Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

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 mM. 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 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-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 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo 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- 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, + . 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.

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. ["C]Uric acid was the principal metabolite found extrarenally, and it accumulated more rapidly in the perfusate than in the urine. Table II  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. 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).  (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 nephrogenie 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) 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 qualitatively 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   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 radioactivities of the renal and hepatic adenine nucleotide pools (ATP, ADP plus AMP) were about one-tenth and one-hundredth, 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).

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 paraaminohippurate (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 extracellular 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  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. (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 ["Clallantoin 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-  (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 AMPstimulated 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.