Comp. Biochem. Physiol., 1971, Vol. 39B, pp. 415 to 421. Pergamon Press. Printed in Great Britain

SHORT COMMUNICATION

UREA BIOSYNTHESIS AND EXCRETION IN FRESH- WATER AND MARINE ELASMOBRANCHS*

L E O N G O L D S T E I N t and ROY P. F O R S T E R

Division of Biomedical Sciences, Brown University Providence, Rhode Island; Department of Biological Sciences, Dartmouth College, Hanover, N. H.

(Received 21 November 1970)

Abs t rac t - -1 . Activity levels of ornithine-urea cycle enzymes in the liver of Potamotrygon, a fresh-water ray, were one-half to one-twentieth those found in marine rays. The rate of incorporation of 14C-bicarbonate into urea by liver slices was also markedly reduced.

2. In contrast to marine elasmobranchs, Potamotrygon did not actively reabsorb 14C-urea in its renal tubules.

I N T R O D U C T I O N

IN CONTRAST to marine elasmobranchs which maintain high concentrations of urea in their body fluids for osmotic balance with sea water (Smith, 1936), the fresh- water stingray Potamotrygon contains only traces of this nitrogenous end-product (Thorson, 1967). In marine elasmobranchs the urea level in body fluids is 350- 400 m M and this constitutes more than one-third of their total osmolarity. Potamotrygon, which has a purely fresh-water habitat, has a urea concentration less than one-hundre th that of the marine elasmobranchs. The urea level in body fluids is the resultant of the difference between the rate of biosynthesis and the rate of excretion of the compound. Thus , in these studies, we measured the urea biosyn- thetic capacity and rate of excretion of urea in Potamotrygon and compared these parameters with those of marine elasmobranchs. We found that the low urea concentration in Potamotrygon is at tr ibutable both to low rates of urea biosynthesis and to loss of the renal tubule 's ability to retain urea.

MATERIALS AND METHODS Small Potamotrygon$ weighing 60-325 g were purchased from Paramount Aquarium,

Ardsley, New York, and Connecticut Aquarium, East Haven, Connecticut. Marine

* Parts of this work were done at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, and the Lerner Marine Laboratory, Bimini, Bahamas.

t Recipient of a U.S.P.H.S. Career Development Award. Positive identification of the rays as Potamotrygon was made by Tyson R. Roberts,

Assistant Curator of Fishes, Museum of Comparative Zoology, Harvard University.

415

416 LEON GOLDSTEIN AND ROY P. FoRsTER

stingrays, Dasyatis americana (3-5 kg) and Urol@hus jamaicemis (100-200 g), were caught offshore in Bimini, Bahamas. Dogfish, Squalus acanthias, weighing 1-2 kg were captured in the vicinity of Mount Desert Island, Maine.

The rates of ammonia and urea excretion were assayed in three Potamotrygon, each kept in 1 1. of distilled water for 4 hr at 24°C. Samples of bath water were taken at the beginning and end of the experimental period and analyzed colorimetrically for ammonia and urea (Brown et aL, 1957; Balinsky & Baldwin, 1961).

The biosynthetic activity of the ornithine urea cycle was assayed in liver slices from freshly killed fish by a radioisotopic technique (Forster & Goldstein, 1966). The activities of the enzymes of the ornithine urea cycle were assayed in homogenates of livers prepared from feshly killed Potamotrygon and marine stingrays. Ornithine carbamoyl transferase (E.C. 2.1.3.3), the arginine synthetase system (E.C. 6.3.4.5) and (E.C. 4.3.2.1) and arginase (E.C. 3.5.3.1) were assayed by colorimetric techniques (Brown and Cohen, 1959). The very low levels of carbamoyl phosphate synthetase (E.C. 2.7.2.5) in Potamotrygon necessi- tated the use of a radioisotopic assay similar to that employed previously to measure low activities of this enzyme in extra-hepatic tissues (Hall et al., 1960; Tatibana & Ito, 1969). The assay mixture contained 0'08 g liver homogenized in 0-32 ml of 1"2 M sucrose, 5/zmoles disodium adenosinetriphosphate, 5/xmoles L-ornithine hydrochloride, 5/zmoles N-acetyl glutamic acid, 10/zmoles MgSO4, 50btmoles NH4HCO3 (gassed to pH 6"8 with CO2), 45/zmoles glycylglycine buffer (pH 7"5), 150 units beef liver ornithine transcarbamylase, 45/zmoles phosphoenolpyruvic acid, 0'75 mg pyruvate kinase (Sigma), 20/zC 14C-NaHCOs (sp. act. 4"7/zC//xM) in a total volume of 1"5 ml which was incubated 15 rain at 38 ° C. Water replaced N-acetylglutamic acid in control tubes. The reaction was stopped by addition of 1"5 ml of ice-cold trichloroacetic acid. The mixture was centrifuged and the clear supernatant fluid was gassed with 12CO2 for 2 hr to eliminate 14CO2. One ml of the supernatant solution was diluted to 5 ml with 0"1 mM L-citrulline and applied to a small Dowex 50(H +) column. The column was washed with 10 ml water and eluted with 5 ml of pyridine in water. The effluent was lyophilized and then redissolved in 0"5 ml water, mixed with 15 ml of scintillation solution and counted in a liquid scintillation counter. The reaction product was identified as 14C-citrulline by gradient elution on a Dowex 50(H +) column.

The rates of total excretion of urea and thiourea were determined using radioisotopic techniques. Approximately 10/zC 14C-urea (sp. act. 4"86/zC/ktM) and 10/xC ~4C-thiourea (sp. act. 2"69/zC//zM) per kg were injected into the caudal vessels of four Potamotrygon and four Squalus. Blood samples were drawn 6 hr (equilibration period) and 22 or 30 hr later. Plasma samples (0"1-0"5 rnl) were either (1) directly mixed with 15 ml scintillation solution and counted in a liquid scintillation counter or (2) treated first with urease to convert 14C- urea (but not 14C-thiourea) to 14CO2, gassed with mCO~ for 2 hr and then mixed with scintillation solution and counted. The percentages of injected x4C-urea and 14C-thiourea that were excreted over a 24 hr period were calculated from these results.

Glomerular filtration rates were evaluated as the inulin clearance on two unanesthetized Potamotrygon weighing 80 and 325 g, respectively. Inulin was injected intramuscularly into four separate sites 6 hr before the first blood collection. A total dose of 0"8 ml of a 5 % solution gave plasma inulin levels that averaged 73 mg O~/o over a 3 hr and 40 min period during which two urine collections were being made in the first fish. A l '0-ml dose in the second gave corresponding plasma levels that averaged 53 mg % in two collections over a 2-hr period. Urine was collected from the cloaca by an indwelling 4 mm O.D. poly- ethylene cannula that had been provided with an expanded bulb near the inserted tip. A "purse-string" ligature around the anus applied distal to the bulb served to secure the cannula which was then fitted with a toy rubber balloon for collecting urine formed during the collection period. A 22-gauge needle was used to withdraw 0"3-ml blood samples at the beginning and end of the experiment from the smaller fish, and 0"6-ml samples were similarly taken at the midpoint of each clearance period from the larger. Blood was drawn

UREA BIOSYNTHESIS AND EXCRETION IN MARINE ELASMOBRANCHS 417

from the tail vessels and powdered heparin in the syringe served as the anticoagulant. Inulin was determined by the resorcinol method following cadmium precipitation of all samples (Schreiner, 1950).

I t w a s impossible to collect entirely uncontaminated urine from these small a n i m a l s . They have no urinary or urogenital papilla and the ureters open separately into the cloaca at a site too far cephalad to be exposed by retraction in the living animal. It is possible that some of the cloacal fluid came from the digestive tract but the following observations suggest that inulin collected in this manner was derived exclusively from the kidneys. Powdered phenol red that served as a marker for water that might have been ingested w a s added to the aerated bath water of the larger fish after the last urine collection had been made. Fourteen hr later no phenol red could be detected anywhere along the gut in this semi- starved animal. The gut was essentially dry and an analysis of the very small amount of fluid that was recoverable (0"2 ml) showed no detectable inulin. It seems that this fish at least did not drink appreciable amounts of water.

RESULTS AND DISCUSSION

T h e rates of ammonia and urea excretion were determined in Potamotrygon and compared to those found in marine elasmobranchs. Th e rates of ammonia excretion for three fresh-water stingrays examined were 920, 935 and 1090 t~moles kg -1 hr -1, respectively. Urea excretion was below the limits of detection, i.e. less than 100 t~moles kg -1 hr -~, in all three fish. The ammoniotelic nature of the fresh- water stingray may be contrasted with the relatively high rates of urea production in such marine elasmobranchs as the skate, Raja erinacea in which the ratio of urea-N to ammonia-N excretion is 5 : 1 (Goldstein et al., 1968).

The urea biosynthetic capacity of liver of Potamotrygon was assessed by determin- ing both the rate of incorporation of 14C-bicarbonate into urea by tissue slices (Forster & Goldstein, 1966) and the activities of ornithine-urea cycle enzymes in tissue homogenates (Brown & Cohen, 1959). Th e rates of incorporation of 14C- bicarbonate into urea were 1.1 x 10 -2 and 3.2 x 10 -3 tLmoles g liver -~ hr -1 in two fresh-water stingrays. Similar experiments with liver slices from the marine elasmobranch Squalus acanthias (Schooler et al., 1966) showed that the rate of 14C-bicarbonate incorporation into urea averaged 3.0 t~moles g-~ hr -1, one hundred times that of Potamotrygon. Another indication of urea biosynthetic capacity is the activity levels of individual ornithine-urea cycle enzymes in the liver. As shown in Table 1 the activity of carbamoyl phosphate synthetase (CPS), the enzyme which initiates the conversion of carbon dioxide and ammonia to urea, in Potamotrygon liver is less than one-tenth the levels in livers of the marine stingrays Dasyatis and Urolophus. The activity of the arginine synthetase system in Potamotrygon is one-half that of the marine stingrays, and the activities of ornithine carbamoyl transferase (OCT) and arginase are one-tenth corresponding activities in the marine species. The level of CPS in Potamotrygon is much lower than in marine elasmo- branchs and similar to that found in the ammonotelic teleost fishes (Huggins et al., 1969).

All marine elasmobranchs examined to date retain high concentrations of urea in their body fluids by actively reabsorbing the compound in the renal tubules subsequent to its filtration at the glomerulus (Smith, 1936; Kempton, 1953).

418 LEON GOLSDTEIN AND ROY P. FORSTER

However, in a freshwater elasmobranch such as Potamotrygon the presence of such a system for the retention of urea would tend to aggravate the problem of osmo- regulating in a hypo-osmotic environment. To determine whether specific retention of urea occurs in Potamotrygon as in marine elasmobranchs we measured the rates of loss of 14C-urea in fresh-water stingrays and compared these rates to those observed in dogfish, Squalus acanthias. As shown in Fig. 1 the rate of excretion of l~C-urea from the body fluids of Potamotrygon was 45-50 per cent of the injected dose per day. This value may be contrasted with the rate of 14C-urea loss in the marine dogfish where only 1-3 per cent of the injected dose was excreted per day. Thiourea, the sulfur analogue of urea, which is not actively reabsorbed by

TABLE 1--ORNITHINE-UREA CYCLE ENZYME ACTIVITIES IN THE LIVERS OF FRESH-WATER (Pota- motrygon) AND MARINE (Dasyatis AND Vrolophus) STINGRAYS

E n z y m e activities (/~moles g liver -1 h r - 0

Species C P S O C T A SS Arg inase

Dasyatis americana 6"5 + 1'3 14,360 + 1420 21"6 + 2"1 34,880 + 820 Urolophusjamaicensis 4.5 8,540 16.5 13,920 Potamotrygon sp. 0"36+0.05 1,600+210 9 " 4 + 0 " 5 4,310+240

CPS = carbamoyl phosphate synthetase; OCT = ornithine carbamoyl trans- ferase; ASS = arginine synthetase system. Values for Dasyatis and Potamotrygon are means + S.E. of four to six fish; values for Urolophus are from one fish.

the renal tubules of elasmobranchs (Clarke & Smith, 1932; Schmidt-Nielsen & Robinowitz, 1964) and has essentially the same volume of distribution (Murdaugh, et al., 1965) was used as a standard of reference to determine to what degree urea might be actively reabsorbed by the renal tubules of Potamotrygon. As shown in Fig. 1 the rate of excretion of 14C-thiourea in the fresh-water stingray was similar to that of 14C-urea indicating that the two compounds are handled similarly by the renal tubules of this elasmobranch. Approximately 15 per cent of the injected x4C-thiourea was excreted per day by Squalus (Fig. 1).

Glomerular filtration rates estimated from inulin clearances averaged 8.3 ml kg -1 hr -1 as determined in four clearance periods in two fresh-water rays. These values are ten to fifteen times those found in small marine skates (Goldstein et al., 1968) and significantly higher than glomerular filtration rates observed in larger marine sharks such as Squalus acanthias (Forster & Berglund, 1956). Of the urea filtered at the glomerulus 85-90 per cent is reabsorbed by the renal tubules in marine elasmobranchs. It was difficult to determine the manner in which urea is handled by the kidneys of Potamotrygon because of the inability to collect urine samples uncontaminated with cloacal contents in these small fish with no urinary bladder. However, in contrast to the findings in marine elasmobranchs, a renal

UREA BIOSYNTHESIS AND EXCRETION IN MARINE ELASMOBRANCHS 419

clearance study done on a single Potamotrygon indicated that only about 50 per cent ofthe filtered urea is reabsorbed by the renal tubules ofthisfish. Thus, acclimatiza- tion to a freshwater environment is accompanied both by increased glomerular filtration rate and decreased tubular reabsorption of urea, both factors leading to elevated rates of renal excretion of the compound. The renal clearance of urea in Potamotrygon is approximately 4 ml kg -1 hr -1 and total body clearance is about 13 ml kg - t hr -1. Thus, the major fraction of urea excreted by Potamotrygon, as in other elasmobranchs (Smith, 1931), leaves via the gills.

,,=, o_ 5C

) -

~: 4c x tt.I

N ,=5,, 2c Q.

IC

POTAMOTRYGON

P, Ps P4

S Q U A L U S

UREA

m TROUREA

t.LLI Sl Sz S3 S4

Fxo. 1. Rates of total excretion of 14C-urea and z4C-thiourea by the fresh-water stingray (Potamotrygon) and the marine dogfish (Squalus acanthias) following intravenous injection of the labelled compounds and their equilibration in the

body fluids.

Our experiments show that the inability of the freshwater elasmobranch, Potamotrygon to maintain the high concentrations of urea characteristic of the marine elasmobranchs is due both to the relatively poor ability of the renal tubules to actively reabsorb the nitrogenous end-product and to the low level of urea bio- synthetic capacity of the liver of this fish. Urea levels in these fresh-water rays are much lower than in marine elasmobranchs, and also far below levels found even in such euryhaline elasmobranchs as Pristis and Carcharinus leucas which are able to enter and live in fresh-water for relatively long periods of time (Smith, 1931; Thorson, 1967). This total loss of the urea habitus suggests that Potamotrygon has probably maintained its fresh-water existence for an extended period of its evolu- tionary history. During this study we incidentally noted the absence of a rectal gland in Potamotrygon where, in contrast to marine elasmobranchs, it would have no assignable function in a fresh-water environment as a sodium chloride secreting gland (Burger & Hess, 1960). This again indicates long-ancestry in a freshwater

420 LEON GOLDSTEIN AND ROY P. FORSTER

habitat for this aberrant member of the Chondrichthyes, a group which appears to have been ocean dwellers since their first appearance in the Devonian (Romer, 1966).

It may be noted that the urea biosynthetic pathway is not entirely absent in Potamotrygon liver and perhaps some level of active renal tubular reabsorption of urea may still exist. Future experiments to determine whether the low levels of both systems might be elevated by transferring the fish to increasing environmental salinities are worth while. Such experiments may shed light on environmental factors that regulate urea biosynthesis and its active cellular transport.

Acknowledgements--This work was supported by NSF Grant GB-8200 and U.S.P.H.S. Grant HE-04457. The Misses Susan A. Schweickert and Deborah Funkhouser provided skillful analytical assistance.

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UREA BIOSYNTHESIS AND EXCRETION I N MARINE ELASMOBRANCHS 421

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Key Word Index---Urea formation in elasmobranch; ornithine-urea cycle; Potamotrygon sp. ; fresh-water elasmobranch; osmoregulation; Dasyatis americana; Urolophus jamaicensis; Squalus acanthias.