ‘C-metabolites. The second kidney was excised and dissected into cortex and papillary section of the inner medulla before freezing in acetone/Dry Ice. A second liver lobe and other tissues were also removed and frozen in acetone/Dry Ice. The radioactive content of tissues, urine, and plasma was determined by techniques previously described (4). Radioactivity was counted by liquid scintillation in Phase Combining System (Amersham/Searle) at an efficiency of 85% for “C. Corrections were made for quenching by adding [“Cltoluene to the scintillation vial and recounting.

Analyses-The analysis of cyclic AMP by a modified protein binding assay has been described (6). Cyclic GMP was measured by the method of Steiner et al. (12) which was modified in that the antibody, succinyl cyclic GMP [‘*“I]iodotyrosine methyl ester complex, was

*R. Coulson, unpublished observation.

added to Millipore filter (0.45 pm) and washed twice with 4 ml of 50 rnM sodium acetate/l5 rnM sodium azide (pH 6.2) at 0” in order to remove the excess unbound ‘2”I-labeled derivative. Perchloric acid extracts of perfusate and urine were titrated to pH 4 to 6 with 10 M KOH and assayed for cyclic nucleotide without further purification.

Inulin was measured by the method of Heyrovsky (13) in perchloric acid extracts of perfusate and urine. During the course of these studies creatinine present in the perfusate at 13 mM was found to have significant effects on the handling of cyclic AMP by the kidney. Thus, inulin was routinely employed as a measure of the glomerular filtration rate in these studies.

For the study of “C-metabolites in kidney and liver, the powdered frozen tissues were extracted into 2 volumes of 1.7 M perchloric acid at 0”, the 4000 x g (20 min) pellet was re-extracted with 0.8 volume of 1.7 M perchloric acid, and the two supernatants were pooled. The perchloric acid-insoluble pellet (containing RNA and DNA) contained less than 4% per kidney or liver of the administered radioactivity. The nature of the radioactivity in this pellet was not further studied. “C-labeled cyclic nucleotides and their “C metabolites were identified in perchloric acid-soluble extracts by separation on columns of Sephadex G-10 and DEAE-Sephadex A-25. Purine bases and purine nucleosides were separated on columns (120 x 1.0 or 120 x 1.2 cm) of Sephadex G-10, eluted with 50 rnM sodium phosphate/l.5 rnM sodium azide (pH 7.0) as described by Sweetman and Nyhan (14). Values for the void volume (VJ and the void volume plus internal volume (V, +

zooo-

F‘ IOOO:

< E 500- c

g '0 Y :: z 100 I " e G 50-

z

! 5

IO-

r

cGMP _

‘Oh7?%------ -J 15 20 25 30

MlflUteS of PerfusIon

FIG. 1. Handling of exogenous cyclic AMP and cyclic GMP by the isolated perfused rat kidney. Kidneys were perfused for 30 min with 50 ml of perfusate containing an initial 0.1 mM cyclic nucleotide. In the curves designated infused CAMP and injused cCMP cyclic nucleotide was infused to maintain a more steady concentration. Upper panel, change in perfusate cyclic nucleotide concentration (log scale) as a function of time. Lower panel, ratio of cyclic nucleotide excreted/cyclic nucleotide filtered (log scale), as a function of time. Glomerular filtration was estimated as the inulin clearance. Each curue represents the mean of 2 to 4 perfused kidneys. In this and subsequent figures the vertical bars indicate * S.E. of the mean except where n = 2 (infused cCMP) when these bars connect the pairs of data points (i.e. indicate the range).


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4960 Renal Metabolism of Exogenous Cyclic Nucleotides

TABLE I

Excretion and metabolism of exogenous cyclic nucleotides by isolated perfused rat kidney

Kidneys were recirculated for 30 min with 50 ml of perfusion fluid containing initially 5 am01 of cyclic nucleotide. See the legend to Fig. 1 with reference to “infused CAMP” and “infused cGMP” for further information. Results are expressed as mean i: S.E. where n = 3 or as individual values where R = 2.

nucleotide

Cyclic AMP 1 3

Cyclic GHP I 2

Total Infuseda cyclic nucleotide cyclic

in perfusate nucleotide

starte end (12.5 min) (27.5 min)

nmol

3640 6560f 10400

2353 +_269

' 4010 4090 9940 3880 4000 9940

Disposition of cyclic nucleotide

filteredb excreted secreted= metabolized d excreted

filtered

863

?I53

480 610

nmol / 15 min / kidney

i580f 7llf 5880 1.8f +246 ?I02 ~326 20.1

3690 3210 6170 3810 3200 6010 6'::

a Total quantity infused into the perfusate wer 15 min. b Glomerular filtration rate estimated from the clearance of inulin. ' Secreted cyclic nucleotide calculated from the difference between filtered and excreted. d Calculated as total loss from perfusate minus total appearing in urine. Intracellular accumulation of cyclic nucleotide

is presumed not to irt?uence this calculation since the perfusate concentration and hence, intracellular space were approximating a steady state.

T Total cyclic nucleotide present in a perfusate volume of 50 ml. Significantly different from values obtained with cyclic GHP, P<O.Ol.

V,) could be obtained from the elution volumes of blue dextran 2000 and acetone, respectively. Typical partition coefficients, K,, (where Kd = (V, - V,)lV,, V, = 41.6 ml, V, = 31.2 ml, V, is the elution volume (in milliliters) of the standard), for some standard compounds on Sepha- dex G-10 were as follows: GTP, 0.20; ATP, 0.24; ADP, 0.38; IMP, 0.38; XMP, 0.42; GMP, 0.67; AMP, 0.81; SH,O, 1.25; [“Clurea, 1.33; inosine, 1.68; [“C]allantoin, 1.72; xanthosine, 2.51; hypoxanthine, 2.64; cyclic GMP, 3.27; cyclic AMP, 3.51; guanosine, 4.20; adenosine, 4.28; xanthine, 4.48; uric acid, 5.03; guanine, 6.10; adenine, 8.19. Allantoin and inosine were not well separated on the G-10 column. When significant amounts of a “C-metabolite appeared in the inosine/allantoin eluate, it was further fractionated by incubating 3 ml of this eluate at room temperature with 0.1 rmol of inosine, 0.4 nmol of EDTA, 1 mg of xanthine oxidase (EC 1.2.3.2), and 20 ag of nucleoside phosphorylase (EC 2.4.2.1) (15). The production of uric acid was followed spectrophotometrically at 293 nm and, upon completion of the reaction, the solution was treated with 100 al of 12 M perchloric acid, titrated to pH 7 with 10 M KOH, and rechromatographed on Sephadex G-10. Similarly, the possibility of [‘Cluric acid being a metabolite was investigated by treating 2 ml of the standard uric acid eluate with 1 ml of 70 mM glycine buffer (pH 9.5), 0.2 amol uric acid and 20 ag uricase (EC 1.7.3.3) at room temperature until the reaction, monitored at 293 nm, was completed (8). The mixture was deproteinized, neutralized, and rechromatographed on Sephadex G-10. Purine 5’.nucleotides were separated on columns (30 x 0.9 cm) of DEAE-Sephadex A-25, eluted with a linear sodium chloride gradient in 50 rnM tris(hydroxymethyl)- aminomethane (pH 8.3) as has been described (16). Good separation of ATP, ADP, AMP, IMP, GTP, GDP, and GMP was achieved over the gradient range 0.1 to 0.5 osmol/kg. Recoveries of “C were consistently in excess of 95% for both G-10 and DEAE columns.

Tissue ATP, ADP, and AMP were measured by standard enzymatic techniques (17, 18). Uric acid was measured in the perchloric acid-soluble extracts of kidney, perfusate, blood, and urine using uricase (8).

Presentation of Results-The results for the “C-metabolite formation are expressed as a percentage of the total administered radioactivity (counts per min). In experiments with the perfused rat kidney, the total renal content of “C was estimated from the total weight of frozen tissue obtained at the end of the perfusion. In the in oioo experiments, the total liver, kidney, and blood contents of “C were estimated from the organ/body weight ratios for bled rats (19) and assuming a plasma volume of 4.5 ml/l00 g of body weight or a whole blood volume of 7.5 ml/100 g of body weight.

Unless indicated otherwise, results are expressed as the mean l S.E. The statistical significance of results was determined with Student’s t test.

RESULTS

Handling of Cyclic AMP and Cyclic GMP by Isolated Perfused Rat Kidney-Cyclic AMP or cyclic GMP, each present in the perfusate at an initial concentration of 0.1 mM and total content of 5 pmol, were rapidly removed from the perfusate (Fig. 1, upper panel). A net transtubular secretion of both cyclic AMP and cyclic GMP was apparent from the greater amount of cyclic nucleotide excreted than filtered (Fig. 1, lower panel).3 Moreover, cyclic GMP was more extensively secreted than was cyclic AMP. In order to obtain more accurate and comparable data for the net secretion and metabolism of these cyclic nucleotides by the perfused rat kidney, cyclic nucleotide was infused into the perfusion medium to maintain the perfusate concentration at about 0.1 mM throughout the perfusion. The results of such experiments are shown in Fig. 1 and Table I. Cyclic GMP was metabolized at about the same rate as cyclic AMP (about 400 nmol/min/kidney) but was excreted at more than 2-fold and secreted at more than 4-fold the corresponding rates for cyclic AMP (Table I). The higher rate of net transtubular secretion of cyclic GMP compared to cyclic AMP (about 200 uersus 50 nmol/min/kidney, respectively) probably accounts for the more rapid disappearance of cyclic GMP from the perfusate.

Production and Lhtributian of “C-Metabolites of Exoge- nous Cyclic [“CjAMP in Isolated Perfused Rat Kidney-Fig. 2 shows the time course for the removal of cyclic [IclAMP from, and the appearance of ‘C-metabolites in the perfusate and the simultaneous accumulation of cyclic [“C]AMP and “C- metabolites in the urine. [“C]Uric acid was the principal metabolite found extrarenally, and it accumulated more rapidly in the perfusate than in the urine. Table II shows the

’ Secretion is the cyclic nucleotide excreted in excess of that amount filtered by the kidney. Thus a ratio greater than 1.0 for cyclic nucleotide excreted/cyclic nucleotide filtered indicates net secretion. In Fig. 1 (lower panel), this ratio was consistently lower when infused cyclic nucleotide curves were compared to the respective noninfused cyclic nucleotide curves.


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PERFUSATE

IO 20 30 40 50 60

18 -

14.

IO-

6-

2 30

2

8 26

22 /

2 ii-

OO IO 20 30 40 50 60 Minutes 0, PerfusIon

FIG. 2. Time course for the distribution of exogenous cyclic [“C]AMP and its “C-metabolites in the per&ate (upper panel) and urine (lower panel) of the isolated perfused rat kidney. Kidneys were perfused with 5 pm01 of cyclic [8- I‘C]AMP (6.6 x IO8 cpm) in 50 ml of perfusate. Urine was recirculated with the perfusate for the first 10 min of perfusion and, thereafter, five consecutive lo-min urine collections were made. Urine and perfusate samples were chromatographed on Sephadex G-10 as described under “Experimental Procedures.” O-0, the sum of radioactivity found in xanthine (X), hypoxanthine (H), inosine (rH), and/or allantoin. Inosine and allantoin were not separated on the Sephadex G-10. Values represent means for two kidneys and vertical bars indicate the ranges between pairs of data points.

distribution of cyclic [“C ]AMP and “C-metabolites after 60 min of perfusion. Total radioactivity was equally distributed among kidney, final perfusate, and urine (10 to 60 min). The renal content of “C was found almost entirely as adenine nucleotides with [“C]ATP accounting for the majority. The final perfusate contained mostly [“C]uric acid, whereas the total radioactivity in urine (10 to 60 min) consisted mainly of unchanged cyclic [“C]AMP and [“Cluric acid.

The incorporation of 20% of the total added radioactivity into renal ATP (Table II) was equivalent to 1 pmol of the adenine moiety from the cyclic AMP being incorporated into the total ATP pool of the rat kidney (normally -2 rmol, see below). Because of this large incorporation, the effect of adding 5 pmol of cyclic AMP to the perfusate was studied on the renal ATP, ADP, and AMP contents. The results (Table III) demonstrate that the addition of 5 rmol of cyclic AMP to the perfusate was sufficient to raise the renal ATP and ADP contents about 2-fold over control values but to have no effect on the AMP content. When urine was collected in these studies about 1 pmol of the perfusate cyclic AMP was lost in the urine (Table 11) and the renal ATP and ADP pools were increased

but to a lesser extent (Table III). From the data in Table III and from the dry weight of the perfused kidneys (0.39 * 0.01 g for 10 kidneys), it could be estimated that the addition of 5 lmol of cyclic AMP to the perfusate increased the total renal ATP content by 1.9 pmol and ADP content by 0.5 Fmol. When urine was collected throughout the perfusion the increases were 1.0 and 0.2 pmol for renal ATP and ADP, respectively. The latter increases are in close agreement with the amount of cyclic AMP incorporated into renal ATP and ADP after perfusion with 5 rmol of cyclic [“C]AMP (Table II). This suggests that the adenine moiety of the cyclic nucleotide was utilized in the synthesis of extra ATP and ADP in addition to being incorporated into the pre-existing ATP, ADP and AMP pools by their normal turnover. The extent of the latter incorporation was demonstrated by a specific radioactivity of

the renal adenine nucleotide pool (ATP, ADP plus AMP) which was about one-half that of the original cyclic [IclAMP added to the perfusate (data not shown).

Production and Distribution of “C-Metabolites of Enoge-

nous Cyclic [Y]GMP in Isolated Perfused Rat Kidney-Fig. 3 shows the time course for cyclic [%]GMP and its “C-metabolites disappearing from and appearing in the perfusate and urine during a kidney perfusion. Table IV shows the distribution of these “C-labeled compounds in kidney, final perfusate, and urine (10 to 60 min) after 60 min of perfusion. The total radioactivity was about equally distributed between kidney,

final perfusate, and urine. In these experiments and analogous to the experiments with cyclic [“CIAMP, renal tissue radioactivity was found mostly as [“Clpurine nucleotide (GTP, GDP, and GMP); perfusate radioactivity was principally as [“C]uric acid, whereas urinary (10 to 60 min) radioactivity was principally as unchanged cyclic [“CIGMP. In one perfused kidney, urine was collected throughout the perfusion (i.e. 0 to 60 min) and this resulted in 1.6-fold more cyclic [“CIGMP appearing in the urine and 0.6-fold less [“Clurate in the final perfusate. However, the renal content and metabolite distribution of radioactivity were unaltered (data not shown).

Production of Uric Acid from Exogenous Cyclic AMP or

Cyclic GMP in Isolated Perfused Rat Kidney-Because of the large metabolism of cyclic [“C]AMP or cyclic [“C]GMP to [‘C]uric acid (Tables II and IV), it was of interest to determine whether these cyclic nucleotides replaced the endogenous substrates for nephrogenic uric acid or whether they were additive to the endogenous synthesis. Table V demonstrates the latter postulate to be correct. The addition of 5 pmol of cyclic AMP or cyclic GMP to the perfusate increased nephro- genie uric acid 2- to 3-fold. The increment in uric acid synthesis showed a very close agreement with the amount of “C-labeled cyclic nucleotide incorporated into [‘C]uric acid (Table V). When urine was allowed to recirculate with the perfusate (i.e. was not collected), the increment in nephrogenic uric acid could account for up to 3 pmol of the 5 pmol of cyclic nucleotide originally added to the perfusate (Table V).

Production and Distribution of “C-Metabolites of Exoge-

nous Cyclic [“CjAMP or Cyclic [“CIGMP in Intact Rat-Fig. 4 shows the multiexponential disappearance curves for plasma radioactivity following the injection of cyclic [i’C]AMP or cyclic [“C]GMP into rats. By inspection of these curves it is apparent that the plasma clearance of radioactivity is qualita- tively similar for both cyclic nucleotides. In Table VI is shown the accumulation of radioactivity in various tissues relative to the plasma, taken 60 min after the “C isotope injection. The radioactivity from both “C-labeled cyclic nucleotides was


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TAEILE II

Production and distribution of “C-metabolites of cyclic [&“CJAMP in isolated perfused rat kidney

Data are from two kidneys as described in legend to Fig. 2. After 60 min of perfusion, the kidneys were clamp-frozen and extracted into perchloric acid. Perchloric acid-soluble extracts of kidney, urine and final perfusate were fractionated and analyzed as described under “Experimental Procedures.”

Unchanged cyclic AMP

Uric acid

ATP

ADP

AMP

Inosi"e/allantoi" a

Xanthine

Hypoxanthine

Total 14C-compounds identified

Total I4 C recovered

I Total "C-compounds

kidney

Tissue distribution of "C-compounds

perfusate total urinary excretion

(IO - 60 mi")

per tent of tot.31 I4 C added to perfusate at zero-time

17.0, 17.5

37.3, 37.3

20.4, 19.9

5.7, 6.5

2.6, 2.4

1.5, 2.9

1.4, 1.0

0.1, 0.6

86.0, 88.1

94.2, 94.4

0 . 0 0 , 0 17.0, 17.5

1.4, 1.2 28.7, 27.3 7.2, a.0

20.4, 19.9 0 , 0 0 , 0

5.7, 6.5 0 9 0 0 9 0

2.6, 2.4 0 , 0 0 . 0

0 , 0.5 1.0, I .a 0.5, 0.6

0 , 0 0 , 0 1.4, I.0

0 > 0 0 , 0 0.1, 0.6

30.1, 30.5 29.7, 29.1 26.2, 28.5

32.0, 32.9 34.3, 31.9 27.9, 29.6

a lnosine and allantoin co-chromatographed on Sephadex G-IO. The I4 C-metabolite located in this position was not further fractionated.

TABLE III

Effect of exogenous cyclic AMP on renal concentration of adenine nucleotides

Isolated rat kidneys were perfused for 60 min. Cyclic AMP, when present, was at an initial concentration of 0.1 mM in 50 ml of perfusate. Urine was either allowed to recirculate with the perfusate or was collected throughout the 60 min of perfusion. After 60 min, kidneys were clamp frozen whilst still being perfused and frozen kidney powder was extracted into perchloric acid. ATP, ADP, and AMP were analyzed in the perchloric acid-soluble extract. Data are presented as the mean i S.E.

Cyclic AMP present

in perfusate

Number recirculated of

kidneys ATP ADP AMP

umol / g dry weight kidney

+a 4 4.14 + 0.23 2.09 + 0.18 0.49 ? 0.04

+ + 3 8.92 t 0.55 b 3.48 2 0.38 ' 0.46 ? 0.10

+ 3 6.30 2 0.44 c 2.42 f 0.13 0.42 t 0.10

a Two controls performed with and two controls performed without urine collection exhibited no difference in their adenine nucleotide contents and therefore these data were pooled.

b Significantly different from respective control value (P<O.OOl).

c Significantly different from respective control value (PcO.02).

most extensively concentrated in the kidney (principally the

cortex) and, to a lesser extent, the liver. Similar results have

been obtained previously with cyclic [3H]AMP (4). A small

amount of the radioactivity from cyclic [%]AMP was found to

accumulate in the heart, small intestine, and lung. In compari-

son to the radioactivity from cyclic [%]AMP, radioactivity

from cyclic [l’C]GMP accumulated less in the kidney but to an

equal extent in the liver. No significant accumulation of

radioactivity from cyclic [r’C]GMP was found in the other

tissues examined.

Tables VII and VIII show the chemical nature of the

radioactivity in whole blood, urine, liver, and kidney, 60 min after the injection of cyclic [r’C]AMP or cyclic [r’C]GMP.

Two-thirds of the total radioactivity injected as cyclic

[‘“C]AMP could be recovered in the sum of urine (0 to 60 min),

kidneys, and liver. The renal and hepatic radioactivity was


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100 -

go-

80-

PERFUSATE

i

-=4 30 F

g 20 x 2 0 2 IO

z 2 O 0 IO 20 30 40 50 60

.\" 40 URINE Total ‘C

X- y/~

30 /Cc c 0 [~V]~GMP

- 0 IO 20 30 40 50 60 Minutes of Perfusion

FIG. 3. Time course for the distribution of exogenous cyclic [“CIGMP and its “C-metabolites in the per&ate (upper panel) and urine (lower panel) of the isolated perfused rat kidney. A kidney was perfused with 5 pmol of cyclic [8-“C]GMP (4 x lo* cpm) in 50 ml of perfusate. Urine was recirculated with the perfusate for the first 10 min of perfusion and, thereafter, five consecutive 10.min urine samples were taken. Urine and per&ate samples were chromatographed on Sephadex G-10 as described under “Experimental Procedures.” 04, the sum of radioactivity found in xanthine (X), inosine (rH), and/or allantoin. Inosine and allantoin were not separated on the Sephadex G-10.

mainly in the form of [“Clpurine nucleotide (ATP, ADP, AMP, and IMP), whereas urinary radioactivity constituted primarily unchanged cyclic [l’C]AMP in the first 30 min and [‘“Clallantoin in the second 30 min after the injection. In comparison to the isolated perfused rat kidney, the relative amount of cyclic AMP incorporated into tissue adenine nucleotide pool was low in the intact rat. Thus, the specific radio- activities of the renal and hepatic adenine nucleotide pools (ATP, ADP plus AMP) were about one-tenth and one-hun- dredth, respectively, that of the original injected cyclic [“Cl- AMP (data not shown). After the injection of cyclic [“CIGMP, four-fifths of the radioactivity could be recovered in the sum of urine (0 to 60 min), kidneys, and liver (Table VIII). In these animals the radioactivity in the urine was twice, and that in the kidney half that found after injection of cyclic [,‘C JAMP. Kidney and liver contained almost all the radioactivity as the [“Clguanine nucleotides (GTP, GDP, and GMP) whereas the urinary radioactivity was mainly unchanged cyclic [‘*C]GMP in the first 30 min and f14C]allantoin in the second 30 min after the injection.

In contrast to the isolated perfused rat kidney, injection of

“C-labeled cyclic nucleotides into the intact rat did not produce any significant quantities of [‘Cluric acid. Likewise, neither the normal uric acid content of the blood (11 + 2 nmol/ml, seven animals) nor the normal urinary excretion of uric acid (1.7 + 0.2 pmo1/60 min, five animals) was increased during the 60-min period following the injection of 3.3 pmol of cyclic AMP or cyclic GMP into intact rats (data not shown).

DISCUSSION

Renal Transport of Cyclic Nucleotides-The experiments reported here with the isolated perfused rat kidney confirm previous observations that exogenous cyclic AMP is rapidly cleared and metabolized by the kidney (6), and extend these observations to show that exogenous cyclic GMP is similarly treated by the kidney. Although it has been suggested that cyclic AMP is dephosphorylated prior to entry into the lymphocyte or thyroid cell (20, 21), there is no evidence that this is a necessary prerequisite for cyclic AMP (or cyclic GMP) entry into the renal cell. On the contrary the evidence suggests that intact cyclic nucleotide and not some circulating metabolite penetrates the cell membrane. Thus, in the isolated perfused rat kidney a transtubular secretory process has been demonstrated whereby intact cyclic AMP and cyclic GMP gain access to the urine (Fig. 1, Table I) and, other than [“C]uric acid, no “C-metabolites were found to accumulate in the perfusate during perfusion with [“C]cyclic AMP or [“C]cyclic GMP (Figs. 2 and 3, and Tables II and IV). The characteristics of cyclic nucleotide flux into the kidney have not been extensively studied. It is possible that renal cyclic AMP influx may occur at the peritubular boundary by an organic acid transport system since cyclic AMP transtubular transport is inhibited by substances such as probenecid (6), and cyclic AMP competes with the renal transport of paraaminohippu- rate (22). In at least two species, rat (Table VI) and dog (23), the kidney cortex appears to be the principal tissue involved in the clearance (and metabolism) of exogenous cyclic AMP and cyclic GMP. Moreover, in both species, exogenous cyclic GMP was more extensively secreted into the urine than was exogenous cyclic AMP (Fig. 1, Table I, and Ref. 5). However, the dog kidney metabolized exogenous cyclic GMP at a greater rate than exogenous cyclic AMP (5), whereas the rat kidney exhibited identical rates of metabolism (Table I).

Renal Metabolism of Cyclic Nucleotides-The majority of the cyclic nucleotide entering the renal cell is metabolized. An estimated rate of metabolism of 400 nmol/min/kidney for both cyclic nucleotides (Table I) was about half the reported cyclic AMP phosphodiesterase activity for rat kidney cortex (24). Since, for reasons previously described (see above), the extra- cellular metabolism of cyclic nucleotides was assumed to be negligible, then it is probable that the [“Cluric acid found in the perfusate and urine during perfusion of the rat kidney with cyclic [“C ]AMP or cyclic [“C]GMP is synthesized in the kidney and passed into the perfusate. The [“C]uric acid accumulated more in the perfusate than the urine, presumably because filtered uric acid is extensively reabsorbed by the rat kidney (25). After addition of 5 pmol of “C-labeled cyclic nucleotide to the perfusate, the amount of “C-cyclic nucleotide incorporated into [“C]uric acid correlated very closely with the increase in nephrogenic uric acid (Table V). Thus, the addition of cyclic AMP or cyclic GMP augmented uric acid


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TABLE IV

Production and distribution of “C-metabolites of cyclic [S-“C]GMP in isolated perfused rat kidney

Data refer to the experiment described in Fig. 3. After 60 min of perfusion the kidney was clamp-frozen and extracted into perchloric acid. Perchloric acid-soluble extracts of kidney, final perfusate, and urine were fractionated and analyzed as described under “Experimental

Procedures.”

Unchanged cyclic GMP

Uric acid

GTP

GDP

GMP

Inosine/allantoin a

Xanthine

Total I4 C-compounds identified

Total I4 C recovered

Total “C-compounds

Tissue distribution of 14

C-compounds

kidney perfusate total urinary excretion

(IO - 60 min)

per cent of total 14 C added to perfusate at zero-time

29.4 0.9 0 28.5

35.2 1.2 28.8 5.2

13.4 13.4 0 0

3.3 3.3 0 0

3.6 2.3 0.8 0.5

1.5 0.3 I .o 0.2

0.9 0.1 0 0.8

87.3

94.5

21.5 30.6 35.2

22.4 35.8 36.3

a lnosine and allantoin co-chromatographed on the Sephadex G-IO. The I4 C-metabolite located in this position was not further fractionated.

TABLE V

Effect of exogenous cyclic nucleotides on the renal synthesis of uric acid

Isolated rat kidneys were perfused for 60 min. Cyclic nucleotides, min) renal content of uric acid contributed only about 5% to the when present, were at an initial perfusate content of 5 amol. Urine was estimated uric production and was not included in these calculations. either recirculated with perfusate throughout the perfusion or was Results are expressed as the mean + S.E. or as individual values when collected for a part (10 to 60 min) of the perfusion. Uric acid production n = 2 or 1. Kidney weights before perfusion, based on the weights of the is expressed as the uric acid present in the final perfusate (at 66 min) nonperfused contralateral kidney, ranged from 1.4 to 2.0 with a mean plus, where applicable, the urinary excretion of uric acid. The latter * S.E. of 1.66 * 0.05 g wet weight (n = 13). accounted for 10 to 30% of total nephrogenic uric acid. The final (60

Cyclic nucleotide Number present in of perfusate kidneys

None

cyc 1

cyc 1

Cycl

Cycl

(control)

c AMP

c AMP

c GMP

c GMP

Urine recirculated

with perfusate

2 b

Uric acid production Metabolism of cyclic nucleotide

mean increment to uric acid a total over control

nmol / 60 min / kidney

1570 ? 150

4470 ? 300 2900

2800 , 3960 1810

3220 i 500 1650

3350 1780

N.M. ’

1870 , 1870

N.M.

1760

c radioactivity of the I4 - C cyclic nucleotide precursor and the radioactivity in See Tables II and IV.

b Two controls without and two controls with urine collection showed no difference in total uric acid production and these data are pooled.

C N.H., not neasured.


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14C(cGMP mjectlon)

I 1 I I I

0 IO 20 30 40 50 60 Minutes After Injection

FIG. 4. Time course of plasma radioactivity following the injection of cyclic [“CIAMP or cyclic [I’C]GMP into the intact rat. Rats were injected intravenously at zero-time with 3.3 mu01 of cyclic (8-“C] AMP (6.9 x 1O’cpm) or 3.3 rmol of cyclic [8-“C]GMP (3.7 x 10acpm), each present in a volume of 0.9 ml. Arterial blood samples (200 ~1) taken at 0.5, 1, 1.5, 3, and 5 min after the injection and at 5- or lo-min intervals thereafter for a total of 60 min. were centrifuged in heparin-treated tubes, and plasma samples (20 ~1) were analyzed for “C by liquid scintillation counting. Throughout the 60 min experiments, a 280 rnM mannitol solution was infused intravenously at 0.2 ml/min. The “C content of the final (60 min) total body plasma was slightly less than 1% of the radioactivity injected as either “C-labeled cyclic nucleotide. Values in each curue represent the means for two animals; vertical bars indicate ranges between pairs of data points.

synthesis but did not, in addition, augment the synthesis of uric acid from endogenous precursor. The latter possibility has been suggested from studies using N6,0*‘-dibutyryl cyclic [SH]AMP in the perfused rat heart (26). Although uricase activity is present in rat kidney homogenates (27), the amount of [“Clallantoin produced by the isolated perfused rat kidney accounted for no more than 3% of the total “C-labeled cyclic nucleotide added to the fusate (Tables II and IV), This is consistent with the perfused dog ki.dney in which [“Clxanthine was a precursor of [“Cluric acid but not [“Clallantoin (28).

In the intact rat, by contrast to the isolated perfused rat kidney, [*‘C]uric acid did not accumulate, whereas [“Clallan- toin was found in both urine and blood after the injection of “C-labeled cyclic nucleotide (Tables VII and VIII). It is possible that, in the intact rat, [“C]uric acid is produced in the kidney and that hepatic uricase converts most of this to [“Clallantoin, in which form it can be more extensively excreted in the urine (29). Production of [‘Clallantoin from cyclic [“C]AMP has been demonstrated in rat liver (30), and this may be the character of an unidentified biliary “C-

TABLE VI

Ratio of tissue to plasma radioactivity after injection of “C-cyclic

nucleotide into the rat

Rats were injected intravenously with 3.3 pmol of cyclic [8-“IAMP (7.1 x lo6 cpm) or 3.3 Fmol of cyclic [8-“CJGMP (3.7 x 10’ cpm). For 60 min after this injection, mannitol was infused intravenously into the rats at a rate of 56 pmol/min. At the end of this period the animals were exsanguinated (by bleeding 5 to 10 ml of arterial blood) and the tissues were rapidly excised, dissected (in the case of the kidney into cortex and papillary section of the inner medulla) and frozen in acetone/Dry Ice. Tissues were homogenized in 5 volumes of distilled water and samples of this (100 ~1) and the final plasma (20 ~1) were counted by liquid scintillation with corrections for quenching. See “Experimental Procedures” for further details.

Tissue Cyclic [“C]AMP” Cyclic [“C]GMPL

cpmlg wet tissue:cpmlml plasma’

Kidney (whole) 218.0 + 41.1 68.5, 119.0 Kidney (cortex) 277.0 + 44.1 106.0, 191.0 Kidney (inner medulla) 23.0 4.2, 6.2 Liver 26.5 i 3.3 19.9, 38.6

Heart 2.4 i 1.3 0.5, 1.1 Brain 0.1 * 0.1 0.2, 0.1 Adipose 0.4 * 0.2 0.2, 0.5 Lung 5.3 * 1.2 0.6, 1.6 Skeletal muscle 0.3 * 0.1 0.3, 0.2 Spleen 1.2 * 0.1 0.6, 1.0 Skin 0.4 * 0.2 0.3, 1.2 Testis 0.8 zt 0.5 0.3, 0.5 Small intestine 2.1 * 0.3 0.8, 1.3

“Mean * S.E. from three animals except kidney (inner medulla) which is the result from a single animal.

b Individual values from two animals.

‘Values for counts per min/ml in the final plasma are indicated in Fig. 4.

metabolite accumulating after perfusion of the rat liver with cyclic [14C]AMP (31). The metabolism of radioactively labeled cyclic AMP in other tissues such as rat liver slices (30), toad bladder (32, 33), and bovine thyroid cells (21) has produced a significant accumulation of radioactivity in inosine. However, in the studies reported herein on the perfused rat kidney and the intact rat, and also in studies with the perfused rat liver (31) and the cultured hepatoma cell (34), inosine was found to be an insignificant product of cyclic nucleotide metabolism.

The extensive metabolism of cyclic AMP and cyclic GMP to their respective purine 5’ nucleotides and, moreover, the significant expansion of the renal ATP and ADP pools by exogenous cyclic AMP in the studies reported herein is of considerable significance with regard to the effects of exogenous cyclic nucleotides on renal function and metabolism. Thus, in cultured renal cells, the transport of thymidine and the ATP pool size were both increased by exogenous cyclic AMP (35). The mechanism for exogenous cyclic AMP- stimulated thymidine transport could, therefore, be via the synthesis of extra ATP. Likewise, other endergonic renal processes such as gluconeogenesis (36) may be stimulated by exogenous cyclic AMP due to an increase in the production of renal ATP.

Although the renal GTP pool was not measured in the studies reported herein, it is known to be about one-tenth the size of the renal ATP pool (37), and it seems probable that some renal effects of exogenous cyclic GMP might be mediated by alterations in the production of renal GTP.


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TABLE VII

Production and distribution of “C-metabolites of ezogenous cyclic [B-“C]AMP in intact rot

Rats were injected intravenously with 3.3 am01 of cyclic [8-“C]AMP injection one kidney and one liver lobe per animal were rapidly excised (6.9 x 10’ cpm). For the next 60 min mannitol was infused intrave- and frozen in acetone/Dry Ice, pulverized, and extracted into 1.7 M nously at a rate of 56 amol/min and two consecutive 30-min urine perchloric acid. Perchloric acid-soluble extracts of urine, blood, kidney samples were collected directly into 1.7 M perchloric acid. Arterial and liver were fractionated as described under “Experimental Proce- blood samples were taken 30 and 60 min after the isotope injection and dures.” Data from two animals are presented. immediately added to 1.7 M perchloric acid. One hour after the

Unchanged cyc I ic AMP

Uric acid

ATP

ADP

AMP

I MP

Allantoin c

Xanthine

Hypoxanthine

Total “C-compounds identified

Total 14C recovered

Total “C-compound! Tissue distribution of 14C-compounds

liver .a kidney a blood b urine urine (O-30 min) (30-60 min)

25.5, 16.2

1.7, 1.3

8.6, 17.6

11.6, 12.1

13.5, 5.2

4.1, 1.2

6.4, 5.8

0.6, 0.7

0.5, 0

72.5, 60.1

74.8, 62.3 1

per cent of total I4 C injected into rat at zero-time

0 , 0 0 , 0 D , 0 24.3, 15.0 1.2, I.2

0 > 0 0.4, 0 0.1, 0 0.7, 0.6 0.5, 0.7

5.9, 7.2 2.5, 10.2 0.2, 0.2 0 . 0 0 , 0

4.2, 4.4 7.3, 7.6 0.1, 0.1 0 , 0 0 , 0

2.1, I.5 11.2, 2.9 0.2, 0.6 0 , 0.2 0 , 0

0.8, 0.1 3.3, I.' 0 , 0 0 . 0 0 , 0

1.5, 0.7 0.8, 0.4 0.5, 0.9 1.4, 1.3 2.2, 2.5

0 , 0 0.4, 0 0 I 0 0.2, 0.6 0 , 0.1

0 , 0 0.4, 0 0 , 0 0.1, 0 0 I 0

14.5, 13.9 26.3, 22.2 1.1, 1.8 26.7, 17.7 3.9, 4.5 9

14.7, 14.6 27.3, 22.6 1.1, I.8 27.3, la.8 4.4, 4.5

a Total tissue weights estimated from organ/body weight data for bled rats (19).

b Data for total blood (estimated as 7.5 ml/l00 g,body weight) obtained 60 min after [ I4 Clcyclic AMP injection. aloDd

obtained 30 min after injection contained about the same amount of radioactivity (about 2% per animal) with a similar composition of radioactive metabolites plus a trace (0.5% per animal) of [14C]cyclic AMP.

C [l4C]allantoin was identified on Sephadex G-IO as described in the “Experimental Procedures”.

TAEILE VIII

Production and dihribution of “C-metabolites of exogenous cyclic [B-“C]GMP in intact rot

Rats were injected intravenously with 3.3 amol of cyclic [%“C]GMP (3.7 x 10’ cpm). The experimental procedure is described in Table VII. The data are from two animals.

Unchanged cyclic GMP

Uric acid

GTP

GDP

GMP

Allantoin ’

Total l4 C-compounds identified

Total 14C recovered

Total l4 C-compounds liver a

Tissue distribution of 14C-compounds

kidney .a blood b urine urine (O-30 min) (30-60 min)

per cent of total I4

C injected into rat at zero-time

44.3, 45.8

1.3, 0.1

13.6, 11.7

10.8, 7.0

3.2, 3.7

7.3, 7.0

80.5, 75.3

81.7, 78.1

0 ,

0 ,

9.1,

5.8,

1.8,

0.7,

17.4,

17.4,

0 0 , 0 0 7 0 -4i3 ,* ;; --1.3, 2.1

0 0 , 0 0 I 0 0.9, 0 0.4, 0.1

6.8 4.0, 4.8 0.5, 0.1 0 0 9 0 , 0

4.0 4.5, 2.9 0.5, 0.1 0 0 I 0 1 0

I.8 1.4, I.0 0 t 0 0 0.6 , 0 , 0.3

0.4 0.2, 0.2 0.4, 0.6 2.7, 2.8 3.3, 3.0

13.0 10.1, 8.9 1.4, 0.8 46.0, 47.1 5.6, 5.5

14.0 10.1, 9.3 1.5, 0.8 46.5, 47.3 6.2, 6.7

z Total tissue weights estimated from the organ/body weight ratio for bled rats (19). Data for total blood (estimated as 7.5 ml/100 g body weight) obtained 60 min after the [ 14C]cyclic GMP injection. Blood obtained 30 min after injection contained about

14 he same amount of radioactivity (about 2% per animal) with a

similar composition of radioactive metabolites. No [ Clcyclic GMP was detected in the blood at either time.

c [14C]allantoin was identified on the Sephadex G-IO as described in “Experimental Procedures”.


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Rend Metabolism of Exogenous Cyclic Nucleotides 4967

Acknowledgments--The author wishes to thank Dr. R. H. demic Press, New York

Bowman, in whose laboratory this work was performed, for his 18. Adam, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer,

constant support and encouragement. Thanks are also ex- H.-U., ed) pp. 573-577, Academic Press, New York

tended to Drs. M. E. Trimble and I. M. Weiner for their 19. Webster, S. H., Liljegren, E. J., and Zimmer, D. J. (1947) Am. J.

Anat. 81, 477-513 invaluable advice. and to Mrs. F. Barnev and Mrs. J. Bowman 20. MacManus, J. P., Whitfield. J. F., and Braceland, B. (1971)

for their excellent technical assistance.

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1976, 251:4958-4967.J. Biol. Chem.

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Metabolism and Excretion of Exogenous Adenosine 3’S ... · significant effects on the handling of cyclic AMP by the kidney. Thus, inulin was routinely employed as a measure of the

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