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Species differences in the effect of parathyroid hormone on cyclic amp production
Comp. Biochem. PhvsioL V o / 67A. pp. 471 to 475
0300-9629/80/1101-0471502.00/0
9 Pergamon Press Ltd 1980 Printed in Great Britain
SPECIES DIFFERENCES IN THE EFFECT OF PARATHYROID H O R M O N E ON CYCLIC AMP P R O D U C T I O N 1. LEWIN, S. TOMLINSON, G. N. HENDY, J. L. H. O'R1ORDAN, 1D. W. PICKARD and 1A. D. CARE Department of Medicine, Middlesex Hospital, London, W1N 8AA and 1Department of Animal Physiology and Nutrition, University of Leeds, Leeds LS2 9JT, England
(Received 11 January 1980) Abstract 1. The effects of injected parathyroid hormone on the renal excretion of cyclic AMP and on its plasma concentration have been studied in chicken, goose, rabbit, pig, calf and man. 2. The amplitude of the peak plasma cyclic AMP response to a single injection of parathyroid hormone showed considerable differences between the species examined especially when a large dose of the hormone was used. The response shown by calves, aged 2 days to 6 weeks, was particularly poor. A sigmoid dose response curve was obtained in a human subject on measuring the maximum plasma cyclic AMP concentration in response to injected parathyroid hormone. 3. In general, there was no obvious relationship between plasma concentrations of cyclic AMP and its excretion rate by the kidneys but in the calf both parameters were low. Similarly, there appeared to be no simple relationship between extracellular cyclic AMP concentration and the phosphaturic response to the administration of parathyroid hormone.
INTRODUCTION Parathyroid h o r m o n e (PTH) stimulates renal adenylate cyclase, initiating the production of adenosine 3'5'cyclic m o n o p h o s p h a t e (cyclic A M P ) which mediates some of the final biological effects of the hormone. In the rat, intravenous injection of bovine parathyroid h o r m o n e (BPTH) is rapidly followed by an increase in urinary cyclic A M P excretion, the peak of which precedes the peak of phosphate excretion (Chase & Aurbach, 1967). In man, it causes an increase in b o t h plasma and urinary cyclic AMP, but the p h o s p h a t u r i c response may be variable (Tomlinson et al., 1974). The calcium and phosphorus requirements of animals vary with species leading to different demands on calcium and phosphorus homeostasis. This might be associated with species differences in control mechanisms. There have been few studies of the effect of B P T H on extracellular cyclic A M P in species other than man. We have, therefore, examined the changes in the cyclic A M P produced following the administration of B P T H to chicken, goose, rabbit, pig, calf as well as man. METHODS
Highly purified bovine parathyroid hormone was prepared for injection or infusion by dissolving the freezedried powder in 5% dextrose solution containing 1~ bovine or porcine serum albumin. Plasma and urinary cyclic AMP and, in some cases, urinary phosphate were measured as previously described (Tomlinson et al., 1974). All animals were fed normal commercial diets, being fasted overnight prior to the day of study but having free access to water.
Man Three normal subjects, aged 21, 23 and 32, fasted overnight and remained at rest during the test procedures. They 471
were encouraged to drink liberal quantities of water to allow frequent urine collection. Bovine PTH was administered via an antecubital vein and blood samples were obtained via a cannula in the contralateral antecubital vein and urine samples collected at suitable intervals.
Chicken and 9oose Anaesthesia was induced in 1 chicken and 3 geese by intramuscular injection of equithesin. Light anaesthesia was then maintained with halothane and oxygen. Normal saline was infused at the rate of 0.1 ml rain -1 (chicken) or 0.4-0.8 ml rain- ' (goose) via a jugular vein catheter to promote a diuresis. BPTH was injected or infused via the same catheter and blood samples were drawn from a catheter in the contralateral jugular vein. Urine samples were obtained via ureteric catheters. Rapid intravenous injections of BPTH were administered to 1 hen and 2 geese. A third goose underwent a 2 hr infusion of BPTH followed immediately by a rapid intravenous injection of the hormone. Rabbit Single, rapid injections of BPTH were administered via a central ear vein to 2 conscious rabbits held within restraining cages. Blood samples were obtained from cannulae in the contralateral central ear veins. Ply Anaesthesia was induced in 2 female pigs with etorphine (Immobilon, Reckitt & Colman, Hull) and maintained with halothane and oxygen. Mannitol (5% in 0.07 M sodium chloride) was infused via an intravenous catheter to maintain a diuresis. A solution of BPTH was injected or infused via the same cannula. Blood samples were drawn from a second catheter in a peripheral vein and urine samples were obtained via an indwelling Foley catheter in the bladder. Calf Anaesthesia was induced with etorphine (10#lkg -1 Large Animal Immobilon, Reckitt & Colman) and maintained with halothane and oxygen. Using female calves the bladder was catheterized via the urethra using a Foley
472
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catheter. Infusions were carried out via a catheter placed in an external jugular vein. In 2 calves an injection of BPTH was administered at the age of 2 days and again at the age of 5 weeks. One of these calves was further studied at the age of 6 weeks when it was infused with BPTH over 2 h followed immediately by a rapid intravenous injection of the hormone.
to 447 nmol/kg/hr in the hour following the injection of PTH. Goose
After an injection of 3.5 units BPTH/kg, the plasma cyclic AMP concentration rose to a peak of 215 nmol/1 from an initial value of 79 nmol/l. Basal urinary cyclic AMP excretion was 21 nmol/kg/hr and increased to 79 nmol/kg/hr in the hour after injection. Phosphate excretion was not measured. In the second goose a dose of 11.8 units PTH/kg caused the plasma cyclic AMP concentration to rise from 76 to 606 nmol/1 and the urinary cyclic AMP excretion to increase from 80 to 292 nmol/kg/hr in the hour after injection but there was no significant increase in phosphate excretion. During an infusion of 6.9 units BPTH/kg/hr (total 50 units) over 2 h to a third goose, plasma cyclic AMP concentration increased from 21 nmol/l to a peak of 53 nmol/l after 100 min and then declined to 29 nmol/1 by the end of the infusion. There was no significant change in urinary cyclic AMP excretion during the infusion. At the termination of the infusion an injection of 2.7 units BPTH/kg (total 10 units) produced an increase in plasma cyclic AMP of 31 nmol/1 and a doubling in urinary cyclic AMP excretion from 5 to 10 nmol/kg/hr (Fig. 2a).
RESULTS
Man
In 2 normal subjects, an intravenous injection of BPTH was followed by a rapid and marked increase in plasma cyclic AMP which reached a peak within 12min, declining rapidly thereafter to reach basal values 60-90 min after injection. In subject SM an injection of 3.8 units PTH/kg produced an increase in plasma cyclic AMP concentration from 18 nmol/1 to 177nmol/l and an increase in urinary cyclic AMP excretion from 4 nmol/kg/lar to 179 nmol/kg/hr in the hour after injection. In subject MD an injection of 4.3 units PTH/kg caused the plasma cyclic AMP concentration to rise from 15 nmol/l to 352nmol/1 and the urinary cyclic AMP excretion to increase from 5 nmol/kg to 319 nmol/kg/hr in the hour after injection. In both subjects ther6 was a significant increase in urinary phosphate excretion (7.5 fold and 4.7 fold, respectively). In a third normal subject (GH), the plasma cyclic AMP response to injection of graded doses of BPTH showed a sigmoid dose response curve (Fig. 1). There was a small increase in plasma cyclic AMP concentration with doses of BPTH up to 1.3 units/kg (total dose of 100 units PTH), followed by a marked increase with 2.6 units/kg (total dose of 200 units PTH) after which the cyclic AMP responses reached a maximum between a PTH dosage of 3.8 units/kg (250 units) and 7.6 units/kg (500 units).
Rabbit
An injection of 2.9 units BPTH/kg into a rabbit caused a small increase in plasma cyclic AMP concentration of 12 nmol/l and in a second rabbit a dose of 4.0 units PTH/kg produced a greater increase of 108 nmol/L Pi#
The injection of 20units BPTH/kg (total dose 500 units) into a pig caused a rapid increase in plasma cyclic AMP concentration from 18 to 78 nmol/l. Urinary cyclic AMP excretion increased from a basal value of 13 nmol/kg/hr to 104 nmol/kg/hr in the hour following injection. However, no significant increase in urinary phosphate excretion occurred. An infusion of 16.7units BPTH/kg (total dose
Chicken
An injection of 20 units BPTH/kg caused the plasma cyclic AMP concentration to rise from a basal level of 31 nmol/l to 540 nmol/1, the peak value being reached 7 min after the injection. Basal urinary cyclic AMP excretion was 38 nmol/kg/hr and this increased
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Fig. 1. The relationship between the maximum plasma concentration of cyclic AMP achieved and the dose of bovine parathyroid hormone .(BPTH) injected intravenously into an adult human volunteer.
Effect of parathyroid hormone on cyclic AMP production 250units) was administered to a second pig over 30 min. There was no significant change in plasma cyclic AMP concentration throughout the infusion but urinary cyclic AMP excretion increased from 2.0 nmol/kg/hr to 7 nmol/kg/hr during the hour after the beginning of the infusion. No significant decline in response was observed during this short infusion in contrast to the blunted response noted in man (Tomlinson et al., 1976). Phosphate excretion increased from the basal level of 8.5/~mol/min to 30 ~mol/min in the 30 rain following the end of the infusion.
473
ing the injection (Fig. 2b). There was no significant change in the plasma concentration of cyclic AMP during this experiment. DISCUSSION
Cyclic AMP acts as an intracellular mediator in the action of a number of peptide hormones and some of this nucleotide may, to a varying degree, escape into extracellular fluids. In the human subjects, injection of BPTH was followed by a rapid rise in plasma cyclic AMP which Calf reached peak values between 5 and 12 min and then In a 2 day calf an injection of 500 units BPTH quickly declined. Urinary cyclic AMP responses were (15.2 units/kg) produced a small rise in plasma cyclic maximal in the first hour after injection. Kaminsky et AMP from 7 to 16 nmol/1. Basal urinary cyclic AMP at. (1970) were able to demonstrate a dose-dependent excretion was 1 nmol/kg/hr and this increased to increase in urinary excretion of cyclic AMP in man 7 nmol/kg/hr in the hour following the injection. In after infusion of PTH. Our study in a normal subject the same animal at 5 weeks of age the same total dose now shows that graded amounts of BPTH given by of BPTH (now equivalent to 8.6 units/kg) failed to injection also produce a dose-related increase in the produce any significant increase in plasma cyclic peak plasma cyclic AMP response, maximal stimuAMP, but there was an increase in urinary cyclic lation of renal adenylate cyclase being attained at a AMP excretion from 1 to 8 nmol/kg/hr in the hour dose of 3.8 units PTH/kg. after injection. On neither occasion was there any sigThe human extracellular cyclic AMP response to nificant change in urinary phosphate excretion. administered BPTH is difficult to compare with those In a second calf, the response to the injection of of other species (Fig. 3) because of possible differences BPTH was essentially similar. At the age of 2 days an in the distribution and metabolism of the injected injection of 500 units BPTH (16.7 units/kg) produced hormone and of the cyclic AMP produced. However, a small rise in plasma cyclic AMP from 5 to 14 nmol/1 in some of the species examined there were similariand an increase in urinary cyclic AMP excretion from ties to man. Injections of BPTH into chicken, goose a basal value of 0.1 nmol/kg/hr to 3 nmol/kg/hr in the and rabbit produced rapid increases in plasma cyclic hour after injection. At 5 weeks the same total dose of AMP with time courses similar to those in the human BPTH (now equivalent to 9.1 units/kg) caused a small subjects. In the chicken and goose these changes were increase in plasma cyclic AMP from 5 to 9 nmol/l and also reflected in an increase in urinary cyclic AMP an increase in urinary cyclic AMP excretion from excretion. In the pig, the peak plasma cyclic AMP 0.4 nmol/kg/hr to 3 nmol/kg/hr in the hour after injec- response occurred slightly later, at 15 rain, and the tion. In this calf, studied again at the age of 6 weeks, decline in concentration was slower but the pattern of an infusion of BPTH was followed by an injection of urinary response was similar to that observed in man. the hormone. The infusion of 1000 units BPTH over The magnitude of the peak plasma cyclic AMP re2 hr (8.3 units/kg/hr) caused an increase in urinary sponse to a single injection of PTH showed considerexcretion of cyclic AMP but this declined towards the. able differences between the species examined (Fig. 3) end of the infusion. At the termination of the infusion and this was most evident in the studies using large an injection of 500 units of the hormone (7.7 units/ doses of the hormone. The cyclic AMP response to kg/hr) caused a further increase in urinary cyclic PTH injection in chicken, goose, rabbit and pig was AMP excretion from an initial value of characterized by relatively large changes in both 0.5 nmol/kg/hr to 1.6 nmol/kg/hr in the hour follow- plasma and urine. In contrast, in the calf, the magniBPTH
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Fig. 2. (a) The effects of an intravenous infusion to an adult goose of a total dose of 50 units bovine parathyroid hormone (BPTH), followed by a single intravenous injection of l0 units BPTH, on the plasma concentration and renal excretion rate of cyclic AMP. (b) The effect of an intravenous infusion to a 6 weeks old calf of a total dose of 1000 units BPTH, followed by a single injection of 500 units BPTH, on the renal excretion rate of cyclic AMP. C.B.P. 67/3A- K
474
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Fig. 3. A comparison between the maximum rise in plasma cyclic AMP concentration produced in response to the amount of bovine parathyroid hormone (BPTH) injected intravenously. tude of the plasma and urinary cyclic AMP responses was comparatively small. Many factors may account for the difference in magnitude of the cyclic AMP responses to PTH injection observed in these species. The volume of distribution of the injected BPTH would influence the concentration of the hormone at the renal receptor sites and may differ in the species examined. This was found to be approximately 30~o of body weight in the calf (Sherwood et al., 1968) but only 8.6~o of body weight in man (Papapoulos et al., 1977). Clearance of the hormone might also differ, and slower metabolism in some species might therefore allow more sustained hormonal action. Differences in plasma membrane interactions with PTH might account for some of the species differences in the magnitude of the extracellular cyclic AMP responses. For example, Martin et al. (1974) found that synthetic 1-34 fragments of bovine and human PTH generated similar amounts of cyclic AMP in renal cortical membrane preparations derived from the chick whereas the bovine fragment was 5 times more potent than the human fragment in preparations from the rat. There appears to be no simple relationship between changes in extracellular cyclic AMP concentration and the phosphaturic response to PTH administration. In the two human subjects shown here, increases in extracellular cyclic AMP were accompanied by increases in urinary phosphate excretion. However, phosphaturia may occur in response to infusions of a low dose of BPTH when no increase in extracellular cyclic AMP is detectable (Walker et al., 1977) and, conversely, the phosphaturic response in man may be insignificant even when large increases in extracellular cyclic AMP occur (Tomlinson et al., 1974). Kaminski et al. (1970) found that infusions of BPTH into a normal human subject continued to produce a dose related increase in urinary cyclic AMP excretion even after phosphaturia had reached a plateau. It therefore appeared that the maximal physiological effect of the
hormone had been achieved by submaximal stimulation of adenylate cyclase, a phenomenon also observed with other hormones. Injections of BPTH into goose and calf failed to cause an increase in phosphate excretion despite detectable changes in extracellular cyclic AMP, although a phosphaturic effect of PTH has been reported in the chicken (Levinsky & Davidson, 1957) and the starling (Clark et al., 1976) after intravenous injection of the hormone. In the cow, Mayer et al. (1966) found that a single injection of 1200 units BPTH had no effect on urinary phosphate excretion but an increase occurred in the same animal after repeated administration of the hormone over several days. The relatively poor cyclic AMP and phosphaturic responses of the rumina n t kidney to BPTH may be related to the fact that the ruminant's salivary glands rather than the kidneys play the dominant role in phosphate excretion in response to BPTH (Clark, et al., 1975). These results show that in some species there can be a dissociation between the urinary excretion of phosphate and cyclic AMP following the administration of PTH. It is possible that this variation reflects specific differences in mineral homeostasis. Acknowledgement--The skilled technical assistance of Mr T. D. Gibson is gratefully acknowledged. We are indebted to the Medical Research Council for financial support.
REFERENCES
BARLINGP. M., ALBANOJ. D. M., TOMLINSONS., BROWNB. L. & O'RIORDAN J. L. H. (1974) A saturation assay method for adenosine 3',5'-cycle monophosphate in plasma and its use in studies of the action of bovine parathyroid hormone. Biochem. Soc. Trans. 2, 453-455. BROWN B. L., ALBANOJ. D. M., EKINS R. P., SGHERZIA. M. & TAMPIONW. (1971) A simple and sensitive saturation assay method for the measurement of adenosine 3',5'-cyclic monophosphate. Biochem. J. 121, 561-562.
Effect of parathyroid hormone on cyclic AMP production CHASE L. R. & AURBACHG. D. (1967) Parathyroid function and the renal excretion of 3',5'-adenylic acid. Proc. natn. Acad. Sci. U.S.A. 58, 518 528. CLARK N. B., BRAUN E. J. & WlDEMAN R. F. (1976) Parathyroid hormone and renal excretion of phosphate and calcium in normal starlings. Am. d. Physiol. 231, 1152-1158. CLARK R. C., FRENCH T. J., BEAL A. M., CROSS R. B. & BUDTZ-OLSEN O. E. (1975) The acute effects of intravenous infusion of parathyroid hormone on urine, plasma and saliva in the sheep. Q. J. exp. Physiol. 60, 95-106. KAMINSKI N. I., BROAOUS A. E., HARDMAN J. G., JONES D. J., BALL J. H., SUTHERLAND E. W. & LIDDLE G. W. (1970) Effects of parathyroid hormone on plasma and urinary Y,5'-monophosphate in man. J. clin. Invest. 49, 2387-2395. LEVINSKY N. G. & DAVIDSOND. C. (1957) Renal action of parathyroid extract in the chicken. Am. J. Physiol. 191, 530-536. MAYER G. P., MARSHAK R. R. & KRONFELD D. S. (1966)
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Parathyroid effects on renal phosphorus excretion in the cow. Am. J. Physiol. 211, 1366-1370. SHERWOOD L. M., MAYER G. P., HAMBERG C. F., KRONFELD D. S., AURBACHG. D. & POTTS J. T. (1968) Regulation of parathyroid hormone secretion: proportional control by calcium, lack of effect of phosphate. Endocrinology 83, 1043-1051. TOMLINSON S., BARLINGP. M., ALBANOJ. D. M., BROWN B. L. & O'RIORDAN J. L. H. (1974) The effect of exogenous parathyroid hormone on plasma and urinary adenosine Y,5'-cyclic monophosphate in man. Clin. Sci. molec. Med. 47, 481-492. TOMLINSON S., HENDY G. N., PEMBERTOND. M. & O'RIORDAN J. L. H. (1976) Reversible resistance to the renal action of parathyroid hormone in man. Clin. Sci. molec. Med. 51, 59-69. WALKER D. A., DAVIES S. J., SIDDLE K. & WOODHEADJ. S. (1977) Control of renal tubular phosphate reabsorption by parathyroid hormone in man. Clin. Sci. molec. Med. 53, 431-438.
0300-9629/80/1101-0471502.00/0
9 Pergamon Press Ltd 1980 Printed in Great Britain
SPECIES DIFFERENCES IN THE EFFECT OF PARATHYROID H O R M O N E ON CYCLIC AMP P R O D U C T I O N 1. LEWIN, S. TOMLINSON, G. N. HENDY, J. L. H. O'R1ORDAN, 1D. W. PICKARD and 1A. D. CARE Department of Medicine, Middlesex Hospital, London, W1N 8AA and 1Department of Animal Physiology and Nutrition, University of Leeds, Leeds LS2 9JT, England
(Received 11 January 1980) Abstract 1. The effects of injected parathyroid hormone on the renal excretion of cyclic AMP and on its plasma concentration have been studied in chicken, goose, rabbit, pig, calf and man. 2. The amplitude of the peak plasma cyclic AMP response to a single injection of parathyroid hormone showed considerable differences between the species examined especially when a large dose of the hormone was used. The response shown by calves, aged 2 days to 6 weeks, was particularly poor. A sigmoid dose response curve was obtained in a human subject on measuring the maximum plasma cyclic AMP concentration in response to injected parathyroid hormone. 3. In general, there was no obvious relationship between plasma concentrations of cyclic AMP and its excretion rate by the kidneys but in the calf both parameters were low. Similarly, there appeared to be no simple relationship between extracellular cyclic AMP concentration and the phosphaturic response to the administration of parathyroid hormone.
INTRODUCTION Parathyroid h o r m o n e (PTH) stimulates renal adenylate cyclase, initiating the production of adenosine 3'5'cyclic m o n o p h o s p h a t e (cyclic A M P ) which mediates some of the final biological effects of the hormone. In the rat, intravenous injection of bovine parathyroid h o r m o n e (BPTH) is rapidly followed by an increase in urinary cyclic A M P excretion, the peak of which precedes the peak of phosphate excretion (Chase & Aurbach, 1967). In man, it causes an increase in b o t h plasma and urinary cyclic AMP, but the p h o s p h a t u r i c response may be variable (Tomlinson et al., 1974). The calcium and phosphorus requirements of animals vary with species leading to different demands on calcium and phosphorus homeostasis. This might be associated with species differences in control mechanisms. There have been few studies of the effect of B P T H on extracellular cyclic A M P in species other than man. We have, therefore, examined the changes in the cyclic A M P produced following the administration of B P T H to chicken, goose, rabbit, pig, calf as well as man. METHODS
Highly purified bovine parathyroid hormone was prepared for injection or infusion by dissolving the freezedried powder in 5% dextrose solution containing 1~ bovine or porcine serum albumin. Plasma and urinary cyclic AMP and, in some cases, urinary phosphate were measured as previously described (Tomlinson et al., 1974). All animals were fed normal commercial diets, being fasted overnight prior to the day of study but having free access to water.
Man Three normal subjects, aged 21, 23 and 32, fasted overnight and remained at rest during the test procedures. They 471
were encouraged to drink liberal quantities of water to allow frequent urine collection. Bovine PTH was administered via an antecubital vein and blood samples were obtained via a cannula in the contralateral antecubital vein and urine samples collected at suitable intervals.
Chicken and 9oose Anaesthesia was induced in 1 chicken and 3 geese by intramuscular injection of equithesin. Light anaesthesia was then maintained with halothane and oxygen. Normal saline was infused at the rate of 0.1 ml rain -1 (chicken) or 0.4-0.8 ml rain- ' (goose) via a jugular vein catheter to promote a diuresis. BPTH was injected or infused via the same catheter and blood samples were drawn from a catheter in the contralateral jugular vein. Urine samples were obtained via ureteric catheters. Rapid intravenous injections of BPTH were administered to 1 hen and 2 geese. A third goose underwent a 2 hr infusion of BPTH followed immediately by a rapid intravenous injection of the hormone. Rabbit Single, rapid injections of BPTH were administered via a central ear vein to 2 conscious rabbits held within restraining cages. Blood samples were obtained from cannulae in the contralateral central ear veins. Ply Anaesthesia was induced in 2 female pigs with etorphine (Immobilon, Reckitt & Colman, Hull) and maintained with halothane and oxygen. Mannitol (5% in 0.07 M sodium chloride) was infused via an intravenous catheter to maintain a diuresis. A solution of BPTH was injected or infused via the same cannula. Blood samples were drawn from a second catheter in a peripheral vein and urine samples were obtained via an indwelling Foley catheter in the bladder. Calf Anaesthesia was induced with etorphine (10#lkg -1 Large Animal Immobilon, Reckitt & Colman) and maintained with halothane and oxygen. Using female calves the bladder was catheterized via the urethra using a Foley
472
I. LEwx~ et al.
catheter. Infusions were carried out via a catheter placed in an external jugular vein. In 2 calves an injection of BPTH was administered at the age of 2 days and again at the age of 5 weeks. One of these calves was further studied at the age of 6 weeks when it was infused with BPTH over 2 h followed immediately by a rapid intravenous injection of the hormone.
to 447 nmol/kg/hr in the hour following the injection of PTH. Goose
After an injection of 3.5 units BPTH/kg, the plasma cyclic AMP concentration rose to a peak of 215 nmol/1 from an initial value of 79 nmol/l. Basal urinary cyclic AMP excretion was 21 nmol/kg/hr and increased to 79 nmol/kg/hr in the hour after injection. Phosphate excretion was not measured. In the second goose a dose of 11.8 units PTH/kg caused the plasma cyclic AMP concentration to rise from 76 to 606 nmol/1 and the urinary cyclic AMP excretion to increase from 80 to 292 nmol/kg/hr in the hour after injection but there was no significant increase in phosphate excretion. During an infusion of 6.9 units BPTH/kg/hr (total 50 units) over 2 h to a third goose, plasma cyclic AMP concentration increased from 21 nmol/l to a peak of 53 nmol/l after 100 min and then declined to 29 nmol/1 by the end of the infusion. There was no significant change in urinary cyclic AMP excretion during the infusion. At the termination of the infusion an injection of 2.7 units BPTH/kg (total 10 units) produced an increase in plasma cyclic AMP of 31 nmol/1 and a doubling in urinary cyclic AMP excretion from 5 to 10 nmol/kg/hr (Fig. 2a).
RESULTS
Man
In 2 normal subjects, an intravenous injection of BPTH was followed by a rapid and marked increase in plasma cyclic AMP which reached a peak within 12min, declining rapidly thereafter to reach basal values 60-90 min after injection. In subject SM an injection of 3.8 units PTH/kg produced an increase in plasma cyclic AMP concentration from 18 nmol/1 to 177nmol/l and an increase in urinary cyclic AMP excretion from 4 nmol/kg/lar to 179 nmol/kg/hr in the hour after injection. In subject MD an injection of 4.3 units PTH/kg caused the plasma cyclic AMP concentration to rise from 15 nmol/l to 352nmol/1 and the urinary cyclic AMP excretion to increase from 5 nmol/kg to 319 nmol/kg/hr in the hour after injection. In both subjects ther6 was a significant increase in urinary phosphate excretion (7.5 fold and 4.7 fold, respectively). In a third normal subject (GH), the plasma cyclic AMP response to injection of graded doses of BPTH showed a sigmoid dose response curve (Fig. 1). There was a small increase in plasma cyclic AMP concentration with doses of BPTH up to 1.3 units/kg (total dose of 100 units PTH), followed by a marked increase with 2.6 units/kg (total dose of 200 units PTH) after which the cyclic AMP responses reached a maximum between a PTH dosage of 3.8 units/kg (250 units) and 7.6 units/kg (500 units).
Rabbit
An injection of 2.9 units BPTH/kg into a rabbit caused a small increase in plasma cyclic AMP concentration of 12 nmol/l and in a second rabbit a dose of 4.0 units PTH/kg produced a greater increase of 108 nmol/L Pi#
The injection of 20units BPTH/kg (total dose 500 units) into a pig caused a rapid increase in plasma cyclic AMP concentration from 18 to 78 nmol/l. Urinary cyclic AMP excretion increased from a basal value of 13 nmol/kg/hr to 104 nmol/kg/hr in the hour following injection. However, no significant increase in urinary phosphate excretion occurred. An infusion of 16.7units BPTH/kg (total dose
Chicken
An injection of 20 units BPTH/kg caused the plasma cyclic AMP concentration to rise from a basal level of 31 nmol/l to 540 nmol/1, the peak value being reached 7 min after the injection. Basal urinary cyclic AMP excretion was 38 nmol/kg/hr and this increased
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Fig. 1. The relationship between the maximum plasma concentration of cyclic AMP achieved and the dose of bovine parathyroid hormone .(BPTH) injected intravenously into an adult human volunteer.
Effect of parathyroid hormone on cyclic AMP production 250units) was administered to a second pig over 30 min. There was no significant change in plasma cyclic AMP concentration throughout the infusion but urinary cyclic AMP excretion increased from 2.0 nmol/kg/hr to 7 nmol/kg/hr during the hour after the beginning of the infusion. No significant decline in response was observed during this short infusion in contrast to the blunted response noted in man (Tomlinson et al., 1976). Phosphate excretion increased from the basal level of 8.5/~mol/min to 30 ~mol/min in the 30 rain following the end of the infusion.
473
ing the injection (Fig. 2b). There was no significant change in the plasma concentration of cyclic AMP during this experiment. DISCUSSION
Cyclic AMP acts as an intracellular mediator in the action of a number of peptide hormones and some of this nucleotide may, to a varying degree, escape into extracellular fluids. In the human subjects, injection of BPTH was followed by a rapid rise in plasma cyclic AMP which Calf reached peak values between 5 and 12 min and then In a 2 day calf an injection of 500 units BPTH quickly declined. Urinary cyclic AMP responses were (15.2 units/kg) produced a small rise in plasma cyclic maximal in the first hour after injection. Kaminsky et AMP from 7 to 16 nmol/1. Basal urinary cyclic AMP at. (1970) were able to demonstrate a dose-dependent excretion was 1 nmol/kg/hr and this increased to increase in urinary excretion of cyclic AMP in man 7 nmol/kg/hr in the hour following the injection. In after infusion of PTH. Our study in a normal subject the same animal at 5 weeks of age the same total dose now shows that graded amounts of BPTH given by of BPTH (now equivalent to 8.6 units/kg) failed to injection also produce a dose-related increase in the produce any significant increase in plasma cyclic peak plasma cyclic AMP response, maximal stimuAMP, but there was an increase in urinary cyclic lation of renal adenylate cyclase being attained at a AMP excretion from 1 to 8 nmol/kg/hr in the hour dose of 3.8 units PTH/kg. after injection. On neither occasion was there any sigThe human extracellular cyclic AMP response to nificant change in urinary phosphate excretion. administered BPTH is difficult to compare with those In a second calf, the response to the injection of of other species (Fig. 3) because of possible differences BPTH was essentially similar. At the age of 2 days an in the distribution and metabolism of the injected injection of 500 units BPTH (16.7 units/kg) produced hormone and of the cyclic AMP produced. However, a small rise in plasma cyclic AMP from 5 to 14 nmol/1 in some of the species examined there were similariand an increase in urinary cyclic AMP excretion from ties to man. Injections of BPTH into chicken, goose a basal value of 0.1 nmol/kg/hr to 3 nmol/kg/hr in the and rabbit produced rapid increases in plasma cyclic hour after injection. At 5 weeks the same total dose of AMP with time courses similar to those in the human BPTH (now equivalent to 9.1 units/kg) caused a small subjects. In the chicken and goose these changes were increase in plasma cyclic AMP from 5 to 9 nmol/l and also reflected in an increase in urinary cyclic AMP an increase in urinary cyclic AMP excretion from excretion. In the pig, the peak plasma cyclic AMP 0.4 nmol/kg/hr to 3 nmol/kg/hr in the hour after injec- response occurred slightly later, at 15 rain, and the tion. In this calf, studied again at the age of 6 weeks, decline in concentration was slower but the pattern of an infusion of BPTH was followed by an injection of urinary response was similar to that observed in man. the hormone. The infusion of 1000 units BPTH over The magnitude of the peak plasma cyclic AMP re2 hr (8.3 units/kg/hr) caused an increase in urinary sponse to a single injection of PTH showed considerexcretion of cyclic AMP but this declined towards the. able differences between the species examined (Fig. 3) end of the infusion. At the termination of the infusion and this was most evident in the studies using large an injection of 500 units of the hormone (7.7 units/ doses of the hormone. The cyclic AMP response to kg/hr) caused a further increase in urinary cyclic PTH injection in chicken, goose, rabbit and pig was AMP excretion from an initial value of characterized by relatively large changes in both 0.5 nmol/kg/hr to 1.6 nmol/kg/hr in the hour follow- plasma and urine. In contrast, in the calf, the magniBPTH
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Fig. 2. (a) The effects of an intravenous infusion to an adult goose of a total dose of 50 units bovine parathyroid hormone (BPTH), followed by a single intravenous injection of l0 units BPTH, on the plasma concentration and renal excretion rate of cyclic AMP. (b) The effect of an intravenous infusion to a 6 weeks old calf of a total dose of 1000 units BPTH, followed by a single injection of 500 units BPTH, on the renal excretion rate of cyclic AMP. C.B.P. 67/3A- K
474
I. LEWINet al. 600 -,7 Goose Chicken*
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Fig. 3. A comparison between the maximum rise in plasma cyclic AMP concentration produced in response to the amount of bovine parathyroid hormone (BPTH) injected intravenously. tude of the plasma and urinary cyclic AMP responses was comparatively small. Many factors may account for the difference in magnitude of the cyclic AMP responses to PTH injection observed in these species. The volume of distribution of the injected BPTH would influence the concentration of the hormone at the renal receptor sites and may differ in the species examined. This was found to be approximately 30~o of body weight in the calf (Sherwood et al., 1968) but only 8.6~o of body weight in man (Papapoulos et al., 1977). Clearance of the hormone might also differ, and slower metabolism in some species might therefore allow more sustained hormonal action. Differences in plasma membrane interactions with PTH might account for some of the species differences in the magnitude of the extracellular cyclic AMP responses. For example, Martin et al. (1974) found that synthetic 1-34 fragments of bovine and human PTH generated similar amounts of cyclic AMP in renal cortical membrane preparations derived from the chick whereas the bovine fragment was 5 times more potent than the human fragment in preparations from the rat. There appears to be no simple relationship between changes in extracellular cyclic AMP concentration and the phosphaturic response to PTH administration. In the two human subjects shown here, increases in extracellular cyclic AMP were accompanied by increases in urinary phosphate excretion. However, phosphaturia may occur in response to infusions of a low dose of BPTH when no increase in extracellular cyclic AMP is detectable (Walker et al., 1977) and, conversely, the phosphaturic response in man may be insignificant even when large increases in extracellular cyclic AMP occur (Tomlinson et al., 1974). Kaminski et al. (1970) found that infusions of BPTH into a normal human subject continued to produce a dose related increase in urinary cyclic AMP excretion even after phosphaturia had reached a plateau. It therefore appeared that the maximal physiological effect of the
hormone had been achieved by submaximal stimulation of adenylate cyclase, a phenomenon also observed with other hormones. Injections of BPTH into goose and calf failed to cause an increase in phosphate excretion despite detectable changes in extracellular cyclic AMP, although a phosphaturic effect of PTH has been reported in the chicken (Levinsky & Davidson, 1957) and the starling (Clark et al., 1976) after intravenous injection of the hormone. In the cow, Mayer et al. (1966) found that a single injection of 1200 units BPTH had no effect on urinary phosphate excretion but an increase occurred in the same animal after repeated administration of the hormone over several days. The relatively poor cyclic AMP and phosphaturic responses of the rumina n t kidney to BPTH may be related to the fact that the ruminant's salivary glands rather than the kidneys play the dominant role in phosphate excretion in response to BPTH (Clark, et al., 1975). These results show that in some species there can be a dissociation between the urinary excretion of phosphate and cyclic AMP following the administration of PTH. It is possible that this variation reflects specific differences in mineral homeostasis. Acknowledgement--The skilled technical assistance of Mr T. D. Gibson is gratefully acknowledged. We are indebted to the Medical Research Council for financial support.
REFERENCES
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Effect of parathyroid hormone on cyclic AMP production CHASE L. R. & AURBACHG. D. (1967) Parathyroid function and the renal excretion of 3',5'-adenylic acid. Proc. natn. Acad. Sci. U.S.A. 58, 518 528. CLARK N. B., BRAUN E. J. & WlDEMAN R. F. (1976) Parathyroid hormone and renal excretion of phosphate and calcium in normal starlings. Am. d. Physiol. 231, 1152-1158. CLARK R. C., FRENCH T. J., BEAL A. M., CROSS R. B. & BUDTZ-OLSEN O. E. (1975) The acute effects of intravenous infusion of parathyroid hormone on urine, plasma and saliva in the sheep. Q. J. exp. Physiol. 60, 95-106. KAMINSKI N. I., BROAOUS A. E., HARDMAN J. G., JONES D. J., BALL J. H., SUTHERLAND E. W. & LIDDLE G. W. (1970) Effects of parathyroid hormone on plasma and urinary Y,5'-monophosphate in man. J. clin. Invest. 49, 2387-2395. LEVINSKY N. G. & DAVIDSOND. C. (1957) Renal action of parathyroid extract in the chicken. Am. J. Physiol. 191, 530-536. MAYER G. P., MARSHAK R. R. & KRONFELD D. S. (1966)
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Parathyroid effects on renal phosphorus excretion in the cow. Am. J. Physiol. 211, 1366-1370. SHERWOOD L. M., MAYER G. P., HAMBERG C. F., KRONFELD D. S., AURBACHG. D. & POTTS J. T. (1968) Regulation of parathyroid hormone secretion: proportional control by calcium, lack of effect of phosphate. Endocrinology 83, 1043-1051. TOMLINSON S., BARLINGP. M., ALBANOJ. D. M., BROWN B. L. & O'RIORDAN J. L. H. (1974) The effect of exogenous parathyroid hormone on plasma and urinary adenosine Y,5'-cyclic monophosphate in man. Clin. Sci. molec. Med. 47, 481-492. TOMLINSON S., HENDY G. N., PEMBERTOND. M. & O'RIORDAN J. L. H. (1976) Reversible resistance to the renal action of parathyroid hormone in man. Clin. Sci. molec. Med. 51, 59-69. WALKER D. A., DAVIES S. J., SIDDLE K. & WOODHEADJ. S. (1977) Control of renal tubular phosphate reabsorption by parathyroid hormone in man. Clin. Sci. molec. Med. 53, 431-438.