Parathyroid extract effects on phosphorus metabolism in Rana pipiens

GENERAL

AND

COMPARATIVE

Parathyroid

ENDOCRINOLOGY

Extract

9,

76-92 (1967)

Effects Ram

J. R. CORTELYOU, Department

of Biological

on Phosphorus

Metabolism

in

pipied

P. A. QUIPSE,

AND

Sciences, De Paul University,

D. J. McWHINNIE Chicago, Illinois 60614

Received July 26, 1966 The influence of mammalian parathyroid extract on the distribution of phosphorus in blood, urine, and soft tissues of amphibians has been investigated. Under normal ambient temperature, parathyroid extract induced a significant decrease in plasma phosphorus. The latter, however, was not accompanied by a simultaneous hyperphosphaturia, as reported for mammals, whether studies were made of total inorganic phosphorus in individual urine samples or in 24-hour total urine output; similarly, no change was observed in levels of urinary 3zP excretion. Rather, phosphorus excretion tended to decrease in extract-treated Rana pipiens, and Triturus viridescens v. became significantly hypophosphaturic. Lack of an elevated urine phosphorus in frogs was not due to inward skin transport from environmental water. In vivo and in vitro skin 32P transfer was not extract-influenced, although it did show temperature dependence. The tendency to hypophosphaturia was associated with a marked increase of in vivo phosphorus incorporation into soft tissues and mitochondria after parathyroid extract addition. It was further found that parathormone effects on the heterothermic amphibian were influenced by the temperature to which the animal was acclimated. In contrast to warmacclimated frogs, those exposed to cold stress responded to hormone administration with a significant hyperphosphaturia, and simultaneously showed a decreased tissue and mitochondrial accumulation of phosphorus. Normal cold-acclimated frogs differed from normal animals maintained at higher temperature by conservation of phosphate through reduced renal release and increased mitochondrial incorporation. It is clear, therefore, that parathormone reverses these cold-induced modifications of phosphorus metabolism. This may provide a rationale for the previously observed degeneration of amphibian parathyroid glands in winter, the increased sensitivity of cold-acclimated frogs to hormone, and the apparent discrepancy between heterotherms and the homoiothermic mammal with respect to parathormone-induced changes in phosphorus distribution. Studies

extract

Responses

on the mechanism of parathyroid action have increasingly revealed

that changes in mineral content fluids subsequent to administration

of phosphorus

metabolism

to

parathyroid extract action are more varied than those of calcium metabolism, and interpretations of the physiological effects are thus rendered more difficult. Although extract-induced dissolution of skeletal mineral leads to decreasedbone phosphorus (Gedalia and Kletter, 1964), hyperphosphatemia does not result, possibly due to a renal effect of the extract. Many investigators have observed that in mammals this hormone causes a decrease of phosphate reabsorption by the kidney (Harrison and Harrison, 1941; Cargill and Witham, 1949; Handler, et al., 1951; Calcagno and Lowe, 1956) resulting in a

of body of exo-

genous parathormone are caused by altered bone and/or kidney metabolism. The ability of parathyroid tissue to elicit localized bone resorption (Gaillard, 1955) suggeststhat the well-established hypercalcemia and hypercalciuria found in extract-treated mammals are caused by increased mobilization of bone calcium. 1 This work was supported by the National Science Foundation, grants G-13403 and GB-1485. 76

PARATHYROID

EXTRACT

typical hypophosphatemia-hyperphosphaturia pattern. These findings have been strengthened by the observation that kidneys of the dog (Talmage et al., 1960) and chicken (Levinsky and Davidson, 1957), infused directly with parathormone, show increased phosphate excretion. Conversely, it appears that parathyroidectomy reduces renal excretion of phosphorus in mammals (Buchanan et al., 1959; Ito et al., 1962; Beutner and Munson, 1960). There have been few studies on the physiology of parathyroids in lower vertebrates, and these have largely dealt with calcium regulation. Some species of fish (Fundulus catenatus) are not sensitive to mammalian parathormone, while the females of other species (F. Icansae) respond to hormone only when previously treated with estrogens (Fleming and Meier, 1961). However, hypercalcemia and hypercalciuria have been reported in hormone- treated reptiles (Clark, 1965). Investigations in these laboratories have demonstrated that amphibians have functional parathyroid glands regulating mineral met’abolism. Rana pipiens responds to gland removal by a decrease in plasma calcium, and an increased urine calcium (Cortelyou el al., 196Oc) ; conversely, hypercaleemia and hypercalciuria appear after injection of hormone (Cortelyou, 1960b, 1962a). Although the foregoing results are in accord with those o’btained in mammals, hormone-induced phosphorus changes in lower vertebrates appear to differ from the mammalian response, with the possible exception of reptiles (Clark, 1965) ; parathormone injection of turtles elicits a typical hyperphosphaturia. Schrire (1941)) however, reported that hormone-treated Xenopus Eaevis responded by an increase in serum phosphorus rather than hypophosphatemia. Regulation of phosphorus metabolism by parathyroids of amphibians has also been previously demonstrated in these laboratories to vary from the mammalian pattern. Cortelyou (1960a, 196210) found that parathyroidectomized R. pipiens showed an increase in renal clearance of phosphorus, as well as hyperphosphatemia. Further, preliminary studies (Cortelyou, 1960b, 1962a)

EFFECTS

OK

Rana

pipiens

a?

have shown that, urine phosphorus does not change significantly subsequent to hormone treatment at, room temperature, contrary to the hyperphosphaturia which is a constant feature of the mammalian response. The fate of the phosphorus mobilized by hormone action in the amphibian must yet be determined. An indication of one of t,he possible distribution sites of inereased phosphorus titers has recently been suggested. It has been shown that i?z vitro paratbormone stimulates phosphorus uptake int’o mitochondria, and that mitochondrial translocation of phosphate under hormone influence may be related to substrate oxidation (Sallis et al., 1963a,b; Rasmussen rt al., 1964). In investigating endocrinologieal control of mineral metabolism in a heterotherm such as the frog, it is also necessary to consider the role of seasonal variation, especially as this relates to temperature. The glands of amphibians have been undergo involution during the wi (Cortelyou, et al., 196Oc; Isono, 1960; Boschwitz, 1961) suggesting a functional degeneration and decreased secretion of hormone. Hence, to comprehensively study the effect of parathyroid hormone in amphibians, phosphorus metabolism must be compared in animals acclimated to both warm and cold temperatures. These investigations were therefore undertaken to (1) determine if exogenous parathormone regulates changes in phosphorus metabolism of amphibians, (2) to trace the fate of phosphorus mobilized by hormone aotion, and (3) to analyze the effects of temperature on the distribution of phosphorus. The results unequivocally demonstrate that, in contrast to homoiotherms, the ultimate pathway parathormone-regulated phosphorus in t heterothermic amphibian is directed by the temperature to which the animal has been acclimated. MATERIALS

AND

METHQDS

Male R. pipiens used in these st,udies were obtained from Oshkosh, Wisconsin, and maintained on a constant-flow water table. Animals were not used for at least 2 days, thus providing time for evacuation of phosphorus containing feces, which

78

CORTELYOU,

QUIPSE,

could contaminate urine and environmental water in subsequent experimental procedures. The frogs used in these studies ranged from 40 to 45 gm in weight. I. Effect of Parathormone Individual Blood

and

on Phosphorus Levels Urine Samples

of

Parathyroid extract (“Paroidin,” Parke-Davis; PTE) was injected into the dorsal lymph sac of frogs. In three groups of animals, 5, 10, and 30 units of hormone, respectively, were administered as a single injection, and at 2, 4, 6, 24, and 48 hours, blood and urine were collected as individual samples. A fourth group received 5 units on alternate days, to a total of 7 injections; blood plasma and urine were collected as individual samples at 24-hour intervals for 14 days. Appropriate controls were used in all experiments. Injection of equal volumes of parathyroid extract vehicle (phenol-glycerolwater) into control animals showed no significant effect on phosphorus levels. Plasma and urine total inorganic phosphorus (Pi) values were determined by the method of Dryer et al. (1957). II.

E$ect

of Parathormone on Phosphorus of %$-Hour Urine Samples

Levels

Because of the unexpected results obtained with individual urine samples, values for total Pi excreted daily were determined. Pilot studies indicated that 24-hour catheterization was unfeasible. Consequently, experiments were designed to determine the Pi released by normal and hormone-treated frogs by measuring its content in a fixed volume of environmental water before and 24 hours after immersion of the animal. Water used in these studies was doubly filtered through analytical grade paper and subsequently through a Millipore filter (type HA, 0.45 p) to remove particulate organic material. The Kitson and Mellon (1944) method designed for detecting of trace amounts of Pi was used in this study. A. Warm-Acclimated Frogs. Frogs were placed in individual containers with a fixed volume of water which had previously been assayed for Pi content; after 24 hours, the abdomen was compressed to evacuate any urine remaining in the bladder. The total volume was filtered and an aliquot was taken for Pi analysis. The difference in Pi level from time zero to 24 hours represents the total net exchange per day. Animals were transferred to freshly filtered water daily for 4 consecutive days; the mean value of these daily samples was considered the normal 24hour Pi loss of frogs through the urinary route. To provide a direct measure of PTE action, control frogs used to establish the normal daily levels of Pi excretion subsequently served as experimental

AND

MCWHINNIE

animals. Each was injected via the dorsal lymph sac with 30 units of PTE at 24-hour intervals for 4-6 days, and simultaneously, environmental water Pi was determined. For 2 days before use, and during the control and experimental periods, the animals were maintained at 18-22°C. A corresponding experiment was done using the urodele, Triturus viridescens viridescens. B. Cold-Acclimated Frogs. The data obtained from the above study did not demonstrate a phosphaturic effect of PTE in amphibians. Consequently, another study was undertaken using the same procedure with, however, a reduction in environmental temperature. Animals were cold acclimated at 68°C for 2 days before use, and were maintained at this temperature throughout the control and experimental periods. Because of increased sensitivity of cold-acclimated frogs to PTE, the dose was reduced to 20 units per day.

III. Effect of Parathyroid Extract Distribution of Radio-Phosphorus

on

To elucidate the influence of PTE on phosphorus metabolism in amphibians, the radioactive isotope, phosphorus-32 (NazH3%P04; high specific activity. ORNL) was used to provide increased sensitivity. Radioactive samples were counted with a NuclearChicago gas-flow, low background counter and a Geiger-Mueller detector with an infinitely thin window. A. Warm-Acclimated Frogs. Frogs maintained at 18-22°C were simultaneously injected with 5 PC of 32P (intraperitoneal) and 20 units of PTE (dorsal lymph sac) ; controls received azP only. The animals were placed into individual containers with a fixed water volume which had previously been assayed for P, content. Samples were taken at 2, 4, 6, 12, and 24 hours for counting. Total Pi was also determined at the end of the 24-hour period. To trace the pattern of 32P distribution within control and PTE-treated frogs, animals were sacrificed at 24 hours. Whole blood samples were hemolyzed with distilled water; liver, kidney, femur, gastrocnemius muscle, dorsal skin, and eyes were isolated, weighed, and digested with nitric acid. Blood and wet-ashed t.issues were appropriately diluted and counted. The data are expressed as total szP released to, environmental water, and as fractional curies of activity per unit wet weight of tissue or per milliliter of blood. B. Cold-Acclimated Frogs. Identical procedures (with omission of the la-hour assay) were used with control and PTE-treated frogs preacclimated and maintained at 68°C throughout the 24-hour experi-

PARATHYROID

EXTRACT

mental period; at t,his time, the animals were sacrificed and tissues were isolated and counted. C. Mitochondrial Incorporation oj Phosphorus. Since parathyroid extract increased 3LP incorporation into soft tissues of warm-acclimated frogs, experiments were designed to determine if isotope had become localized in mitochondria. Both warmand cold-acclimated animals which had received simuhaneous injections of 5 MC of 32P and 20 unit,s of PTE were sacrificed at 24 hours. Livers were isolated and weighed, and a 25% homogenate was prepared with a Kontes Dual1 homogenizer in cold, buffered (pH 7.4) 0.25 M sucrose. Aliquots were taken from t#he init,ial homogenate for determination of protein content and radioactivity. After sedimentation of debris and nuclei by centrifugation at 9OOg, 4”C, for 20 minutes, the supernatant fluid was re-centrifuged at 20,000 g, 4”C, for 30 minutes. Three layers were clearly apparent : (1) an upper, protein-rich, tan layer of mitochondria, as determined by Janus Green B stainability; (2) a thin, protein-deficient, milky white midlayer (Janus Green B negative) ; and (3) a small, black, lower layer of remaining debris. The mitochondrial layer was isolated and washed twice in fresh aliquots of buffered sucrose; it was then lysed and appropriate dilutions were taken for assays of radioactivity and protein content. Protein concentrations were determined with the method of Lowry et al. (1951), as modified by Oyama and Eagle (1956). In an effort to account for the higher level of mitochondrial 32P in cold-acclimated normal frogs when compared with those maintained at. higher temperatures, glycogen levels of liver and gastrocnemius muscle were det,ermined using the method of Carroli et al. (1956). IV.

Sk&

Transport

of Phosphorus

8. Whole Organ&r&. Since no hyperphosphaturia occurred in PTE-treated, warm-acclimated frogs, attention was t,urned t,o the possibility that urine Pj excreted into environment,al water may be reabsorbed through the skin; this was assessed by determining changes in the s2P content of labelled environmental water through a 24-hour period. To trace the fate of 32P absorbed through the skin, tissues were isolated and counted. Only warmacclimated frogs were used in this experiment. Normal and PTE-treated (20 units) frogs were placed into wat,er of known Pi content containing 35 ~?c of QP. At 24 hours, a second aliquot was used to determine the change in total P,. Animals were then sacrificed and tissues were isolated and counted. B. Isolated Skin. In recognition of the role of skin in ion transport, studies were done to determine the net movement of 32P across skin isolated from

EFFECTS

ON

&ina

pipie7ZS

19

normal and PTE-treated frogs. Both warmand cold-acclimated animals, injected with 20 units of PTE, were sacrificed after 24 hours and skinned with care to avoid permeability changes due l;o mechanical damage. Dorsal and ventral skin isolates were mounted over the end of a 2.0-cm diameter tube? all manipulations being accomplished under Ringer’s solution Artificial cells containing 4.0 ml of Ringer’s solution were so assembled that the out’side skin surface was immersed in 3ZP-labelled (I pC/25 ml; wat,er. Cell assemblies were incubated for 3 hours: movement of s?P t,hrough the skin was evaluaLed by determining the radioactivity of 0.2ml aliquots taken from the Ringer’s solution in&e the ccl! every 30 minutes. All solutions were pie-equilibrated to, and main.tained at, the acclimation temperatures of the 2 groups of frogs throughout the duration of the experiment. Data obtained from all experiments were analyzed by use of the Student’s t test. RESULTS

I. E$ect of Parathyroid Extract on f%osLevels of Individual Blood and Urine Samples. Plasma and urine Pi levels were determined in numerous individual samples collected from normal R. pipiens and from those which had received either a single hormone dose of 5, 10, or 30 units, or repetitive dosesof 5 units. In all cases,hypophosphatemia was observed, without a concomitant increase in urine phosphorus. As shown in Table I, 5 and IO units of PTE caused significant decreases (31 and 387& respectively) in plasma Pi levels within 2 hours, which persisted through the 4%hour experimental period. Similarly, a slight, but significant hypophosphatemia occurred after administration of 30 units. When 6 units were injeizted on alternate days (7 injections), the plasma Pi concentration remained significantly below the contro1 value (Table 2). The data clearly indicate that whether small or large hormone doses as a single acute injection or repetitive injections are used, individual urine samples collected at various times show no significant change in Pi concentration. II. Effect of Parathgroid Extract on Phosphoms Levels of 24-Hour Urine Samples. Since the previous study on individual frog urine samplesshowed no change, or a slight decreasein phosphorus excretion after treatphorus

80

CORTELYOU,

PLASMA

AND

URINE

PHOSPHORUS

QUIPSE,

Rana

IN

AND

TABLE pipiens

Phosphorus

Hours after PTH injection

5 units

MCWHINNIE

1 AFTER

A SINGLE

(mean mg/lOO

PTH

10 units

INJECTION

OF PAROIDIN

ml zk SE)

PTH

30 units

PTH

Plasmah Control 2 4 6 24 48

(19)” (11) (10) (12) (14) (9)

3.94 2.75 2.83 2.49 2.54 2.38

zk 0.21 + 0.10 f 0.15 + 0.14 z!z 0.16 f. 0.14

(11) (12) (10) (10) (10)

2.44 2.56 2.41 2.64 2.99

+ k + f f

0.12 0.11 0.11 0.17 0.13

(10) (10) (10) (10) (19)

3.00 2.71 3.37 3.08 3.69

* zb + + f

0.21 0.10 0.39 0.21 0.33

+ IL * + 5

0.32 0.40 0.19 0.40 0.29

(6) (7) (15) (12) (10)

2.27 2.56 1.99 2.04 2.28

f k + ix f

0.44 0.47 0.27 0.23 0.03

Urinec Control 2 4 6 24 48

(32) (10) (12) (10) (10) (6)

2.28 2.23 2.46 2.43 2.45 1.72

* Number of animals. b All differences significant c No differences significant.

31 -t 3~ f + zk

0.14 0.27 0.18 0.15 0.02 0.25

(11) (10) (11) (9) (13)

at the 5’$?? level

or less, except

ment with hormone, the tot.al daily loss of phosphorus was measured. In this study, individual frogs were maintained at 18-22°C in a constant volume of water which was renewed daily. The net increase in phosPLASMA

AND

URINE FIVE

PHOSPHORUS UNITS

2.62 2.62 1.93 2.38 2.73

IN

ADMINISTERED Phosphorus

Time

in days

Controls 1 2 3 4 5 6 7 9 10 11 12 13 14 a Number of animals. b All differences significant c No differences significant,

dose at 48 hours.

phorus content between time zero and 24 hours was taken as the normal daily loss of urinary phosphorus in frogs. The mean value obtained through 4 days was 35.25 pg. Subsequently, these same animals treated

TABLE 2 Rana pipiens

OF PAROIDIN

the 30-unit

DURING ON (mean

REPETITIVE ALTERNATE mg/lOO

ml zt SE)

Plasmas

(19)Q (5) (9) (9) (11) (5) (11) (3) (4) (5) (5) (5) (7) (5)

3.93 2.93 2.77 2.89 2.13 2.70 2.59 4.22 2.03 1.88 2.26 1.87 2.23 2.53

INJECTIONS DAYS

Urinec

* 0.21 f 0.73 5 0.11 k 0.31 zk 0.25 f 0.37 + 0.33 IO.97 rk 0.28 2~ 0.02 + 0.29 Ik 0.20 + 0.39 + 0.28

at the 5’% level or less, except except for day 6.

(25) (5) (36) (33) (23) (11) (12) (9) (16) (15) (14) (17) (11) (20)

for day

7.

2.39 3.24 2.36 2.09 2.13 2.31 4.94 2.07 2.36 2.10 2.35 2.22 1.65 2.60

AI 0.14 f 0.40 3~ 0.17 Ik 0.22 f 0.16 + 0.35 f 0.15 AZ 0.15 zk 0.25 zk 0.16 3~ 0.31 rk 0.25 z!z 0.16 zk 0.12

OF

PARATHYROID

INFLUEWE

EXTRACT

EFFECTS

Rana

pipiens

T,4BLE 3 OF TEMPERATURE ON THE RELEASE OF PHOSPHCBRUS TO ENVIRONMENTAL BY Runa pipiens DURING DAILY INJECTION OF P~ROIDIN ,~g phosphorus/24

Acclimation temperature

So. animals

1%22°C

18

6-8°C

ozi

4

Q Probability of the significance b Number of samples through

Normal

klleansof4

days)

35.25

31 0.60 Wb 30.91 + 3.95 (23)

with 30 units of PTE per day for 4 days released a mean of 31.68 pg. This difference in daily phosphorus loss before and during treatment was not statistically significant (Table 3). Thus, whether measured by individual urine samplesor by total loss per day, frogs do not show a parathyroid extractinduced hyperphosphaturia when maintained at ambient temperatures, but rather a tendency to hypophosphaturia. To evaluate whether the lack of a parathyroid extract = induced hyperphosphaturia is characteristic of amphibians in general, or is a response unique to anurans, the urodele Triturus viridescens v. was also studied. The individual normal daily phosphorus loss to environmental water was measured for 9 salamanders, and was 7.19 =I=0.51 pg, the loss through 4 days during daily injection of 20 units of PTE was 4.25 rrt 0.10 pg, and the difference was highly significant (p = .0005). The response of both anurans and urodeles to mammalian parathyroid extract was essentially the same since t#here was no hyperphosphaturia observed after treatment. Further, in both amphibian groups, there was a decrease in daily urine phosphorus excretion with PTE treatment, although this was only statistically significant for the urodele. In a similar study, frogs were maintained at reduced temperature (S-S’C) and again the daily change in phosphorus level of the environmental water was measured. Through 6 days the normal phosphorus loss of low temperature-acclimated frogs was 30.91 pg. Subsequent daily PTE treatment (20 units) of these same frogs elicited a

Wam~i.

hours &SE Normal + PTH (means of4-6 days) 31.68

i 0.13 (65) 43.35 llc 4.18 (24

of the difference. 4 to 6 days from the number

81

of animals

0.20 0.025 __----

indicated.

significant hyperphosphaturia through 6 days. These data are also presented in Table 3. The mean daily phosphorus loss was increased to 43.35 pg per animal. Through this study, the influence of environmental temperature on the physiological action of a mammalian hormone on a heterothermic species became apparent, and was included in subsequent experiments III. E$ect of Parathyroid Extract on tribution of Radio-Phosphorus. The results of the preceding experiments are in conformity with those obtained when urinary phosphate clearance was followed with radio-pbQsphorus. Both warm- and cold-acclimated frogs were injected with 32P only, or with isotope and PTE; at various imervals the radioactivity of the environmental water was measured. As shown in Table 4 for both control and PTE-treated animals of eit)her temperature history, there was a gmdual increase ‘of activity through 24 hours, indicating the continuing release of urinary 32P. Comparison of the levels of radiophosphorus excretion between warm-acclimated normal frogs and those which received PTE demonstrated that there was no signif?cant elevation in the latter group, thus confirming the previously observed lack of a PTE-induced hyperphosphaturia. Conversely, it is evident that the cold-aoclimated animals responded differently; as early as 4 hours and continuing through 24 hours, there was a significant increase in %lP clearance when compared with control. frogs. Analyses of total inorganie phosphorus content in the environmental water at 24 hours showed that cold-acclimated frogs

82

CORTELYOU,

QUIPSE,

TABLE INFLUENCE

OF TEMPERATURE ON SIMULTANEOUS INJECTION Total

Time

in hours

THE RELEASE OF ISOTOPE nanoouries

(12)b (12) (12) (9) (11)

271.38 236.15 244.70 322.51 328.75

(11) (11) (11) (11)

112.85 141.28 199.88 365.34

a Probability of the significance b Number of animals.

4 OF a2P TO ENVIRONMENTAL AND PAROIDIN INTO Rana f Norm4

+ IL if +

78.83 53.33 55.83 77.33 75.88

+ I? + +

acclimation, (14) (14) (14) (11) (14)

Cold 2 4 6 24

MCWHINNIE

32P released

NOiTUl

Warm 2 4 6 12 24

AND

46.89 23.36 32.81 50.34

acclimation, (11) (11) (11) (11)

WATER

AFTER

pipiens

SE + 20 units

PTH

P”

2 63.49 I!I 19.92 rk 80.81 f 94.13 z!z 87.62

0.40 0.40 0.20 0.45 0.30

+ f + f.

0.10 0.05 0.10 0.005

I&22°C 198.46 199.15 323.02 331.75 398.02 6-8°C 253.31 304.57 343.83 668.95

86.95 83.04 90.64 76.95

of the difference.

under PTE influence were hyperphosphaturic. In contrast, treated warm-acclimated animals did not show this response. These differences were similar in direction and magnitude to those presented in Table 3; thus the data are not included here. In light of the observed hypophosphatemia and lack of hyperphosphaturia in warm-acclimated frogs treated with PTE, an intraorganismic site of localization of phosphorus released from bone was sought. Tissues and whole blood were isolated at the termination of the 32P excretion study (24 hours), and their radioactivity was determined. It can be seen in Table 5 that there was an accumulation of isotope within tissues of both control and PTE-treated frogs. However, tissues of the latter group were increased in activity approximately 2-fold over control values, and the differences were significant at the 5y0 level or less, except for muscle and whole blood. The lack of change in 32P content of blood probably indicates that the isotope had equilibrated between tissues and body fluid in both controls and treated animals considerably earlier than 24 hours. The increase in femur activity is interpreted to represent a differential in the sites of isotope incorporation and PTE-induced demineralization resulting

in an apparent increase in bond radioactivity. When tissues of the cold-acclimated frogs were similarly studied (Table 5), it was found that they also accumulated isotope. Tissues of PTE-treated animals consistently showed less 32P activity than did controls, although the differences only approach statistical significance. These data stand in direct contrast to those obtained with warmacclimated animals under parathyroid extract influence, which showed much larger 32P tissue incorporation than did controls. The data presented in Tables 4 and 5 have been summarized in an isodiametric display given in Fig. 1. This permits the simultaneous demonstration of phosphorus shifts with respect to loss through the urinary route (Fig. 1, A) and its tissue accumulation (Fig. 1, B). Additionally, it allows comparison of parathyroid extract and temperature influences on this distribution. To determine if mitochondria might be the PTE-sensitive intracellular site of isotope accumulation within the soft tissues of frogs, these organelles were isolated from liver homogenates of control and treated animals, both warm- and cold-acclimated. At 24 hours after injection of PTE and S?P, or ZsP only, animals were sacrificed and their livers were

PARATHPROID

IXFLUEXCE -

EXTRACT

EFFECTS

TABLE OF TEMPERATURE ON THE TISSUE SIMVLTANEOUS INJECTION OF ISOTOPE Nanocuries

Tissue

32P/gm

RWla

5 DISTRIBUTIOX AND Pa~o~uriv

wet wt. l

Normal

ON

p@k?&S

OF 33P sy 24 HOURS 1sli0 Rana pipiens

AFTER -_____.

SE

Normal

+ 20 units

PTH

-

Warm

83

acclimation,

P

1%22°C ___-

Liver Kidney Skin Eye Femur Muscle Blood

(15’lb (13) (15) (12) (15) (15) (13)

429 09 286.91 120.90 16.22 244.24 39.46 18.85

+ + i +I f 2 +

101.39 50.50 20.06 3.44 41.41 9.72 2.43

(18) (15) (18) (15) (18) (18) (14) Cold

Liver Kidney Skin Femur Muscle Blood a Probability b Number

(11) (11) (11) (11) (11) (10)

190.65 167.51 128.62 271.16 11.99 19.66

of the significance of animals.

zk F + + k i

96.33 11.20 12.34 30.30 1.41 3.35

acclimation, (11) (10) (11) (11) (11) (11)

767.36 591.0’7 243.60 29.09 398.15 44.33 18.38

Ik 7.45 i 146.94 5 64.64 + 6.81 AZ 94.50 5 9.25 jr 1.29

0.0005 0.025 0.0‘25 0.05 0.05 0.33 0.45

F I!Z rt k i *

0.20 0.05 0.15 Q.15 0.20 0.45

.--

6-8°C S9.76 125.74 109.12 232.28 9.16 19.10

13.33 16.66 12.12 19.34 2.57 1.19

of the difference.

weighed, homogenized, and appropriately centrifuged. Analyses of the protein content and 32P activity of both the initial homogenat,es and the mitochondrial fractions were done. The protein concentration (pg protein/pg wet weight) of 54 samples of frog liver homogenate was 16.07% f 0.38, and there were no significant differences between control and PTE-treated frogs, or between those which were warm- or cold-acclimated. Of the total liver cell protein, 570 is present in the mitochondria of warmacclimated frogs and approximately 3% in cold-acclimated frogs. In either group no significant difference was observed between the mitochondrial protein content of normal and treated animals (Table 6). If, however, one compares the percentage of liver protein that is mitochondrial in warm- and coldacclimated frogs, the difference is highly significant (p = .0005). It is thus clear that although exogeneous PTE does not cause a change in the total level of mitochondrial protein in frog liver, acclimation to low temperature induces an approximately 40y0 decrease in this comnonent.

The increased s2P accumulation in soft tissues of PTE-treated, warm-a~~li~lated frogs is clearly within mitochondria as shown in Table 6. There was a highly significant, and almost 2-fold increase in the specific activity of mitochondrial lysates of liver from PTE-injected frogs when compared with controls. These data are consistent with the observation that the decreased plasma phosphorus, and the slight hypophosphaturia, are accompanied by an increased tissue incorporation of this ion. Conversely, it can be seen that when similar studies were done with cold-acclimated animals, -there was a highly significant decrease in 3:! incorporation of the liver mitochondria of the PTE-treated group, as compared with controls. As previously shown, such. ani.mals are hyperphosphaturic and have a decreased 32P accumulation in various tissues. It may also be noted in Table 6 that t’here was a marked difference in the specific activities of liver mit80chondria from warmand cold-acclimated normal R. pipiens. When one compares the percentage of mitochondrial activity of warm-a&mated frogs with that of animals maintained at low km-

84

CORTELYOU,

QUIPSE,

AND

MCWHINNIP

PTH 0

NORMAL

FIG. 1. An isodiametric presentation of the effect of temperature on the response of phosphorus metabolism in Runa pipiens to mammalian parathyroid extract. Column height represents magnitude of radioactivity. (A) The net change in 32P activity of environmental water, reflecting urinary phosphorus loss, at various intervals after administration of PTE to warm- and cold-acclimated frogs. (B) Incorporation of 3zP into various tissues of warm- and cold-acclimated frogs 24 hours after PTE treatment.

it is apparent that the 32P level of the latter group increased over 2-fold, and the difference was significant at the 0.05% level. It is probable that this difference reflects physiological changes in energy metabolism related to temperature acclimation. As shown in Table 7, frogs maintained at

perature,

6-8°C had significantly higher tissue glycogen levels than warm-acclimated animals. The evaluation was based on a “two-tailed” analysis of Student’s t test since it was not possible to predict the direction of carbohydrate changes in the heterothermic amphibian as a result of exposure to low temperature.

PARATHYRO~D

EXTRACT

85

EFFECTS oiv Rana pipiens

TABLE

6

IP*'FLUENCE OF TEMPERATURE ON THE MITOCHONDRIAL ACCUMUL~~TION OF 32P BY 24 SIMULTANEOUS INJECTION OF ISOTOPE AND PAROID~N INTO Rana pipiens

Warm-acclimated Controls +PTH Cold-acclimated Controls +PTB a Number 6 Probability

f& Mitochondrial activity 3~ SE (nanocuries/mg mitochondrial protein) (nanocuries/mg initial homogenats protein)

yO Mitochondrial protein =t SE (mg mitoehondrial protein/ mg initial homogenate protein)

Condition

(17)a (8)

5.35 4.99

+ 0.61 + 0.46

0.30*

(12) (14)

2.68 3.00

i. 0.22 zk 0.26

0.20

(7) (7)

42.13 76.49

+ 2.99 IL 8.22

0.000.5

(8) (11)

99.02 57.26

It 6.78 F 3.70

0.0005

(B-8°C)

of samples. of significance

of the difference.

IV. Skin Transport of Phosphorus. The establishment of parathyroid extract-induced hyperphosphaturia in cold-acclimated frogs, and the lack of this responsein those maintained at higher temperatures, was based upon studies of total Pi and 32Prelease to environmental water; the values obtained therefore represent the net exchange of phosphorus between the organism and the external environment. It was consequently considered necessary to determine if the slight hypophosphaturia observed in warmacclimated PTE-treated frogs was due to a large inward transport of phosphorus through the skin. This possibility was assessedby maintaining animals in water of known Pi content labelled with 32P;warmacclimated animals, 12 normal and 12 PTEtreated, were used in this study. At time zero, 2, 4, 6, 12, and 24 hours, the radioactivity of the environmental water was measured, and at 24 hours, the net change in Pi of the water and radioactivity of wetashed liver, kidney, skin, femur, and eye were determined. The results showed that radiophosphorus

was absorbed by frogs and was subsequently incorporated into tissues as the 32Pactivity of the environmental water gradually clined. At -time zero, approximately 27 PC were present in the water; this decreased to 24 PC! through 24 hours. At no time were significant differences (p = .30-.45) in transport of a2Pnoted between normal frogs and those which received PTE. When tissue radioactivity was determined at 24 hours, was similarly found that while they d inc0rporat.e isotope, no significant differences (p = .15-.45) existed between treated and normal frogs. It is thus clear that although phosphorus is t,ransported from environmental water through frog sk.in to ultimately become incorporated into tissues, the magnitude of transport is the same in control and experimental frogs, and is not PTEinfluenced. Therefore, the lack of a hyperphosphaturic response, and t,he tend~~~~ to hypophosphaturia in warm-a~el~mated~ PTE-treated animals, cannot be explained by an increased inward transport of phosphorus originally released via the urinary route. Further, analyses of inorganic phos-

TABLE

7

INFLUENCE OF TEMPERATURE ON TISSUE GLYCOQEN LEVELS IN NORMAL Rana ‘% Tissue glyeogen i SE (mg glycogen/mg

Acclimation temperature 1%22°C 6--8°C a Number b Probability

-

(IS-22’C)

Liver

pipiens

-__-

Muscle

2.62

+ 0.49 (11P 4.22 zk 0.58

of samples. of the significance

dry weight)

of the difference

0.056

based

0.47

+ 0.04 (23)

1.05

10.11

on a “two-tailed”

test.

~.OOL

86

CORTELYOU,

QUIPSE,

phorus in the same environmental water at the end of 24 hours showed that no significant change in concentration occurred from time zero, as previously indicated in Table 3; the data were similar in magnitude and direction, and hence are not included here. The results of the above experiment are supported by studies done on skin isolates from warm- and cold-acclimated frogs; ‘both dorsal and ventral skin isolates from normal and PTE-treated animals were used. No differences were observed with skin pieces isolated from dorsal and ventral surfaces of the frogs. It was found that although 32P was transported across skin to the nonradioactive Ringer’s solution inside the cell throughout 3 hours, none of the differences between sampling intervals were significant. This may have been due to the absence of a continuous diffusion gradient which internal tissue incorporation of the isotope would develop. Although the magnitude of transport was temperature-sensitive, skin isolates from warm-acclimated frogs having a greater net transport capacity than those from coldacclimated animals, no significant differences existed between the normal and PTEinjected groups. Therefore, it is unlikely that animals under parathyroid extract influence at higher temperatures fail to become hyperphosphaturic because of increased skin reabsorption of phosphorus. DISCUSSION

The administration of mammalian parathyroid extract has significant effects on the pattern of phosphorus distribution and metabolism in the amphibian, R. pipiens. Hypophosphatemia occurs rapidly after a single injection of 5, 10, or 30 units of PTE, or 5 units given repeatedly. These results are more clearly similar to the mammalian response than are those obtained with other lower vertebrates. Schrire (1941), for example, reported that Paroidin-treated Xenopus laevis were hyperphosphatemic, although she observed an initial depression of plasma phosphorus. It is of interest to note that Clark (1965) was unabIe to demonstrate any change in plasma phosphorus levels of

AND

MCWHINNIE

turtles which had received as much as 200 units of parathyroid extract. The smallest amount of PTE employed in these studies, 5 units, elicited a significant decrease in plasma phosphorus in 2 hours, which persisted through 48 hours. Previous studies, however (unpublished results), have shown that plasma calcium is not affected by this low dose. It would thus appear that the frog has a differential response to hormone with respect to blood levels of these two mineral ions, and that the threshold for observation of gross phosphorus changes is lower than that for calcium. Buchanan (1961) reported a similar observation in mammals, wherein 10 units of parathyroid extract produced a phosphatemic responsewhile a larger dose was required to induce a clear calcemic effect. If one were to anticipate a parallel response of amphibians and mammals to PTE, the decreasedblood phosphorus would be reflected by an increase in its urinary release. Studies with mammals (Talmage and Krainte, 1954; Bartter, 1954) have demonstrated this effect, which has been explained as a hormonally induced inhibition of phosphate reabsorption (Albright and Ellsworth, 1929); and Nicholson (1959) suggested that the action is directly on the distal tubule of the kidney. However, there are data which contraindicate this interpretation. Foulks and Perry (1959b) observed only a negligible elevation of filtered phosphate load after parathyroid extract administration, and suggestedthat phosphorus was shifted from extracellular fluid compartments into cells. Further, these investigators (Foulks and Perry, 1959a) found little change in regulation of phosphate excretion after parathyroidectomy. It was consistently found in this study, that unlike mammals, parathyroid extract does not increase phosphorus loss through the urinary route in frogs maintained at ambient temperature. Neither individual urine samples nor the total daily urine output showed hyperphosphaturia. Rather, there was a tendency to hypophosphaturia, with a small and constantly observed decrease in urinary phosphorus. That this response is not found in anurans alone is

PARATHYROID

POSTULATE

EXTRACT

EFFECTS

OK

&ULCl

1 Decreased

Increased PTN induces reabsorption

pipiens

tubular Of Pi

‘i

POSTULATE PTH prevents reabsorption

2

Increased

tubular of Pi

Increased P:

Decreased PI.

AMPHIBIAN RESPONSE

Decreased 'i

Increased 'i

K \ No significant to hypophosphaturia, Hypophosphaturia,

change, r.

tendency R. pipiens; viridescens.

Increased 'i

Fro. 2. A diagrammatic scheme representing two possible effects of parathyroid extract on kidney (K). Expected changes in blood and urine phosphorus (Pi) levels are shown as they would occur under conditions of hormone administration (PTH) or gland ablation (PTX). The anticipated responses are compared with those obtained in amphibians. (Data for phosphorus changes in parathyroidectomised R. pipiew taken from Cortelyou, 1962b.)

demonstrated by results obtained with treated T. viridescens v. In the latter, urine phosphorus levels were significantly below those of normal salamanders. The absenceof a grossphosphaturic effect in amphibians after treatment with parathyroid extract was confirmed by tracing urinary releaseof phosphorus with the corresponding radioisotope. In no case was the excretion level of szP in treated frogs at ambient temperature greater than isotope clearance from normal animals. The effect of parathyroid extract on the anuran mesonephrosremains ambiguous. In an effort to elucidate whether there is a physiological effect on the amphibian “kidney,” certain alternates are available as shown in Fig. 2. Postulate 2 conforms to results obtained with mammals, and the third diagram depicts amphibian responses

to hormone addition and gland ablation. Postulate 1 is included only to offer an alternate which cannot as yet be definitely excluded. It is doubtful that hormone induces phosphate reabsorption (not,e Postulate 1) since following PTE treatment, urine phosphate does not significantly fall and plasma levels are markedly decreased; further, after parathyroidectomy of frogs both hyperphosphatemia and hyperphosphaturia are observed (Cortelyou, 1960a, 1962b) ; decreasedplasma phosphorus would be expected if there was an increased reabsorption. It is more likdy that the elevated urine phosphorus in parathyroprivic frogs reflects the increased plasma phosphate load which may in turn be due to a lack of hormone-dependent tissue incorporation. It is equally doubtful that parathormow

88

CORTELYOU,

QUIPSE,

acts t,o prevent phosphate reabsorption in amphibians (Fig. 2, note Postulate 2), as traditionally suggested in mammals, since parathyroid extract addition does not cause hyperphosphaturia, whereas parathyroidectomy does (Cortelyou, 1962b). However, a phosphaturic response could be ascribed to the mesonephrosif one were to assumethat under hormone influence the stability of urine phosphorus levels, in light of the observed hypophosphatemia, represents a net increase in phosphate clearance. It must be cautioned here, however, that a tendency to hypophosphaturia was shown for the frog, and there was a significant decrease in salaurine phosphorus of PTE-treated manders. Hence, without postulating any effect of parathyroid extract on the amphibian mesonephros, urine phosphorus could fall simply due to the decreasedplasma phosphorus load. Since parathyroid hormone induces dissolution of bone mineral (Chang, 1951; Bollet et al., 1963), and since in amphibians PTE administration is followed by hypophosphatemia and a tendency to hypophosphaturia, the question arises as to the fate of phosphorus mobilized from bone. The results of these experiments clearly indicate that soft tissues of the frog act as a phosphate reservoir. Subsequent to treatment, there was a marked increase in the phosphorus accumulation of liver, kidney, skin, and eye. These observations are in conformity with the reports of Tweedy and Campbell (1944), Bartels (1954), and Egawa and Neuman (1964) that parathyroid hormone increases transport and cellular uptake of phosphorus. More recently, Sallis et al. (1963a) reported that the hormone exerts a phosphate-accumulating effect on isolated mitochondria. Similar results were obtained in this investigation with in viva studies on frogs; mitochondria isolated from livers of PTE-injected animals incorporated twice as much radiophosphorus as those of normal frogs. It would thus appear that phosphorus mobilized from bone in PTE- treated amphibians is released to plasma, but is so rapidly accumulated intracellularly and then by mitochondria, that hypophospha-

AND

MCWHINNIE

temia results and there is no increased phosphorus excretion. Although it may be argued that the lack of an observable hyperphosphaturia in the aquatic frog is due to skin reabsorption of phosphorus voided via urine into environmental water, this suggest,ion is untenable since there was no difference found between normal and treated frogs as regards, (1) the inorganic phosphorus level of the water, (2) the rate of isotope disappearance from environmental water, and thus the rate of absorption through the skin of intact animals, and (3) the rate of 32P transport through skin isolates. The lack of an increased tissue accumulation of 32Pin PTE-treated frogs exposed to isotopically labelled water may be explained by the time sequence used in this experiment. Frogs were given parathyroid extract and immediately placed into labelled water. If it is assumedthat this treatment rapidly causesmobilization of unlabelled bone phosphorus, and that this is in turn rapidly accumulated in soft tissues, a tissue phosphorus level may be reached in the early hours of the experiment which metabolically precludes further significant incorporation. On the other hand, the 32Pin the environment must not only be transported through skin but must be distributed throughout circulation before becoming available to soft tissues, which would, in this view, have already become phosphate saturated. However, the normal process of secular exchange between the stable and radioactive isotopes of phosphorus would occur to the same extent in tissues of both normal and PTEtreated frogs. Hence by 24 hours, although tissuesof both groups would be radioactive, the magnitudes of isotope accumulation would be similar. The data presented here are in conformity with this interpretation. Studies of temperature effects on the physiological response of frogs to parathyroid extract have shed considerable light on the regulation of mineral metabolism in heterotherms and provide a rational basis for the apparent discrepancy from the mammalian pattern. It has long been observed in these laboratories that change of

PARATHYROID

EXTRACT

season provokes morphological variation in the parathyroid glands of frogs. During winter (Cortelyou el al., 196Oc) or after exposure of anurans to low temperature (Von Brehm, 1964)) the degenerative changes and vacuolation of parathyroid cells may suggest there is decreased functional activity of the glands, and hormone production may fall. Further, it has been observed that PTE-induced changes in frog bone metabolism are season sensitive (Cortelyou, 1965). It was assumed that temperature is the operative factor in seasonally induced changes of parathyroid function, alad the influence of cold on hormone effects in amphibians was studied. From the data obtained, it is clear that cold acclimation profoundly varies the distribution of phosphorus in PTE-treated frogs. A marked hyperphosphaturia occurs as manifested by an increase in daily urine phosphorus levels, and by the increased rate of renal release of 32P~ Conversely, these frogs show a decreased uptake of phosphorus into soft tissues, and subsequently into mitochondria. It is evident that under normal temperatures, phosphorus liberated from skeletal elements under PTE action is ultimately incorporated into these organelles; low temperature acclimation inhibits t’he mechanism involved in such accumulation, which could provide a plasma phosphate load sufficient to cause hyperphosphaturia. The response to cold temperature, and therefore probably season, i.e., decreased parathyroid hormone production, could be cozsidered a favorable physiological adaptation of the heterothermic amphibian. If the parathyroid glands maintained their normal production of hormone during winter months, under which conditions cellular and mitochondrial incorporation of phosphorus is inhibited and the animals become hyperphosphaturic, an excessive loss of this mineral ion could occur with concomitant imbalances in energy metabolism. This concept’ is evidenced by the observation that cold-acclimated frogs showed extreme sensit.ivity to parathyroid extract; injection of 30 units was lethal to animals maintained at 668°C in contrast to those which were warm-acclimated,

EFFECTS

ON

&Vla

pipiel1.S

89

An influence of temperature ~1: phosphorus metabolism of normal amphibians was also apparent. Cold-acclimated normal frogs excreted slightly less inorganic phosphorus or saP than did normal warm-acelimated animals. This may represent an attempt to conserve phosphate under cold stress, whereas homoiotherms show increased urinary phosphorus under these aonditions (Beaton, 1963). The decreased rate of 32P transport in the cold across skin isolated from frogs kept at 6~3°C is interpreted as a low temperature effect of diffusion rates and/or metabolic activity supporting active transport. In addition, although gross studies of soft tissue a?P accumulation and total liver protein showed little difference between normal warm- an cold-acclimated frogs, mitochondria isolated from livers of the latter incorporated twice as much phosphorus and contained less protein than animals maintained at higher t,emperature. That these changes are associated with variations in carbohydrate metabolism as a result of cold exposure is evidenced by the increased glycogen in tissues of normal frogs held at low temperature. The elevated glycogen of gastrocnemius muscle probably represents decreased glycogenolysis related to cessation of motor activity in the cold. However, the increased liver glycogen: which was associated with an iuereased mitochondrial 32P incorporation and decreased mitochondrial prot’ein eoment, is more difficult to explain and may be due to either increased glycogen synthesis and/or decreases in rates of its catabolism. n’umerous studies on mammals have shown tha,t thermogenic responses t,o cold exposure include increased (1) protein catabolism, (2) oxidat’ion of fat’ty acids, (3) Krebs cycle oxidations, (4) ATP production, and (5) synthesis of mitoehondrial ~bospbol~~id in liver (cf. review by Smith and Noijer, 1962). These metabolic alterations in coldstressed mammals are regulated in large part by hormonal deployment of alternate substrates from protein and lipid catabolism into energy and heat-yielding pathways, with a consequent sparing of carbohydrate. It has also been observed thal;

90

CORTELYOU,

QUIPSE,

glucose-14C is more rapidly incorporated into glycogen in cold- than in warm-acclimated animals (Depocas et a.Z., 1957). It would thus appear that elevated glycogen levels in livers of frogs maintained in the cold may represent both an increased synthesis and a sparing action mediated by utilization of other reserves. Similar elevation of liver glycogen in heterotherms has been reported in several species of cold-acclimated fish (Dean and Goodnight, 1964). The increased 32P incorporation in liver mitochondria of cold-exposed frogs may be associated with an increase of base triphosphates through elevated activities of Krebs cycle and electron transport chain, and/or an increased mitochondrial B-oxidation of fatty acids, or with phospholipid synthesis. It would be tempting to suggest, since cytidine triphosphate increases markedly during the early hours of cold acclimation in mammals (Rossiter and Nicholls, 1957), that uridine triphosphate levels might also rise in cold-acclimated frogs and contribute via UDP-glucose to the elevated liver glycogen. In summary, this investigation has shown that phosphorus metabolism in amphibians is under parathyroid control, and that the fate of phosphorus mobilized by hormone action is influenced by the temperature to which the animal is acclimated. At ambient temperature, PTE-treated frogs become hypophosphatemic as phosphorus is incorporated into soft tissues, and ultimately mitochondria; urinary release of phosphorus tends to decrease. Conversely, PTE-treated frogs maintained in the cold demonstrate inhibition of tissue and mitochondrial accumulation of phosphorus, and hyperphosphaturia. Skin reabsorption of phosphorus voided in urine to environmental water can occur, but the rate of transport in normal frogs and those under PTE influence is identical, and thus cannot account for the net phosphorus changes observed. These effects of exogenous parathyroid extract upon temperature-inare superimposed duced changes in phosphorus metabolism. Low temperature-acclimated normal frogs respond to cold stress by (1) conservation of phosphorus through decreased renal

AND

MCWHINNIE

excretion, (2) increased mitochondrial incorporation of phosphorus, and (3) increased tissue glycogen levels; the latter probably represents the heterothermic attempt t.o spare glycogen reserves at the expense of increased lipid utilization and synthesis. Parathyroid hormone reverses the coldinduced physiological adjustments in phosphorus metabolism. REFERENCES F., AND ELLSWORTH, R. (192:9#). S’tudies on the physiology of the parathyroid glands. I. Calcium and phosphorus studies on a case of idiopathic hypoparathyroidism. J. Cl&. Invest. 7, 183-20’1. BARTELS, E. D. (1954). Action of the parathyroid hormone (a survey). Actn Endocrinol. 15, 71ALBXQHT,

81. BARTTER,

F. C. (1954). The renal action of the parathyroids. J. Clin. Endocrinol. Metab. 14,

826-827. BEATON,

J. R. (1963). Phosphorus metabolism in cold-exposed rats. Can. J. Biochem. Physiol. 41,

2209-2214. BEUTNER,

E. H., AND MUNSON, P. L. (1960). Time course of urinary excretion of inorganic phosphate by rats after parathyroidectomy and after injection of parathyroid extract. Endo-

crinology B~LLJST,

66,

6~10-616.

A. J., HANDY, J. R., AND PARSON, W. (1963). Effect of parathyroid hormone administration on bone composition in guinea pigs. Proc. Sot. Exptl. Biol. Med. 112, 868-871. BOSCHWITZ, D. (1961). Parathyroid glands of Bufo viridis Laurentis. Herpetologica 17, 19% 199. BUCHANAN, G. D. (1961). Parathyroid influence on renal excretion of calcium. In “The Parathyroids” (R. 0. Greep and R. V. Talmage, eds.), pp. 334352. Thomas, Springfield, Illinois. BUCHANAN, G. D., KRAINTZ, F. W., AND TALMAGE, R. V. (19591). Renal excretion of calcium and phosphate in the mouse as infhrenced by the para.thyroids. Proc. Sot. Exptl. Biol. Med. 101, 306-309. CALCAGNO,

P. L., AND LOWE, C. U. (1956). Studies on pa.rathyroid extract in normal subjects and a patient with pseudohypoparathyroidism. Ann.

N.Y. Acad. CARCILL, W.

Sci.

64,

456462.

H., AND WITHAM, A. C. (1949). Parathyroid hormone and the tubular reabsorption of glucose and phosphate in man. Federation Proc. 88, 21-28. CARROLL, N. V., LONGLEY, R. W., AND Ron, 5. H. (1956). The determination of glycogen in liver

PARATHYROID

EXTRACT

and muscle by use of anthrone reagent. J. Biol. Chem. 220, 583-593. CHBNG, H. Y. (1951). Grafts of parathgroid and o’ther tissues to hone. Anat. Record 111, 2S-48. CLARK, N. B. (196’5). Experimental and histological studies of the parathyroid glands of freshwater turtles. Germ. Comp. Endocrinol. 5, 297312. CORTELYOU, J. R. (1960a). Plasma and urine phospborus changes in totally parathyroidectomized Rnna pipiens. hat. Record 137, 346. CORTELYOU, J. R. (1960b). The effects of commercially prepared parathormone on calcium and phosphorus levels in unoperated Rana pipiens. bat. Record 138, 341. CORTELYO~, J. R., HIBNER-OWERKO, il., AND, MULROY, J. (196Oc). B!ood and urine calcium changes in totally parathyroideetomized Rana pipiens. Enclo&nologu 166, 441-450. CORTELPOU, J. R. (1962a). Changes in plasma and urine calcium and phosphorus of Rana pipiens subsequent to different dose levels of Paroidin. Am. Zool. 2, 405. CORTELY~U, J. R. (1962b). Phosphorus changes in totally parathyroidectomized Rnna pipiells. Endocrinology ‘70, 618-621. CORTELYOU, J. R. (1965). Parathormone influenced metabolism in amphibia. Ezcerpta Med. Intern. Congr. S-r. 99, 101. I~E~N, J. M., AND GOODNIGHT, C. J. (1964). A comparative study of carbohydrate metabolism in fish as affected by temperature and exercise. Physiol. Zool. 37, 280-299. n~pocas, P., MA~LEoD, G. K., AND HART, J. S. (1957). St,udies on glucose metabolism of warmand cold-acclimated rats at -5°C. Rev. Can. Biol. 116, 83-95. DRYER, R. L., TAMMES, A. R., AND ROUTH, J. L. (1957). The determination of phosphorus and phosphatase with N-phenyl-p-phenylenedinmine. J. Biol. Chem. 225, 177-183. &XWA, J., AND KEUMPIN, W. F. (1964). Effect of parathyroid hormone on phosphate turnover in bone and kidney. Endocrinology 72, 370-376. FLEMING, W. R., END MEIER, A. (1961). The effect of mammalian parathormone on the s?rum calcium levels of Fund&s kansae and Fundulus catenatus. Comp. Biochem. Physiol. ‘2, 1-7. FOULKS, J. G., .4ND PXRRY, F. A. (19159a). Renal excretion of phosphate following parathfroidectomy in the dog. Am. .I. Physiol. 196, 554-560. FOCLKS, J. G., AND PERRY, F. il. (1959b). Mechanism of the phosphaturic action of acute intravenous injection of parathyroid extracts in the dog. Am. .I. Physiol. 196, 561-566. G.4ILLARD, P. J. (19%). Parathyroid gland tissue

EFFECTS

m

and bone 154-169.

Rana in

pipiens

vi&o.

Exptl.

91 Cell

Rex.

Suppl.

3,

I., ASD KLETTER, M. (1964). Effect* of parathyroid hormone on bone composition in rats. Endocrinology ‘74, 165-16’9. HANDLER, P., COHN, D. V., AND DEMAKIA, TV. (19851). Effect of parathyroid extract on the renal excretion of phosphate. Sm. J. Physioi. 265, 4X4441. HARRISON, H. E., ATD HARRIRON, N. C. (10-11) The renal excretion of inorganic phosphate in relation to the action of Vitamin D and parsthyroid hormone. .J. Cli~z. Invest. 20, 47-55. ISONO, H. (1960). Histological study of the parathyroid gland in the toad (B~jo 71uEgaris jnponicus). Acta Schol. Med. Gifu 8, 277-293. ITO, Y.? TSURUFUJI, S., AND SHIKIT~, M3. (196% The effects of parathyroidectomy on p1~osphoru.c reabsorption in the renal tubules in rats. E&docrinol. Jrrpon. ‘9, 181-186. KITSON, R. E., .~SD PVIELLOX, M. G. (1944). Fur-ther studies of the molybdenum blue rca~%ion Ind. Eng. Chem. Anal. Ed. 16, 466-469. LEVINSKY, N. G., AND DAv~nsos, U. G. ilY57j Renal action of parathyroid extrizcc in i’r~ chicken. Am. J. Pky.siol. 191, 530-536. LOWRY, 0. EL, ROSEBROUGH, IT. J., FARR, LY, L., AXD R.~NDALL, R. J. (1951), Protrin measurement with the Folin phenol reagent. 1. t?iol Chem. 193, 265-275. NICHOLSON, T. F. (19589). The mode aud site oi renal action of parathyroid extract in the do? Can. J. Biochena. Physiol. 37, 11%-117. OYAMA, V. 1.: AXD EAGLE, Ii. (1956). Mrasurerncnr of cell growth in tissue culture with phenol rcagent. Proc. Sot. Ezptl. Biol. Med. 91, 3G-307. RASMUSSEN, H., &LIZ, J., Fax;c, M., D~Lr-cs. Ii.: AND YOUNG:, R. (1964) Parathyroid hormone ant! anion uptake of isolated mitochondria. Blidocrinology 74, 388-394. ROSSITER, R. J., AND NICHOLLS, D. (1957). RIGSphorus metabolism of the adrenal gixctls of rats exposed to a cold environment. Rev. Calr. Bioi. 116, 249-268. SALLIS, j., D~I,r;c.i, H., AXD R.4~mss~;~, Ii. (1963a). Parathyroid hormone-dependent U;Jtake of inorganic phosphate by mitochondria. J. BioZ. Chent. 238, 409%4102. S4LLI6, J., DELIUCA, H.. 4ND ~ASML’SXES, &I. (1963b). Parathyroid hormone stimulation of phosphate uptake bs liver mito~chondria. niothem. Biophys. Rc.~. Comnaun. 10, 266-270. SCHRIRE, V. (1941). Changes in plasma inorganic phosphate assoriated with endocrine activity ix1 Xerloplts Eaevis. S. African J. Med. Scz. 16, 1-5. SXITH, R. E., .SND HOIJER, D. J. (1962). MetabGEDALIA,

92

CORTELYOU,

QUIPSE,

olism and cellular function in cold acclimation. Physiol. Rev. 42, 60-142. TALMAGE, R. V., AND KRAINTZ, F. W. (19.54). Immediate changes in phosphate excretion following parathyroidectomy in the rat. Proc. SOC. Exptl. Biol. Med. ‘85, 416-419. TALMAGE, R. V., WIMER, L. T., AND TOFT, R. J. (1960). Additional evidence in support of McLean’s feedback mechanism of parathyroid action on bone. Clin. Orthoped. 17, 195-294.

AND

MCWHINNIE

W. R., AND CAMPBELL, W. W. (1944). The effects of parathyroid extract upon the distribution, retention and excretion of labelled phosphorus. J. Biol. Chem. 1.54, 339-347. VON BREHM, H. V. (1964). Morphologische Untersuchungen an Epithelkorperchen (Glandulae Parathyroideae) van Anuren. Z. Zellforsch. Mikroskop. Anat. 61, 376-400. TWEEDY,