Autotransplantation of avian parathyroid glands: An animal model for studying parathyroid function

GENERAL

AND

COMPARATIVE

Autotransplantation

ENDOCRINOLOGY

76,451460

(1990)

of Avian Parathyroid for Studying Parathyroid

Glands: An Animal Function

Model

JUDITHA. COLE,LEONARDR. FORTE,PAMELAK. THORNE,RICHARDE. POELLING, AND WILLIAM J. KFMUSE Departments

of Pharmacology and Anatomy, Harry S. Truman Memorial

School Veterans

of Medicine, University of Missouri-Columbia, Hospital, Columbia3 Missouri 65212

and

Accepted March 17, 1989 The parathyroid glands of chickens were autotransplanted and the return of parathyroid function following transplantation was determined. Parathyroidectomy (PTX) resulted in a marked hypocalcemia (5.2 2 0.2 mg/dl) 4 hr following PTX. Plasma calcium (PCs) had declined to 4.3 ? 0.2 mg/dl 24 hr after PTX. Parathyroid glands were transplanted subcutaneously 24 hr after removal and 24 hr later, PCs had risen to 8.6 2 0.5 mg/dl. Seven days after PTX, PCs increased to 10.3 ? 0.2 mg/dl and by 14 days was indistinguishable from control levels (10.8 2 0.2 mg/dl vs. 11.0 ? 0.2 mg/dl, respectively). When chicks with transplanted glands were fed a low Ca (0.08%) diet for 2 weeks they were able to maintain plasma PCs at levels comparable to control birds. Removal of the transplanted glands resulted in marked decreases in PCs (from 9.7 2 0.3 to 5.6 * 0.8 mg/dl), in the fractional excretion of phosphate, in urine CAMP, and in renal 25OH-vitamin Dx-la-hydroxylase activity. Stepwise reductions in PCs and lo-hydroxylase activity were produced in partially PTX and fully PTX chicks by removing part or all of the parathyroid tissue. These data suggest that the transplanted parathyroid tissue was the major source of circulating PTH and that it may be possible to produce different degrees of acute hypoparathyroidism by varying the amount of transplanted parathyroid tissue removed surgically. Chickens with transplanted parathyroid glands thus provide a convenient animal model in which to study parathyroid function in an avian species. 0 1989 Academic Press, Inc.

excretion and hypocalcemia which is reversed by the administration of PTH (Kisse1 and Wideman, 1985). In birds, urine phosphate excretion increased following PTH administration whereas phosphate excretion decreases sharply after PTX (Levinsky and Davidson, 1957; Martindale, 1973; Prashad and Edwards, 1973; Clark and Wideman, 1977). The effects of PTX on calcium and phosphate excretion can be somewhat variable which is thought to be due, at least in part, to the presence of varying amounts of accessory parathyroid tissue (Dudley, 1942; Hurst and Newcomer, 1969; Feinblatt et al., 1973). In order to use an avian species to examine PTH effects on renal function, we thought it would be desirable to have an experimental model where PTH could be

Parathyroid hormone (PTH) is an important regulator of plasma calcium and phosphate levels in birds (Levinsky and Davidson, 1957; Martindale, 1973; Prashad and Edwards, 1973; Clark et al., 1976; Wideman et al., 1980). Birds possess two pair of parathyroid glands lying along the carotid arteries, posterior to each thyroid gland (Clark and Wideman, 1977). Embryologitally, the avian parathyroids arise from the ventral portions of the third and fourth pharyngeal pouches. The anlagen of the parathyroid glands move by relative growth of neck structures until they lie next to or just posterior to the thyroid gland (Schrier and Hamilton, 1952). Plasma calcium levels in adult chickens range from 9 to 10 mg/dl and removal of the parathyroid glands (PTX) results in a marked increase in urine calcium 451

0016~6480/90

$1.50

Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved,

452

COLE ET AL.

eliminated with reasonable certainty by parathyroid ablation. In chickens, surgical PTX becomes increasingly difficult with age because the parathyroid glands migrate deep into the thoracic cavity (Schrier and Hamilton, 1952). However, chickens provide an excellent animal in which to examine PTH regulation of renal function since their kidneys possess PTH receptors coupled positively to adenylate cyclase (Nissenson and Arnaud, 1979; Forte et ul., 1983; Pines et ul., 1983), PTH-regulated calcium and phosphate reabsorption (Clark et uf., 1976), and 25OHD-la and -24 hydroxylase activities (Henry et ui., 1974; Rost et ul., 1981). Autotransplantation of the parathyroid glands could provide a way to circumvent the problem of gland accessibility in mature chickens. Autotransplantation of parathyroid glands is not a new technique; parathyroid glands from experimental animals and humans have been successfully autotransplanted following shortterm organ culture or cryopreservation with complete restoration of normal gland function (Wells, 1974; Brennan et uZ., 1978; Leight et ul., 1978). In addition, Kissel and Wideman (1985) transplanted the parathyroid glands of 10 to 21-day-old chicks into the inner thigh muscles and showed that both plasma-ionized calcium and the fractional excretion of phosphate decreased within an hour following the removal of the transplanted parathyroid tissue, indicating that the transplanted glands were the primary source of circulating PTH. In the present study, we describe a transplantation method which allows us to evaluate the effectiveness of the initial parathyroidectomy prior to transplantation and that subsequently permits us to predict successful parathyroidectomy following removal of the transplanted parathyroid tissue. MATERIALS

AND METHODS

Animuls. One-day-old male white Leghorn chicks (Gullus gc/-

/us) were purchased from the Colonial Poultry Farm (Pleasant Hill, MO). Upon arrival, chicks were fed starter grower diet (Purina, St. Louis, MO) and at 5 days of age were switched to a low calcium (0.08%) diet for 6 days to induce parathyroid gland hypettrophy. Food was removed 12 hr prior to surgery at 12 days of age.

Experiment& (I) Transplantation glandularfunction.

Procedures and evaluation

of the return

of

Chicks were anesthetized with 2.5 ml DIAL/kg body weight (25 mg/ml diallybarbituric acid, 200 mg/ml monoethylurea, and 200 mg/ml urethane). Feathers on the neck and chest were removed and chicks were injected with lidocaine prior to making a 2-cm incision in the neck. The parathyroid glands were located, surgically removed with the aid of a dissecting microscope equipped with tiber optic illumination, and the tissue was placed on ice in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 p&ml streptomycin. The incision was then closed with wound clips and chicks were placed under a heat lamp to maintain body temperature. Parathyroid glands were transferred to a single well of a 24-multiwell plate containing 1 ml of DMEM supplemented as described above. Plates were placed in a desiccator, gassed with 95% OJ5% CO*, and cultured for 24 hr at 37”. Blood samples were obtained 4 and 24 hr following PIX by puncturing a wing vein and collecting the sample in a heparinized capillary tube. Following the 4-h blood sample, chicks received an intraperitoneal injection of 0.5 ml calcium gluconate (25 mg calcium/ml) and 0.2 ml of 100 pmol/ml 1,25(OH)* vitamin Ds and were fed a slurry of chick diet via a feeding tube. The cultured glands were transplanted following the 24-h blood sample by making a small incision in the skin of the right wing and placing the parathyroid glands under the skin. Following transplantation, chicks were injected with calcium gluconate and were force-fed three times a day until transplants were determined to be functional (usually 2-3 days following transplantation as determined by an increase in plasma calcium to >7 mg/dl). Chicks were returned to a starter grower diet until used in experimental procedures (usually 6-8 weeks after gland transplantation). (2) Renal c/em-awe. Ten 8-week-old chickens (6 weeks after gland transplantation) were used to evaluate the effects of transplant removal on renal phosphate and CAMP excretion. Chickens were anesthetized with 2.5 ml/kg DIAL, and a carotid artery and jugular vein were cannulated with PE50 tubing. Mannitol (2.5%) was infused via the jugular vein to induce an osmotic diuresis. Blood samples were collected from the carotid artery and urine samples were collected from the ureters by the method of Wideman and

PARATHYROID

GLAND

Braun (1982). Glomerular liltration rate was estimated by the clearance of carboxy [‘4C]inuhn. A priming dose of 1 lKi [i4C]inulin in 2.5% mannitol was delivered via the venous cannula and was followed by the constant infusion of 0.06 &i/ml at a rate of 0.4 ml/ mm/kg. A 60-min (- 60 to 0 min) equilibration period was followed by a 60-min (O-60 min) control period. At 60 min, the transplanted parathyroid glands were removed from five birds, while five birds served as controls. Urine samples were collected every 20 min and blood samples were drawn at 30 and 90 min. (3) Effects of partial and complete parathyroidectomy on plasma calcium hydroxylase activities.

and renal

25OH

vitamin

Ds-

Eight-week-old chickens (6 weeks after transplantation of the parathyroid glands) were separated into three groups based on their initial responses to parathyroidectomy. Group 1 consisted of four birds with transplanted glands to serve as controls. Since there is currently no radioimmunoassay for avian PTH, we evaluated the effectiveness of initial PTX by changes in plasma calcium and separated the remaining birds into groups 2 and 3. Group 2 was composed of three birds whose plasma calcium averaged 6.6 * 0.2 mg/dl 4 hr after PTX and were thought to possess residual parathyroid tissue. Group 3 was composed of four birds whose plasma calcium averaged 5.4 2 0.2 mg/dl4 hr after PTX and were considered to have no residual parathyroid tissue. Four weeks later all three groups of birds were placed on a low calcium (0.08%) diet for 2 weeks to increase renal 25(OH)Ds-la-hydroxylase activity. At the end of 2 weeks, an initial blood sample was obtained and the transplanted glands were removed from birds in groups 2 and 3. Twenty-four hours later, a tinal blood sample was obtained by heart puncture, the birds were sacriticed, and their kidneys were removed to determine 2SOHDs-la- and 250HD3-24R-hydroxylase activities.

Analytical

Procedures

of renal 25OH vitamin DJ-icx-hydroxylase Renal 250HD3-lo-hydroxylase activity was determined using mitochondrial fractions as described previously (Martz et al., 1985). Briefly, kidneys from 8-week-old male chickens were removed following experimental treatment, a 15% (w/v) homogenate was prepared in 0.25 M sucrose, 15 a Tris-acetate, and 1.9 m Mg acetate (pH 7.4), and mitochondria were isolated by centrifugation. A l-ml aliquot of the mitochondrial preparation was added to glass scintillation vials containing 75 w succinate. The vial was flushed 60 set with 100% Oz and 300 ng of the substrate, 25(OH)-[26(27)-mefhy/-3H]-cholecalciferol (500 cpm/ ng), was added in 20 p,l of ethanol. Vials were incubated for 10 min at 37Oand the reaction was terminated by the addition of 8 ml methanol:chloroform (2:l). The Assay activity.

453

TRANSPLANTATION

lipid fraction was extracted by the method of Bligh and Dyer (1959), evaporated under Nz, resuspended in 1 ml hexane:chloroform:methanol (9: 1: 1), and passed through a 3-cm column of Sephadex LH-20 equilibrated with the same solvent system. Samples were then evaporated under Nz and dissolved in hexane: methylene chloride:methanol (160:29: 11) and chromatographed on a Zorbax-SIL column at 1.O ml/min. One minute fractions were collected for measurement of ‘H-labeled 25-OHD metabolites. Calcium, phosphorus, and cyclic AMP. Plasma calcium was measured using the atomic absorption spectrophotometry. Urine and plasma phosphorus were assayed calorimetrically by the method of Weisman and Pileggi (1974). Cyclic AMP in plasma and urine was measured by radioimmunoassay according to the method of Steiner et al., (1969). Data analysis. All data represented in illustrations or tables are means 2 standard error. The data were analyzed using analysis of variance (ANOVA) and mean values were compared using least significant differences when ANOVA F values indicated there were significant differences (P < 0.05) between the means.

Materials All reagents were of analytical quality and were obtained from standard suppliers. Carboxy [‘4C]inulin (spec act 3.4 mCi/g) was from ICN (Costa Mesa, CA) and 25(OH)-[26(27)-methyl-3H]cholecalciferol (54 mCi/mg) was purchased from Amersham (Arlington Heights, IL). Unlabeled vitamin D3 metabolites were the generous gift of Dr. Milan R. Uskokovic, Hoffman-LaRoche (Nutley, NJ).

RESULTS Evaluation of the Return of Parathyroid Function

The effects of parathyroid gland removal and subsequent transplantation on plasma calcium levels are shown in Fig. 1. PTX chicks were significantly hypocalcemic compared to controls at 4 hr (5.2 % 0.2 mg/ dl vs 9.3 + 0.2 mg/dl, respectively). By 24 hr, plasma calcium levels in PTX chicks had declined to 4.3 2 0.2 mg/dl, despite the administration of calcium gluconate and I ,25(OH)zDS. Parathyroid glands were transplanted following the 24-hr blood sample and by 24 hr after transplantation (48 hr after PTX), plasma calcium has risen to 8.6

454

COLE ET AL.

of pycnotic nuclei or central regions of necrosis . Evaluation

1, 4h24h4f3D

I 7d

14d

TIME

1. The effects of parathyroidectomy and transplantation on plasma calcium. Chicks were fed a low Ca diet for 6 days prior to PTX to induce parathyroid gland hypertrophy. Chicks were PTX at 12 days of age and blood samples were obtained 4, 24, and 48 hr and 7 and 14 days following PTX. Control chicks were returned to the starter-grower diet at 24 hr. Values are means * SEM; n = 7 for controls, n = 27 at 4 hr, n = 26 at 24 hr, and n = 21 at 48 hr in PTX chicks. Significant differences from time-matched controls are indicated by asterisks (p ~0.01). FIG.

? 0.5 mg/dl. Seven days following PTX, plasma calcium further increased to 10.3 2 0.2 mg/dl and was not significantly different from that of controls, indicating that the transplanted glands were functioning equivalent to controls. Visual inspection of the transplanted glands at this time indicated that they were encapsulated and well vascularized. Two weeks following surgery, plasma calcium levels in chicks with transplanted glands were indistinguishable from controls (10.8 ? 0.2 ml/d1 vs 11.0 & 0.2 ml/ dl, respectively). In several animals, the transplanted parathyroid tissue was removed 6 weeks following autotransplantation and the histological appearance was evaluated. Normal chicken parathyroid tissue consists of cords of cells, surrounded by connective tissue containing numerous blood vessels (Fujii and Isono, 1972). Only one cell type, the chief cell is present. Light microscopic examination of the transplanted glands revealed the typical appearance of avian parathyroid glands with nests of parathyroid cells surrounded by connective tissue (Fig. 2). There was no evidence

of Transplant

Function

The function of the transplanted glands was further evaluated by placing the chicks on a low Ca diet and subsequently removing the transplanted tissue. Four weeks after transplanting the glands, three groups of chickens were placed on a low calcium diet for 3 weeks. Group 1 served as controls (birds with parathyroid glands in their normal anatomical position in the thoracic cavity), groups 2 were shams (birds with transplanted parathyroid glands), and group 3 consisted of chickens whose transplanted glands were subsequently removed. Plasma calcium levels were comparable in all three groups of birds before starting the low calcium diet (data not shown) and there was no difference in plasma calcium among any of the three groups after 2 weeks on the low calcium diet (Table 1). Visual inspection of the transplanted glands at this time indicated a marked hypertrophy of the tissue in response to the low Ca diet. Thus, the ability of the chicks with transplanted glands to maintain plasma calcium while on a low calcium diet suggests they have an adequate source of PTH. To determine if the transplanted glands were the major source of PTH, the transplanted glands from one group of chicks were removed and the effect on plasma calcium was determined at 4,8, and 24 hr. Removal of the transplanted parathyroids resulted in a progressive drop in plasma calcium from initial values of 9.7 2 0.3 to 5.6 ? 0.8 mg/dl at 24 hr. Plasma calcium in control animals and sham chicks at 24 hr was 8.8 ? 0.3 and 8.7 ? 0.2 mgldl, respectively (Table 1). The removal of transplanted parathyroid glands resulted in a lower la-hydroxylase activity (I .03 2 0.64 pmol/mg x min-‘), which is marginally significant I’ < 0.06) when compared to controls or chickens with intact transplanted glands (2.35 ? 0.36 and 2.70 ? 0.39

PARATHYROID

GLAND

TRANSPLANTATION

4s5

FIG. 2. Histological appearance of autotransplanted parathyroid glands. Parathyroid transplants were removed 8 weeks following transplantation. Glands were tixed in 2% glutaraldehyde, dehydrated in alcohol, and embedded in plastic. Sections (1.5 uM) were stained with toluidine blue. Final magnification, 1375x.

pmol/mg X min-‘, respectively; Table 1). Removal of the transplanted glands also resulted in an increase in 24R-hydroxylase activity (0.67 2 0.30 pmol/mg x min ‘) compared to controls and shams (0.13 ? 0.03 and 0.07 ? 0.02 pmol/mg x min-‘, respectively; Table 1). We next examined the effects of the removal of transplanted parathyroids on the fractional excretion of phosphate (FEP) and CAMP excretion. There was no difference in the FEP between control birds and chickens with transplanted glands during the I-hr control clearance period. Following removal of the transplanted glands, the FEP declined at each 20-min urine sample and at 60 min reached a value signiticantly lower (12.2 2 3.0%) than in time-matched control birds (31.3 ? 6.5%) (Fig. 3A). Initially, there was no difference in the rate of

CAMP excretion (Fig. 3B). However, in the clearance period prior to PTX, significantly less CAMP was excreted by transplanted birds (130 * 30 pmol/min) than by controls (230 * 20 pmol/min). Following the removal of the glands, CAMP excretion continued to decline and was significantly lower than control birds for the first 20 min following PTX. Excretion of CAMP remained consistently lower than, although not different from, controls for the duration of the experiment. Effect of Varying Degrees of Hypoparathyroidism on Plasma Calcium and Renal 2.5-Hydroxylase Activities

We were also interested in determining varying degrees of hypoparathyroidism

if

456

COLE ET AL. TABLE

EFFECT

OF THE REMOVAL

1

OF TRANSPLANTED PARATHYROID TISSUE 25oHD-HYDROXYLASE ACTIVITIES

Plasma calcium (mg/dl) Initial Intact Sham PTX

4

hr

8

hr

9.9 2 0.4 9.7 It 0.3

6.9 2 0.4

hr

1-Hydroxylase

8.8 2 0.3 8.7 2 0.5 5.5 L 0.8**

6.5 zt 0.7

CALCIUM

25OHD-hydroxylase 24

10.3 ? 0.1

ON PLASMA

2.4 2 0.5 2.7 2 0.4

1.0 & 0.6

AND

RENAL

(pmol/mg X mm’) 24-Hydroxylase 0.13 0.07 0.67

k 0.03 2 0.02 * 0.30

No&. Experiments were performed in g-week-old male chickens 6 weeks following transplantation of the glands. All chicks were fed a low calcium diet for 2 weeks prior to transplant removal. Intact, chickens with their parathyroid glands in the normal anatomical position; Sham, chickens with transplanted glands; and PTX, chickens with their transplanted glands removed. Plasma calcium was determined in PTX chickens at 0,4, 8, and 24 hr after removal of the transplanted glands and at 0 and 24 hr in intact and sham birds. 25OHD-hydroxylase activities were determined in a renal mitochondrial preparation 24 hr following PTX. Values are means ? SEM; n = 4 for each group (**F < 0.01 compared to intact chickens).

could be produced by transplant removal. Eight-week-old chicks with residual parathyroid tissue (partially PTX) and chicks with no residual tissue (100% PTX) (see Table 2) had their transplanted glands removed and plasma calcium and renal 25OHD-hydroxylase activities were determined 24 hr later. Plasma calcium in control birds was 8.8 * 0.2 mg/dl while removal of the parathyroid glands in both groups of

0-A 0

-1 20

40

60 80 Time lmnl

100

120

parathyroidectomized birds was significantly lower (6.2 ? 0.2 and 3.1 ? 0.4 mg/dl in partially and 100% PTX birds, respectively) (Table 2). Renal 25OHD-hydroxylase activities in these three groups of birds also reflected a change in parathyroid status. The lo-hydroxylase activity in 100% PTX chickens was significantly lower than in controls (0.33 & 0.09 and 4.22 ? 0.41 pmol/ mg X min’, respectively), while partially

o.oL~..m-Ap!x .l 0

20

40

60 tlo Time lmd

100

120

FIG. 3. The effect of removal of transplanted parathyroids on the fractional excretion of phosphate (FEP) and the rate of CAMP excretion. Clearance studies were performed on g-week-old male chickens with transplanted parathyroid glands as described under Materials and Methods. Following a 1-hr equilibration period, three 20-min urine samples were collected for control values. At 60 min, transplanted parathyroid glands were removed (PTX) and the effects of PTX were examined for an additional three 20-min clearance periods, Values are means ? SEM, n = 5. Asterisks indicate values significantly different from time-matched controls, P < 0.05. (A) Effects of transplant removal on FEP. (B) Effects of transplant removal on urine CAMP (nmol/min).

PARATHYROID

GLAND

TABLE

EFFECT

OF RESIDUAL

Controls Part PTX 100% PTX

2

PARATHYROID TISSUE 2soHD-HYDROXYLASE

PCs after transplantation tmg/dll

457

TRANSPLANTATION

ON PLASMA ACTIVITIES

PCs after transplant (rnddl)

CALCIUM

removal

AND RENAL

25OHD-hydroxylase (pmol/mg x min-

‘)

4 hr

48 hr

Initial

24 hr

1-OHase

24-OHase

10.6 ? 0.3 (7) 6.6 zk 0.2 (4)* 5.4 2 0.2 (4)**

10.9 * 0.2 9.2 2 0.4* 7.8 k l.l**

8.4 2 0.3 9.3 2 0.3 8s 2 0.5

8.8 ci 0.2 6.2 2 0.3** 3.1 2 0.4**

4.2 5 0.4 2.0 2 1.4* 0.2 2 0.1**

0.64 2 0.17 0.13 ? 0.05* 0.85 k 0.43

NOW. Parathyroid glands were removed, cultured, and transplanted as described under Materials and Methods. Chicks 4-hr plasma calcium levels greater than 6 mg/dl were considered to be incompletely PTX (part PTX). At 8 weeks, chickens switched to a low calcium diet for 2 weeks prior to transplant removal. The effects of transplant removal on plasma calcium renal 25OHD-hydroxylases (OHases) were then evaluated in intact, partially PfX, and 100% PTX birds 24 hr after removal. Values are means ? SEM. Numbers in parentheses are the number of animals/group. Signiticant differences control are indicated by * (P < 0.05) and ** U’ < 0.01).

PTX chicks displayed an intermediate activity (2.01 ? 0.81 pmol/mg x min-‘; Table 2). The changes in plasma calcium and Ia-hydroxylase activity following transplant removal are consistent with the PTX groups representing varying degrees of hypoparathyroidism. These data indicate that short-term, stepwise decreases in circulating PTH can be produced by removing part or all of the functioning parathyroid tissue. DISCUSSION

A classical way of examining the effects of decreased circulating PTH on renal function is to eliminate the source of the hormone by removing the parathyroid glands. This study indicates that removal of the parathyroid glands from chicks and their subsequent transplantation to an ectopic position does not affect the bird’s ability to maintain normal calcium homeostasis. Kisse1 and Wideman (1985) have shown that chicken parathyroid glands transplanted within the thigh muscle were vascularized and encapsulated when examined histologically 10 weeks after transplantation. However, they did not report the time course for reestablishment of gland function following transplantation. In the present study, the transplanted glands appeared to be functioning within 48 hr following transplantation and appeared to be fully functional by

with were and gland from

1 week (based on the return of plasma Ca to control levels). The relatively rapid return of function may be the result of the marked hypertrophy and hyperplasia of the glands induced by 6 days on a low Ca diet prior to their removal. Two weeks on a low calcium diet did not compromise the ability of birds with transplanted glands to maintain plasma calcium, indicating that the glands were fully functional. The transplanted glands were clearly the primary source of PTH since their removal resulted in a marked decrease in plasma calcium to levels less than 6 mg/dl within 24 hr. In birds, PTH regulates renal phosphate transport by inhibiting net phosphate reabsorption and by stimulating net phosphate secretion (Clark et al., 1976: Laverty and Dantzler, 1982). These are direct effects of PTH since Wideman and Braun (198 1) have shown that FEP is greater than the FE3*P in “P-labeled birds following administration of PTH. In the present study, there was no difference in the excretion of phosphate between control and PTX birds prior to the removal of the transplanted glands. The removal of the transplanted glands resulted in a marked and persistent decrease in phosphate excretion which was consistent with the effects of mX in normal birds (Clark and Wideman, 1977) or those with parathyroid glands transplanted to an ectopic site (Kissel and Wideman (1985). The effects of

458

COLE

PTX on CAMP excretion were less clear. In birds, physiological manipulations which decrease plasma PTH are accompanied by a decrease in urinary CAMP excretion, while increases in PTH result in increased CAMP excretion (Pines et ul., 1983). Chase and Aurbach (1967) have shown in rats that exogeneous PTH produced dramatic increases in CAMP excretion while PTX decreased urinary CAMP excretion. In this study, the removal of the transplanted parathyroid tissue resulted in a decrease in the rate of CAMP excretion and these values were consistently lower in PTX animals than in intact controls. However, CAMP excretion was also lower in animals with transplanted glands prior to their removal. The difference in CAMP excretion between control and PTX birds does not appear to be the result of moving the parathyroid glands to an ectopic position since both groups of birds had transplanted parathyroid tissue. Furthermore, FEP in the two groups of birds was not significantly different until the transplanted glands were removed. In birds, CAMP excretion does increase in response to exogeneous PTH and the infusion of calcium chelators, but these are manipulations which markedly elevate circulating PTH. A number of studies have indicated that CAMP may not be the only mediator of PTH actions on renal function since PTH appears to regulate phosphate excretion or plasma calcium without affecting CAMP excretion or renal content of CAMP (Nissenson et ui., 1982; Puschett, 1982). Therefore, in birds, it is possible that endogenous PTH is responsible for a small percentage of basal CAMP excretion under normal conditions and that removal of the glands may cause only a modest and insign&ant decrease in the excretion rate of CAMP. In addition to its role in the regulation of plasma calcium and phosphate, PTH regulates the activity of the renal 25OH-vitamin Dj-lo-hydroxylase (Garabedian et af., 1972; Fraser and Kodidek, 1973: Henry et

ET

AL.

al., 1974; Henry and Norman, 1984; Baksi and Kenny, 1979) and may regulate the activity of 250HD3-24-hydroxylase (Baksi and Kenny, 1979; Rost et ul., 1981; Henry, 1985). Transplantation of the parathyroid glands did not appear to affect the regulation of either Ia-hydroxylase or 24hydroxylase activity. However, removal of the transplanted tissue did result in a marked decrease in lo-hydroxylase activity and an increase in 24-hydroxylase activity. In addition, it was possible to produce varying degrees of hypoparathyroidism by the removal of the transplanted glands in birds presumed to have residual parathyroid tissue or those thought to be without residual tissue. The 24-hydroxylase data are less consistent in terms of the effects of patial and 100% PTX, While 24hydroxylase activity in 100% PTX birds was greater than in controls, 24-hydroxylase activity was lower in partially PTX birds. Whether this reflects a PTHindependent regulation of 24-hydroxylase activity or a biphasic effect of PTH on 24hydroxylase activity remains to be shown. Nevertheless, the differences in plasma Ca and la-hydroxylase activities do suggest that a reproducible degree of acute hypoparathyroidism may be produced in birds by removing portions of the transplanted parathyroid. However, measurement of circulating PTH in these models would be required to confirm this postulate. It would most likely be difficult to produce longterm differences in parathyroid status since compensatory growth of the remaining parathyroid tissue is likely to occur in partially parathyroidectomized birds. In summary, transplantation of the parathyroid glands of chicks provides a convenient avian model in which to study PTH regulation of renal function. The transplantation of the glands does not compromise calcium homeostasis or renal function and provides for an initial evaluation of the effectiveness of the initial surgical PTX for each animal prior to removal of autotrans-

PARATHYROID

GLAND

planted parathyroids. The ability to produce reductions in circulating PTH would provide a valuable means for examining the acute effects of hypoparathyroidism on renal PTH receptor characteristics, adenylate cyclase activity, 25OHD-hydroxylase activities, and subsequent changes in renal transport functions. REFERENCES Baksi, S. N., and Kenny, A. D. (1979). Acute effects of parathyroid extract on renal vitamin D hydroxylase in Japanese quail. Pharmacology 18, 16% 174. Bligh, E, G., and Dyer, W. J. (1959). A rapid method of total lipid extraction and puritication. Canad. J. Biochern. 37, 91 l-919. Brennan, M. F., Brown, E. M., Sears, H. F., and Aurbach, G. D. (1978). Human parathyroid cryopreservation: In vitro testing of function by parathyroid hormone release. Ann Surg. 187, 87-90. Broadus, A. E. (1981). Nephrogenous CAMP. Recent Prog.

Harm.

Res.

37, 667-701.

Chase, L. R., and Aurbach, G. D. (1967). Parathyroid function and the renal excretion of 3’,5’-adenylic acid. Proc, Natl. Acad. Sri. USA 58, 518-525. Clark, N. B., Braun, E. J., and Wideman, R. F., Jr. (1976). Parathyroid hormone and renal excretion of phosphate and calcium in normal starlings. Amer. J. Physiol. 231, 1152-l 158. Clark, N. B., and Wideman, R. F., Jr. (1977). Renal excretion of phosphate and calcium in parathyroidectomized starlings. Amer. J. Physiol. 233, Fl38-F144. Dudley, J. (1973). The development of the uhimobranchial body of the fowl Galius domesticus. Amer. J. Anat.

7t,

6567.

Forte, L. R., Langeluttig, S. G., Biellier, H. V., Poelling, R. E., Magliola, L., and Thomas, M. L. (1983). Upregulation of kidney adenylate cyclase in the egg-laying hen: Role of estrogen. Amer. J. Physiol. 245, E273-E280. Forte, L.R., Langeluttig, L. R., Poelling, R. E., and Thomas, M. L. (1982). Renal parathyroid hormone receptors in the chick: Down regulation in secondary hyperparathyroid animal models. Amer. J. Physiol. 242, El54-E163. Feinblatt, J. D., Raisz, L. G., and Kenny, A. D. (1972). Secretion of avian ultimobranchial calcitonin in organ culture. Endocrinology 93, 227-234. Fraser, D. R., and Kodicek, E. (1973). Regulation of 25hydroxycholecalciferol-1-hydroxylase activity in kidney by parathyroid hormone. Nature @ondon) 241, 163-166.

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459

Fujii, H., and Isono, H. (1972). Ultrastructural observations on the parathyroid glands of the hen (Gallus domesticus). Arch. Histol. Japan. 34, 155-

,LC Gara ri.e ran, M., Hohck, M. F., DeLuca, H. F., and Boyle, L. T. (1972). Control of 25hydroxycholecalciferol metabolism by parathyroid glands. Proc. Natl. Acad. Sci. USA 66, 1673-1676. Harper, J. F., and Brooker, G. (1975). Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after secondary acetylation by acetic anhydride in aqueous solution. J. Cyclic Nucleotide Res. 1, 207-218. Henry, H. L. (1985). Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism is mimicked and enhanced by forskolin. Endocrinology 116, 503-510. Henry, H. L., Midgett, R. J., and Norman, A. W. (1974). Regulation of 25-hydroxyvitamin D3I-hydroxylase in viva. J. Biol. Chem. 249, 75847592. Henry, H. L., and Norman, A. W. (1984). Vitamin D: Metabolism and biological actions. Annu. Rev. Nutr.

4, 493-520.

Hurst, J. G., and Newcomer, W. S. (1969). Functional accessory tissue in ultimo-brancial body of chickens. Proc. Sot. Exp. Biol. Med. 132, 55-557. Kissell. R. E., and Wideman, R. F., Jr. (1985). Parathyroid transplants and unilateral renal delivery of parathyroid hormone in domestiic fowl. Amer. J. Physiol. 249, R732-R739. Laverty, G., and Dantzler, W. H. (1982). Micropuncture of supeficial nephrons in avian (Sturnus vulgaris) kidney. Amer. J. Physiol. 243, F561-F569. Leight, G. S., Parker, G. A., Sears, H. F., Marx, S. J., and Terrill, R. E. (1978). Experimental cryopreservation and autotransplantation of parathyroid gland: Technique and demonstration of function. Ann. Surg. 188, 16-21. Martz, A., Forte, L. R,, and Langeluttig, S. G. (1985). Renal CAMP and 1,25(OH)*Dr synthesis in estrogen-treated chick embryos and hens. Amer. J. Physiol. 249, E626-E633. Pines, M., Polin, D., and Hurwitz, S. (1983). Urinary cyclic AMP excretion in birds: Dependence on parathyroid hormone activity. Gen. Camp. Endocrinol.

49, 90-96.

Puschett, J. B. (1982). Are all of the renal tubular actions of the parathyroid hormone mediated by the adenylate cyclase system? Miner. Electrolyte Metab. 7, 281-284. Rest, C. R., Bible, D. D., and Kaplan, R. A. (1981). In vitro stimulation of 25-hydroxycholecalceferol ld-hydroxylation by parathyroid hormone in chick kidney slices: Evidence for a role of adenosine 3’,5’-monophosphate. Endocrinology 108, 1002-1006.

460

COLE ET AL.

Schrier, J. E., and Hamilton, H. L. (1952). An experimental study of the origin of the parathyroid and thymus glands in the chick. J. Exp. 2001. 119, 165-182. Steiner, S. L., Kipnis, D. M., Utiger, R., and Parker, C. (1969). Radioimmunoassay for the measurement of adenosine 3’,5’-cyclic phosphate. Proc. Natl. Acad. Sci. USA 64, 367-373. Weisman, N., and Pileggi, U. J. (1974). Zn “Clinical Chemistry: Principles and Techniques,” 2nd ed., pp. 720-723. Harper and Row, New York. Wells, S. A., Jr. (1974). The transplanted parathyroid gland: Evaluation of cryopreservation and other environmental factors which affect its function. Surgery 75, 49-55.

Wideman, R. F., Jr., Clark, N. B., and Braun, E. J. (1980). Effects of phosphate loading and parathyroid hormone on starling renal phosphate excretion. Amer. J. Physiol. 239, F233-F243. Wideman, R. F., Jr., Clark, N. B., and Braun, E. J. (1980). Effects of phosphate loading and parathyroid hormone on starling renal phosphate excretion. Amer. J. Physiol. 239, F233-F243. Wideman, R. F., Jr. and Braun., E. J. (1981). Stimulation of avian renal phosphate secretion by parathyroid hormone. Amer. J. Physiol. 241, F263F272. Wideman, R. F., Jr., and Braun, E, J. (1982). Ureteral urine collection from anesthetized domestic fowl. Lab. Anim. Sci. 32, 298-301.