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Protein secretion from parotid glands of vitamin D deficient and glucocorticoid-treated rats
631
Biochimica et Biophysica Acta , 538 (1978) 631--634 © Elsevler[North-Holland Biomedical Press
BBA Report BBA 21465
PROTEIN SECRETION FROM PAROTID GLANDS OF VITAMIN D DEFICIENT AND GLUCOCORTICOID-TREATED RATS
ALAN TENENHOUSE and GOODWILL AFARI
Department of Pharmacology, McGill University, Montreal, Quebec (Canada) (Received August 22nd, 1977)
Summary The rate of isoproterenol stimulated secretion of protein from parotid glands of vitamin D deficient rats and rats treated with methylprednisolone was increased compared to the secretory response of tissue from control rats. It is suggested that the increased secretory response is secondary to a decreased capacity of mitochondria from the tissue of these animals to take up and store Ca~+; i.e. the mitochondria are less efficient buffers of Cytoplasmic Ca2÷. Under these conditions any process, such as protein secretion, which requires an increased cytoplasmic Ca2÷ concentration will operate more effectively.
Several investigators have proposed that an increased cytoplasmic [ Ca2+] is essential for protein secretion from the parotid gland [1--4]. It has also been suggested that one of the functions of mitochondria is to maintain the concentration of cytoplasmic Ca2÷ constant. It is possible therefore that the increased cytoplasmic [Ca:÷] associated with secretion results from an alternation in the calcium buffering capacity of mitochondria. As first demonstrated by Engstrom and DeLuca [5] the rate and extent of Ca2÷ accumulation by isolated mitochondria from liver, kidney and intestine of vitamin D deficient rats is greatly reduced compared with mitochondria from vitamin D replete rats. Kimberg and Goldstein [6] demonstrated that treatment of vitamin D replete rats with cortisone caused a significant reduction in the rate of Ca2÷ accumulation in subsequently isolated liver mitochondria; the mitochondria from cortisone treated rats were indistinguishable from those of vitamin D deficient rats. In the experiments described in this report vitamin D deficient rats and rats treated with methylprednisolone were used to test the hypothesis that the
632 alteration in Ca 2÷ metabolism existing under these conditions resuits in an altered secretory response of parotid gland° Weanling Holtzman rats were housed in metabolic cages in the dark and fed a vitamin D deficient diet (Raehitogenic Diet AOAC, No. t1077~ iCN Nutrition Biochemical Corporation). The eontrot group of rats, of the same strain, age and sex, housed in the regular animal quarters, were fed ~he same diet as the experimental group supplemented with 100 IoUo vitamin D per week. The animals were used 21--26 days after being placed on the vitamin D deficient diet. At that time there was no gross difference be~ween the control and vitamin D deficient groups; serum [Ca ~+] and phosphate were normal and the b o d y weight of the animals within each group varied between 55--75 g° To study the effect of g|ucocortieoid on parotid gland function, Holtzrnan rats (150--170 g) were injected, subcutaneously, with methylprednisolone, 1 rag/day for 7 days. Protein seereti.on from parotid gland was measured as previously described [41. Zymogen protein was labelled in vitro b y incubating parotid gland slices in Krebs=Ringer bicarbonate buffer containing carrier-free [3H]leueine for 5 min followed by a 2-h ~chase' incubation in Krebs-l~inger bicarbonate buffer containing 1.0 mM [~H]leueine. Secretion was measured b y following the rate of release of [3 H] protein from tissue slices superfused with Krebs-Ringer bicarbonate buffer containing 10 p N isopro~erenol. ~esutts are expressed as the percentage change of 3H-labelled protein o u t p u t into the superfusate with exposure of the tissue to seeretagogue. The release of 3H-labelled protein from rat parotid gland m response to isoproterenol is illustrated in Fig. 1. The tissue from vitamin D replete animals responded rapidly to the isoproterenol; the rate of protein release was maximal
300"
T
2o0
~
z
e
o
100
,/ 10
2G
30
40
MINUTES Fig. 1. The effect o f i s o p r o t e r e n o ] ( 1 0 -5 M ) o n ~H-lahelled p r o t e i n ~elease f r o m t h e pasotid gland o f vitamin D-deficient (e) and vitamin D-repleted (*) rats. Superfusion medium was K~ebs-Ringer buffe~ c o n t a i n i n g 1 0 -3 M C a 2+. T i m e ' 0 ' is t h e t i m e the drug r e a c h e d t h e tissue c h a m b e r . P r o t e i n r e l e a s e is e x p r e s s e d as the p e r c e n t greater t h a n b a s a l r e l e a s e , E a c h p o i n t is t h e m e a n -~ S . E . o f a t least three e x p e r ~ iments.
633 within 6--8 rain (120--140% greater than basal). This maximum rate was maintained approx. 10 min then declined to the basal rate approx. 35 min after superfusion with isoproterenol was stopped. The response of the tissue from vitamin D deficient animals was greatly increased. The m a x i m u m rate of secretion, 350% greater than basal was reached within 12--14 min, maintained for 10 min, then decreased b u t was still approx. 75% greater than the basal secretion rate 40 min after superfusion with isoproterenol was stopped. The characteristics of the two responses were similar; the major difference was that in the vitamin D deficient tissue the initial rapid increase in secretion rate persisted for approximately twice as long, reaching a maximum secretion rate 2.5--3.0-times that reached with tissue from vitamin D replete rats. The secretory response to isoproterenol of parotid gland from rats treated with methylprednisolone is shown in Fig. 2. In the tissue from untreated 200
03 tu ..J Lu n-
100
z
LU kO rr a. z
e
10
20
30
40
50
60
MINUTES Fig. 2. T h e e f f e c t o f i s o p z o t e z e n o l (10 -s M) o n 3H-labelled p r o t e i n s e c r e t i o n f r o m p a r o t i d gland of u n t r e a t e d rats ( o ) a n d rats t r e a t e d w i t h 1 r a g / d a y m e t h y l p r e d n i s o l o n e fo~ 7 d a y s (e).
animals the rate of protein release increased to a maximum 85% greater than the basal secretion rate whereas in tissue from steroid treated animals the m a x i m u m rate of secretion was approx. 170% greater than the basal secretion rate. From these experiments it is clear that vitamin D deficiency alters the secretory response of rat parotid gland to isoproterenol. The rate of secretion of 3H-labelled protein release in response to isoproterenol is increased whereas the basal secretion rate is unaffected b y vitamin D deficiency. Although the role of vitamin D in protein secretion from the parotid gland is n o t known, based on the observations reported here and on the k n o w n effects of vitamin D on mitochondrial Ca 2. metabolism [7,8] a working hypothesis has been developed. It is postulated as has been done by several authors, that cytoplasmic [Ca 2. ] must increase, for protein secretion from the parotid gland to occur. The mitochondrial Ca 2÷ uptake process functions to prevent an increase in cytoplasmic [Ca 2+] and so antagonizes the effect of secretagogues. This mitochondrial Ca 2÷ uptake process is vitamin D dependent so that in vitamin D deficient animals the ability of mitochondria to take up Ca 2÷ is impaired. In this situation cytoplasmic [Ca 2÷] is more readily increased. Any process which depends on such an increase will operate more effectively. Glucocorticoid treatment of rats has been shown to alter the ability of isolated
634 mitochondria to take up Ca 2+ t5~; the mitochondria from cortisone treated rats were indistinguishable from those of vitamin D deficient rats. The observation that glucocorticoid treatment of rats alters the secretory response of ~he parotid gland similar to that seen in vitamin D deficiency tends further suppor~ to the hypothesis that mitochondrial Ca 2+ transport and storage is an impor° rant aspect of the contro] of secretion from this organ, This work was supported by grants from the Medical l%esearch Council of Canada and The Cystic Fibrosis Foundation (U.S.A.}° References 1 2 3 4 5 6 7 8
Selinger, Z. a n d Naim, E. ( 1 9 7 0 ) Biochim. Biophys. A c t a 20S. 3 3 5 - - 3 3 7 Rasmussen, H.~ G o o d m a n , D.B.P. a n d Tenenhouse0 A. ( 1 9 7 2 ) CRC Crit. Rev. Biochem. 10 9 5 - - 1 4 3 Berridge, M.J. ( 1 9 7 5 ) Adv. Cyclic N u c l e o t i d e Res. 6, 1 - - 9 8 Afari, G., T e n e n h o u s e , A. a n d Klein, J. ( 1 9 7 7 ) Can. J. Physiol. P h a r r n a c o L 55, 4 1 9 - - 4 2 6 E n g s t r o m , G°W, a n d De L u c a , H . F . (-1964) B i o c h e m i s t r y 3 , 2 0 3 - - 2 0 9 K i m b e r g , D.V. and G o l d s t e i n , S.A. (1967) E n d o c r i n o l o g y 8 0 , 8 9 - - 9 8 De L u c a , H.F. ( 1 9 7 4 ) Fed. Proc. ~3, 2 2 1 1 - - 2 2 1 9 R a s m u s s e n , H. a n d Bordier, P. ( 1 9 7 4 ) i n The P h y s i o l o g i c a l a n d Cellular Basis o f M e t a b o l i c Bone Disease, p p . 2 0 7 - - 2 4 9 ~ Williams a n d Wilkins Co.~ B a l t i m o r e
Biochimica et Biophysica Acta , 538 (1978) 631--634 © Elsevler[North-Holland Biomedical Press
BBA Report BBA 21465
PROTEIN SECRETION FROM PAROTID GLANDS OF VITAMIN D DEFICIENT AND GLUCOCORTICOID-TREATED RATS
ALAN TENENHOUSE and GOODWILL AFARI
Department of Pharmacology, McGill University, Montreal, Quebec (Canada) (Received August 22nd, 1977)
Summary The rate of isoproterenol stimulated secretion of protein from parotid glands of vitamin D deficient rats and rats treated with methylprednisolone was increased compared to the secretory response of tissue from control rats. It is suggested that the increased secretory response is secondary to a decreased capacity of mitochondria from the tissue of these animals to take up and store Ca~+; i.e. the mitochondria are less efficient buffers of Cytoplasmic Ca2÷. Under these conditions any process, such as protein secretion, which requires an increased cytoplasmic Ca2÷ concentration will operate more effectively.
Several investigators have proposed that an increased cytoplasmic [ Ca2+] is essential for protein secretion from the parotid gland [1--4]. It has also been suggested that one of the functions of mitochondria is to maintain the concentration of cytoplasmic Ca2÷ constant. It is possible therefore that the increased cytoplasmic [Ca:÷] associated with secretion results from an alternation in the calcium buffering capacity of mitochondria. As first demonstrated by Engstrom and DeLuca [5] the rate and extent of Ca2÷ accumulation by isolated mitochondria from liver, kidney and intestine of vitamin D deficient rats is greatly reduced compared with mitochondria from vitamin D replete rats. Kimberg and Goldstein [6] demonstrated that treatment of vitamin D replete rats with cortisone caused a significant reduction in the rate of Ca2÷ accumulation in subsequently isolated liver mitochondria; the mitochondria from cortisone treated rats were indistinguishable from those of vitamin D deficient rats. In the experiments described in this report vitamin D deficient rats and rats treated with methylprednisolone were used to test the hypothesis that the
632 alteration in Ca 2÷ metabolism existing under these conditions resuits in an altered secretory response of parotid gland° Weanling Holtzman rats were housed in metabolic cages in the dark and fed a vitamin D deficient diet (Raehitogenic Diet AOAC, No. t1077~ iCN Nutrition Biochemical Corporation). The eontrot group of rats, of the same strain, age and sex, housed in the regular animal quarters, were fed ~he same diet as the experimental group supplemented with 100 IoUo vitamin D per week. The animals were used 21--26 days after being placed on the vitamin D deficient diet. At that time there was no gross difference be~ween the control and vitamin D deficient groups; serum [Ca ~+] and phosphate were normal and the b o d y weight of the animals within each group varied between 55--75 g° To study the effect of g|ucocortieoid on parotid gland function, Holtzrnan rats (150--170 g) were injected, subcutaneously, with methylprednisolone, 1 rag/day for 7 days. Protein seereti.on from parotid gland was measured as previously described [41. Zymogen protein was labelled in vitro b y incubating parotid gland slices in Krebs=Ringer bicarbonate buffer containing carrier-free [3H]leueine for 5 min followed by a 2-h ~chase' incubation in Krebs-l~inger bicarbonate buffer containing 1.0 mM [~H]leueine. Secretion was measured b y following the rate of release of [3 H] protein from tissue slices superfused with Krebs-Ringer bicarbonate buffer containing 10 p N isopro~erenol. ~esutts are expressed as the percentage change of 3H-labelled protein o u t p u t into the superfusate with exposure of the tissue to seeretagogue. The release of 3H-labelled protein from rat parotid gland m response to isoproterenol is illustrated in Fig. 1. The tissue from vitamin D replete animals responded rapidly to the isoproterenol; the rate of protein release was maximal
300"
T
2o0
~
z
e
o
100
,/ 10
2G
30
40
MINUTES Fig. 1. The effect o f i s o p r o t e r e n o ] ( 1 0 -5 M ) o n ~H-lahelled p r o t e i n ~elease f r o m t h e pasotid gland o f vitamin D-deficient (e) and vitamin D-repleted (*) rats. Superfusion medium was K~ebs-Ringer buffe~ c o n t a i n i n g 1 0 -3 M C a 2+. T i m e ' 0 ' is t h e t i m e the drug r e a c h e d t h e tissue c h a m b e r . P r o t e i n r e l e a s e is e x p r e s s e d as the p e r c e n t greater t h a n b a s a l r e l e a s e , E a c h p o i n t is t h e m e a n -~ S . E . o f a t least three e x p e r ~ iments.
633 within 6--8 rain (120--140% greater than basal). This maximum rate was maintained approx. 10 min then declined to the basal rate approx. 35 min after superfusion with isoproterenol was stopped. The response of the tissue from vitamin D deficient animals was greatly increased. The m a x i m u m rate of secretion, 350% greater than basal was reached within 12--14 min, maintained for 10 min, then decreased b u t was still approx. 75% greater than the basal secretion rate 40 min after superfusion with isoproterenol was stopped. The characteristics of the two responses were similar; the major difference was that in the vitamin D deficient tissue the initial rapid increase in secretion rate persisted for approximately twice as long, reaching a maximum secretion rate 2.5--3.0-times that reached with tissue from vitamin D replete rats. The secretory response to isoproterenol of parotid gland from rats treated with methylprednisolone is shown in Fig. 2. In the tissue from untreated 200
03 tu ..J Lu n-
100
z
LU kO rr a. z
e
10
20
30
40
50
60
MINUTES Fig. 2. T h e e f f e c t o f i s o p z o t e z e n o l (10 -s M) o n 3H-labelled p r o t e i n s e c r e t i o n f r o m p a r o t i d gland of u n t r e a t e d rats ( o ) a n d rats t r e a t e d w i t h 1 r a g / d a y m e t h y l p r e d n i s o l o n e fo~ 7 d a y s (e).
animals the rate of protein release increased to a maximum 85% greater than the basal secretion rate whereas in tissue from steroid treated animals the m a x i m u m rate of secretion was approx. 170% greater than the basal secretion rate. From these experiments it is clear that vitamin D deficiency alters the secretory response of rat parotid gland to isoproterenol. The rate of secretion of 3H-labelled protein release in response to isoproterenol is increased whereas the basal secretion rate is unaffected b y vitamin D deficiency. Although the role of vitamin D in protein secretion from the parotid gland is n o t known, based on the observations reported here and on the k n o w n effects of vitamin D on mitochondrial Ca 2. metabolism [7,8] a working hypothesis has been developed. It is postulated as has been done by several authors, that cytoplasmic [Ca 2. ] must increase, for protein secretion from the parotid gland to occur. The mitochondrial Ca 2÷ uptake process functions to prevent an increase in cytoplasmic [Ca 2+] and so antagonizes the effect of secretagogues. This mitochondrial Ca 2÷ uptake process is vitamin D dependent so that in vitamin D deficient animals the ability of mitochondria to take up Ca 2÷ is impaired. In this situation cytoplasmic [Ca 2÷] is more readily increased. Any process which depends on such an increase will operate more effectively. Glucocorticoid treatment of rats has been shown to alter the ability of isolated
634 mitochondria to take up Ca 2+ t5~; the mitochondria from cortisone treated rats were indistinguishable from those of vitamin D deficient rats. The observation that glucocorticoid treatment of rats alters the secretory response of ~he parotid gland similar to that seen in vitamin D deficiency tends further suppor~ to the hypothesis that mitochondrial Ca 2+ transport and storage is an impor° rant aspect of the contro] of secretion from this organ, This work was supported by grants from the Medical l%esearch Council of Canada and The Cystic Fibrosis Foundation (U.S.A.}° References 1 2 3 4 5 6 7 8
Selinger, Z. a n d Naim, E. ( 1 9 7 0 ) Biochim. Biophys. A c t a 20S. 3 3 5 - - 3 3 7 Rasmussen, H.~ G o o d m a n , D.B.P. a n d Tenenhouse0 A. ( 1 9 7 2 ) CRC Crit. Rev. Biochem. 10 9 5 - - 1 4 3 Berridge, M.J. ( 1 9 7 5 ) Adv. Cyclic N u c l e o t i d e Res. 6, 1 - - 9 8 Afari, G., T e n e n h o u s e , A. a n d Klein, J. ( 1 9 7 7 ) Can. J. Physiol. P h a r r n a c o L 55, 4 1 9 - - 4 2 6 E n g s t r o m , G°W, a n d De L u c a , H . F . (-1964) B i o c h e m i s t r y 3 , 2 0 3 - - 2 0 9 K i m b e r g , D.V. and G o l d s t e i n , S.A. (1967) E n d o c r i n o l o g y 8 0 , 8 9 - - 9 8 De L u c a , H.F. ( 1 9 7 4 ) Fed. Proc. ~3, 2 2 1 1 - - 2 2 1 9 R a s m u s s e n , H. a n d Bordier, P. ( 1 9 7 4 ) i n The P h y s i o l o g i c a l a n d Cellular Basis o f M e t a b o l i c Bone Disease, p p . 2 0 7 - - 2 4 9 ~ Williams a n d Wilkins Co.~ B a l t i m o r e