- Email: [email protected]
Modulation of kidney 25-hydroxyvitamin D3 metabolism by 1α,25-dihydroxyvitamin D3 in thyroparathyroidectomized rats
Lifa Sciaaces Vol. 19, pp . 1943-1948, 1976 . Printad in tha D .S .A,
Pergamon Prass
MODUIATION OF KIDNBY 25-HYDROXYVITAMIN D3 METABOLISM BY 1a,25-DIHYDROXYVITAMIN D3 IN THYROPARATHYROIDECIbMIZED RATS John L. Omdahl Department of Biochemistry, School of Medicine University of New Mexico, Albuquerque, Nex Mexico 87131 (Received is final fora November 8, 1976) Vitamin D depleted rats which are given 25-hydroxyvitaain D3 and fed a high caici~-low phosphorus diet prod ce 1a,25-dihydroxyvitaain3fi-D3 fma the precursor 25-hydroxyvitamin-~-D3, Supplementation of these rats with 1a,25-dihydroxyvi~anin D3 results in an induced synthesis of 24,25-dihydroxyvitamin- -D3 and a nearly total suppression of Ia,25-dihydroxyvitaain- 31i-D3 synthesis . The inductive action of 1a,25-dihydroxyvitaain D3 can be effected in approximately 3 hours, occurs in the absence of thyroid and parathyroid tissue and is inhibited by actinomycin D. It appears that 1a,25-dihydroxyvitamin D3 effects its action via a transcriptional process which can occur independently of parathyroid hormone's and calcitonin's influence on kidney 25-hydroxyvitamin D3 metabolism . The vitamin D3 metabolite aost active in calciua transport processes is la, 25-dihydroxyvitamin D3 (1a,25-(OH)2D3) (1,2) . Parathyroid hormone (PTH) promotes 1a,25-(OH)2D3 synthesis by stimulating the kidney-aitochondrial la hydroxylation of 25-hydroxyvitamin D3 (25-OHD3) (3-5) . This hydroxylation reaction is modulated, in part, via a calcitm-feedback mechanism wherein PTH secretion is altered in response to changes in the ambient calcite concentration (6,7), The negative feedback aspect of the control syste® is lost in vitamin D deficiency and appears dependent upon the presence of 1a,25-(Otn 2Dg . Specifically, 1a,25-(OH)2D3 couples the feedback action by suppressing its own synthesis and inducing the production of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a metabolite which has a law biological activity (8-11) . How 1a,25(OH)2D3) effects its actions is presently unclear . Since PTH appears to antagonize the inductive action of 1x,25-(OH)2D3 (8) it is possible that the metabolite's action could be accomplished indirectly via suppression of PTH secretion. Alternatively, 1x,25-(OH)2D3 could be acting independently of PTH similar to the metabolite's direct target-organ function in the intestine (12) . Experiments in this study were designed to evaluate the possible indirect involvement of PTH or calcitonin (CT) . Accordingly, the metabolite's action was examined by using thyroparathyroidectomized (TPTX) rats so as to evaluate a possible PTH or Cf involvement . Additionally, a transcriptional requirement for the metabolite's in vivo action was investigated using actinonycin D, an inhibitor of a-RNAsyn~sis . METHODS idecto - IYeanling ale rats (Holtznan) were fed as adequate Th o wrath caiciua 0 .47 percent -p osphorus (0 .3 percent) vitamin-D-deficient diet for three weeks (13), subsequently dosed with 625 peoles of 25-hydroxyvitaain D3 and switched to a low-caici~n (0 .08 percent) low-phosphorus (0 .1 percent) diet 1943
1944
Modulation of 25-oHD3 Metabolism
Vol . 19, No . 12
for 48 hours prior to blunt surgical thyroparathyroidectomy using a light ether anaesthesia. Five hours post surgery rats were given 5 I .U . of parathyroid extract (Eli Lilly) with operational success verified at 24 hogs by a 2 .5 mg/dl drop in total serum calcium . TPTX rats were given bidaily thyroxine replacement (5 Mcgm), intraperitoneally . Sham and TPTX animals were placed on a highcalcium (0 .8 or 1 .2 percent) low-phosphorus (0 .1 percent) diet for 7 days and given 1a,25-(OH)2D3 chronically (130 pmoles daily, intraperitoneally) or acutely (2 .4 pmoles, 8 1/2 hours before sacrifice, intravenously) as described in the Tables . Carrier vehicle was propylene glycol :ethanol (7 :3) or ethanol : water (95 :5) for the chronic and acute studies, respectively . In Vivo 25-OH- 3fi-D Metabolism - Renal 25-OH- 31i-D3 metabolism was assessed by giv ng an ntravenous in action of purified metabolite (30 pmoles of 25-(26, 27-3H)-OHD3, 10 .7 Ci/mmole ; Amersham-Searle) at 6 or .12 hours prior to sacri Rats were killed by exsanguination with serum fice, as noted in the Tables . used for measurement of calcium (14) and phosphorus concentrations (15) and 25-OH- 3H-D3 metabolites (16) . Separation of 25-OH- 3f{-D3 metabolites was effected by column chromatography (1 .3 x 17 cm, 2 .5 ml fractions) of serer lipid extracts (17) using Sephadex LH-20 as the support phase and a chloroform : hexane (7 :3) mixture for column equilibration and elution. Periodate cleavage of vicinal hydroxyl groups (40 ul of 5$ (w/v) NaI04, pH 3, added to 0 .4 ml sample in methanol, roam temperature for 5 hours) was used to determine the presence of 24,25-(OH)2- 3I-1-D3 and 25,26-(OH)2-3I-I-D3 . Chromatographic fractions were dried under forced air, dissolved in a toluene scintillation cocktail (17) and the radioactivity measured with a Beclanan (LS-245) liquid scintillation spectrophotometer . Vitamin D Metabolites - Crystalline 25-OHD3 (provided by Dr . DeLuca, a, - ~ 2D3 (gift of Hoffmami-LaRoche) were spectrophotoWiscons n an metrically quantitated through the use of the molar extinction coefficient for vitamin D3 (a265 = 18,200) . RESULTS Rats fed a high-calcium low-phosphorus diet for 7 days metabolized 25-OH3i-I-D3 to only 1a,25-(OH)- 3f{-2D3 . A diametric response was obtained in rats given daily 1a,25-(OH)2D3 supplements wherein only 24,25-(Od-n -3fi-2D3 was detectable (Table I) . The chronic ability of 1a,25-(Otl)2D3 to suppress its own synthesis and stimulate 24,25-(Ofi)2D3 production was also demonstrable in the absence of PTH and CT (i .e . using TPTX rats) . No significant difference in the switch over from 1x,25-(OH)2D3 to 24,25-(OH)2D3 production was observed between TPTX and sham animals (Table I) . Rats whose thyroid and parathyroid axis were removed showed the same potential for 1a,25-(OH)2D3 induced 24-hydroxylase and suppressed la-hydroxylase activity as did sham animals . Such results indicate that 1a,25-(OH)2D3 does not effect its inductive-suppressive action via PTH or CT . Although TPTX carrier rats had a lower serum calcium than sham controls there was not a difference in the animals serum 1a,25-(OH)-3ü-2D3 levels . In contrast, serer 24,25-(OH)2- 3fi-D3 concentration was enhanced in TPTX-carrier compared to sham-carrier animals suggesting that PTH may act as an antagonist to 1x,25-(OH)2D3 directed induction of 24-hydroxylase activity, as suggested by the results of Tanaka et al . (9) . Prior to this study the shortest reported time span between 1x,25-(OH)2D 3 dosage and subsequent alteration in 25-OHD3 metabolism has been 9 hours at a 1a,25-(OH)2D3 dosage of 125 pmoles (10) . In contrast we ass able to effect an action for 1a,25-(OH)2D3 at approximately 3 hours following a dosage of 2 .4 pmoles (Table II) . This is the shortest time lag reported wherein . a diametric
Vol. 19, No . 12
Modulation of 25-0®3 Matabolien
1945
response in 25-OHD3 metabolism can be effected by either horaonal or metabolite treataent. Developaent of such an anisal aodel facilitates the in vivo vse of actinouycin D for evaluating a possible transcriptional involvement the metabolite's action . Since previous studies (10,18) have used extremely high TABLE I THYROPARATHYltOIDEC1nMY AND CHRONIC IN VIVO 1a,25-(OH)2D3 PROI~TED CHANGES IN 3H-25-(OH)D3 I~TAHOLISM AND SERi~! CALCIUM CONCENTRATIONS After seven days an a high-calcite (0 .8~) lox-phosphorus (0 .14) diet, the rats were given 30 paoles of 25-(26,27- 311)-OHD3 (10 .7 Ci/aaole) intravenously (30 u1, 954 ethanol) and killed 12 hours later with the serum analyzed as de scribed in the text . Values are the mean t the standard deviation with the number of observations given in parentheses . Group Sham Sham TPTX TPTX
(carrier) (1a,25) (carrier (1a,25)
(5) (5) (4) (5)
24,25-(OH)-3t1-2D3 (dpa/nl)
Serum Parameters 1a,25-(OH)-31t-2D3 (dpn/nl)
0 4928 t 1318 1818 t 458 4970 t 1118
3034 t 1040* 0 2142 t 842 0
Calcium (ag/dl) 8.7 8.8 6.4 9 .2
t t t t
0.5 0.4 0.61 0.2
* (p < 0 .01) carrier compared to 1a,25-(OH)2D3 in sham and TPTX groups for all serum metabolite data . (p < 0 .01) carrier compared to 1a,25-(OH)2D3 in TPTX group . TABLE
II
ACUTE 1a,25-(OH)2D3 INDUCED ALTERATIONS IN 25-OH- 3ti-D3 I~TABOLISM AND THE AFFECT OF ACTINOMYCIN D Rats xere fed a high-calcium (1 .24) low-phosphorus (0 .14) diet for 7 days before experimentation, as per Methods . Actinomycin D (1 up/gm body xeight) or carrier (1 :1 ; ethano1 :0 .15 M NaCl) was given intravenously (i .v .) 1 hour prior to an i .v . dose of 1x,25-(OH)2Dg 2 .4 nmoles) or carrier xhich was followed at 2.5 hours by an i.v . 25-OH-~-D3 injection as described in Table I . Rats xere killed 6 hours after the 25-OH-3li-D3 injection. Values are the aeon t the standard deviation with number of observations given in parentheses. Äctina~ycin D Treatment
Group Sham Shan TPTX TPTX TPT7C TPTX
(carrier) (1a,25) (carrier) (1a,25) (carrier) (1a,25)
(6) (4) (6) (7) (4) (7)
+ +
Serum (dpa/ml) 24,25-(OH)2- 3FI-D3 1a,25-(OH) 2-311-D3 0* 3648 t 1352 20o t z8o 3910 t 772 0* 800 t 210
2228 t 1322i 430 t 573 142o t 75a* 0 874 t 4365 120 t 240
* (p < 0 .01) carrier compared to 1x,25 in sham and 1YTX groups . i (p < 0 .05) carrier compared to 1a,25 in st~aa group . 5 (p < 0 .02) carrier compared to 1a,25 in TPTX/actinomycin D group.
1946
Modulation of 25-0HD3 Metabolism
Vol. 19, No . 12
levels of actinomycin D, it seemed appropriate to reevaluate the antibiotic's action at a concentration which inhibits RNA synthesis (19) but is non-toxic under the employed experimental conditions . Although use of a lower actinomycin D treatment did not completely block the 1x,25-(0~ 2D3 prompted switchover in metabolite production there was nearly an 80~ inhibition in 24,25(OH)2- 3fi-D3 induction (Table II) . In addition, 1a,25-(OH)2- 3Fl-Dg production was maintained to a limited extent in actinomycin D treated rats whereas a complete suppression was observed in control animals (minus actinomycin D) (Table II) . Incomplete blockage of the metabolites action is most likely attributable to the use of a lowered actinomycin D level . An in vitro model system which is more desirable for studying the affects of transcriptional inhibitors, is currently being developed in order to study this subject more extensively . DISCUSSION Kidney activation of 25-(l-IDg to 1a,25-(OH)2D3 controls to a significant degree the maintenance of the ionized calcium pool at a concentration which permits optimal functioning of calcium-dependent cellular processes . Excessive calcium intake in the presence of phosphorus results in the suppression of lahydroxylase and induction of 24-hydroxylase activities (6,~ , which effects a diminution in calcium similation and retention due to lowered levels of 1a,25(~)2D3 synthesis . The physiological role for 24,25-(~ 2D3 under such conditions is not presently clear although the 24-hydroxylation may signify a preparatory step for 25-OEID3 excretion similar to the formation of adrenal steroid glucuronic-acid conjugates . Modulation of the switch-over from la-hydroxylase to 24-hydroxylase activity can potentially involve alterations in levels of PTI~ CT, calcium, phosphorus and 1a,25-(OH)2D3. From this study it is now evident that the cellular modulatioy process can function independently of PTH and CT involvement. Furthermore, the ability to effect the inductive/suppressive actions of 1a,25-(OFn 2D3 within a short time span (approximately 3 hours, Table II), suggests that the metabolite may be acting directly on kidney enzyme synthetic/degradative processes . Alluding to such a possibility are the results of Chen et al . in which 1a,25-(OH)2D3 was observed to stimulate kidney nuclear-RNA synthesis (19) . A detailed description of other factors (e .g . calcium and/or phosphorus) involved in the metabolite's prompted kidney action must await further experimentation. Also suggested from this study is the requirement for a transcriptional step in the molecular mechanism for the inductive-suppressive action of 1a,25(OH)2Dg . MacIntyre et al . (10,18) have shown a similar inhibition in chicks treated with actinomycinD or a-aminitin, an RNA polymerase inhibitor . Although their results are convincing, the experimental model did not allow for stating that the action of 1a,25-(OIL 2D3 or the transcription inhibitors was effected independently of PTH and CT . Such an insight is obtainable from the present study, wherein actinomycin D was used in TPTX rats . It seems reasonable, therefore, to use as a working hypothesis the concept that 1a,25-(OH)2D3 prompts a change in kidney cell nuclear directed m-RNA synthesis which results in stimulated and suppressed assemblage of the mitochondrial 25-OHD3-24hydroxylase and la-hydroxylase enzyme systems, respectively . Although cellular and organelle concentrations of calcium and phosphorus could conceivably alter enzyme activity at the nuclear or enzyme level, it seems unlikely that the ions L se could effect such de novo protein synthesis as demonstrated in this study . It is evident theréfore, that 1a,25-(OH)2D3 occupies an important role in the renal modulation of 25-OHD3 metabolism .
Vol . 19, No . 12
Modulation of 25-0HD3 Metabolism
1947
ACKNOWLEDGMENTS The technical assistance of Vicky Aschenbrermer and Lucy Hunsaker, and manuscript assistance of B . Dershem and M . Samaha is acknavledged . This study was supported with funds from the Nutrition Foundation (8502) and the National Institutes of Health (AM-16905) . REPERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
J . L . OMDAHL, and H .F . D~ELUCA, Physiol . Rev . 53, 327-372 (1973) . M .F . HOLICK, A . KLEINER-BOSSALLER, H . K . SCHNOES, P .M . KASTEN, I .T . BOYLE, and H .F . DELUCA, J . Biol . Chem . 248, 6691-6696 (1973) . M . GARABEDIAN, M . F . HOLICK, H . F . DELUCA, and I .T . BOYLE, Proc . Nat . Acad . Sci . USA 69, 1673-1676 (1972) . b~F. ~GSÉTt, and E . KODICEK, Nature 241, 163-166 (1973) . E .B . MAfYER, J . BLACKHOUSE, L .F . HILL, G .A . LOMB, P . DBSILVA, C .M . TAYLOR, and S .W . STANBURY, Clin . Sci . Mol . Med . 48, 349-365 (1974) . I .T . BOYLE, R .W . GRAY, andd H.FDELUCA, . Proc . N_at . Acad . Sci . USA 68, 2131-2134 (1971) . J . L . OMDAHL, R .W . GRAY, I .T . BOYLE, J . KNUTSON, and H .F . DELUCA, Nature 237, 63-64 (1972) . Y.TANAKA, and H .F . DELUCA, Science ~183r 1198-1200 (1974) . Y . TANAKA, R .S . LORENC, and H .F . DELUGA, Arch . Biochem . Bio s . 171, 521-526 (1975) . I . MACINTYRE, K .W . COISTON, I .M.A . EVANS, E . IAPEZ, S .J . MACAULEY, J . PIEGNOUX-DEVILLE, E . SPANOS, and M . SZEIKE, Clin . Endo . 5 (Suppl .), 85-5 - 95-5 (1976) . R . G . LARKINS, S .J . MACAULEY, and I . MACINTYRE, Nature 252, 412-414 (1974) . P . F . BRUMBAUGH, and M .R . HAUSSLER, J . Biol . Chem . 249 1258-1262 (1974) . T . SUDA, H .F . DELUCA, and Y . TANAKA, qtr-T0,~49-1051 (1970) . J .L . OMDAHL, M . F . HOLICK, T . SUDA, Y . TANAKA, and H .F . DELUCA, Biochemistry 10, 2935-2940 (1971) . P .S . CHEN, JR ., T .Y . TORIBARA, and H . WARNER, Anal . Chem . 28, 1756-1758 (1956) . M .F . HOLICK, and H .F . DELUCA, J . ~Li i~d Res . 12, 460-465 (1971) . P .F . NEVILLE, and H .F . DELUCA, Biochemist 5, 2201-2207 (1966) . I .M.A . EVANS, K .W . COISTON, L . GA~ Î. MACINI'YRE, Clin . Sci . Mol . Med . 48, 227-230 . (1975) . . T C . CHEN, and H .F . DELUCA, Arch . Biochem . Biophys . 156, 321-327 (1973) .
Pergamon Prass
MODUIATION OF KIDNBY 25-HYDROXYVITAMIN D3 METABOLISM BY 1a,25-DIHYDROXYVITAMIN D3 IN THYROPARATHYROIDECIbMIZED RATS John L. Omdahl Department of Biochemistry, School of Medicine University of New Mexico, Albuquerque, Nex Mexico 87131 (Received is final fora November 8, 1976) Vitamin D depleted rats which are given 25-hydroxyvitaain D3 and fed a high caici~-low phosphorus diet prod ce 1a,25-dihydroxyvitaain3fi-D3 fma the precursor 25-hydroxyvitamin-~-D3, Supplementation of these rats with 1a,25-dihydroxyvi~anin D3 results in an induced synthesis of 24,25-dihydroxyvitamin- -D3 and a nearly total suppression of Ia,25-dihydroxyvitaain- 31i-D3 synthesis . The inductive action of 1a,25-dihydroxyvitaain D3 can be effected in approximately 3 hours, occurs in the absence of thyroid and parathyroid tissue and is inhibited by actinomycin D. It appears that 1a,25-dihydroxyvitamin D3 effects its action via a transcriptional process which can occur independently of parathyroid hormone's and calcitonin's influence on kidney 25-hydroxyvitamin D3 metabolism . The vitamin D3 metabolite aost active in calciua transport processes is la, 25-dihydroxyvitamin D3 (1a,25-(OH)2D3) (1,2) . Parathyroid hormone (PTH) promotes 1a,25-(OH)2D3 synthesis by stimulating the kidney-aitochondrial la hydroxylation of 25-hydroxyvitamin D3 (25-OHD3) (3-5) . This hydroxylation reaction is modulated, in part, via a calcitm-feedback mechanism wherein PTH secretion is altered in response to changes in the ambient calcite concentration (6,7), The negative feedback aspect of the control syste® is lost in vitamin D deficiency and appears dependent upon the presence of 1a,25-(Otn 2Dg . Specifically, 1a,25-(OH)2D3 couples the feedback action by suppressing its own synthesis and inducing the production of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a metabolite which has a law biological activity (8-11) . How 1a,25(OH)2D3) effects its actions is presently unclear . Since PTH appears to antagonize the inductive action of 1x,25-(OH)2D3 (8) it is possible that the metabolite's action could be accomplished indirectly via suppression of PTH secretion. Alternatively, 1x,25-(OH)2D3 could be acting independently of PTH similar to the metabolite's direct target-organ function in the intestine (12) . Experiments in this study were designed to evaluate the possible indirect involvement of PTH or calcitonin (CT) . Accordingly, the metabolite's action was examined by using thyroparathyroidectomized (TPTX) rats so as to evaluate a possible PTH or Cf involvement . Additionally, a transcriptional requirement for the metabolite's in vivo action was investigated using actinonycin D, an inhibitor of a-RNAsyn~sis . METHODS idecto - IYeanling ale rats (Holtznan) were fed as adequate Th o wrath caiciua 0 .47 percent -p osphorus (0 .3 percent) vitamin-D-deficient diet for three weeks (13), subsequently dosed with 625 peoles of 25-hydroxyvitaain D3 and switched to a low-caici~n (0 .08 percent) low-phosphorus (0 .1 percent) diet 1943
1944
Modulation of 25-oHD3 Metabolism
Vol . 19, No . 12
for 48 hours prior to blunt surgical thyroparathyroidectomy using a light ether anaesthesia. Five hours post surgery rats were given 5 I .U . of parathyroid extract (Eli Lilly) with operational success verified at 24 hogs by a 2 .5 mg/dl drop in total serum calcium . TPTX rats were given bidaily thyroxine replacement (5 Mcgm), intraperitoneally . Sham and TPTX animals were placed on a highcalcium (0 .8 or 1 .2 percent) low-phosphorus (0 .1 percent) diet for 7 days and given 1a,25-(OH)2D3 chronically (130 pmoles daily, intraperitoneally) or acutely (2 .4 pmoles, 8 1/2 hours before sacrifice, intravenously) as described in the Tables . Carrier vehicle was propylene glycol :ethanol (7 :3) or ethanol : water (95 :5) for the chronic and acute studies, respectively . In Vivo 25-OH- 3fi-D Metabolism - Renal 25-OH- 31i-D3 metabolism was assessed by giv ng an ntravenous in action of purified metabolite (30 pmoles of 25-(26, 27-3H)-OHD3, 10 .7 Ci/mmole ; Amersham-Searle) at 6 or .12 hours prior to sacri Rats were killed by exsanguination with serum fice, as noted in the Tables . used for measurement of calcium (14) and phosphorus concentrations (15) and 25-OH- 3H-D3 metabolites (16) . Separation of 25-OH- 3f{-D3 metabolites was effected by column chromatography (1 .3 x 17 cm, 2 .5 ml fractions) of serer lipid extracts (17) using Sephadex LH-20 as the support phase and a chloroform : hexane (7 :3) mixture for column equilibration and elution. Periodate cleavage of vicinal hydroxyl groups (40 ul of 5$ (w/v) NaI04, pH 3, added to 0 .4 ml sample in methanol, roam temperature for 5 hours) was used to determine the presence of 24,25-(OH)2- 3I-1-D3 and 25,26-(OH)2-3I-I-D3 . Chromatographic fractions were dried under forced air, dissolved in a toluene scintillation cocktail (17) and the radioactivity measured with a Beclanan (LS-245) liquid scintillation spectrophotometer . Vitamin D Metabolites - Crystalline 25-OHD3 (provided by Dr . DeLuca, a, - ~ 2D3 (gift of Hoffmami-LaRoche) were spectrophotoWiscons n an metrically quantitated through the use of the molar extinction coefficient for vitamin D3 (a265 = 18,200) . RESULTS Rats fed a high-calcium low-phosphorus diet for 7 days metabolized 25-OH3i-I-D3 to only 1a,25-(OH)- 3f{-2D3 . A diametric response was obtained in rats given daily 1a,25-(OH)2D3 supplements wherein only 24,25-(Od-n -3fi-2D3 was detectable (Table I) . The chronic ability of 1a,25-(Otl)2D3 to suppress its own synthesis and stimulate 24,25-(Ofi)2D3 production was also demonstrable in the absence of PTH and CT (i .e . using TPTX rats) . No significant difference in the switch over from 1x,25-(OH)2D3 to 24,25-(OH)2D3 production was observed between TPTX and sham animals (Table I) . Rats whose thyroid and parathyroid axis were removed showed the same potential for 1a,25-(OH)2D3 induced 24-hydroxylase and suppressed la-hydroxylase activity as did sham animals . Such results indicate that 1a,25-(OH)2D3 does not effect its inductive-suppressive action via PTH or CT . Although TPTX carrier rats had a lower serum calcium than sham controls there was not a difference in the animals serum 1a,25-(OH)-3ü-2D3 levels . In contrast, serer 24,25-(OH)2- 3fi-D3 concentration was enhanced in TPTX-carrier compared to sham-carrier animals suggesting that PTH may act as an antagonist to 1x,25-(OH)2D3 directed induction of 24-hydroxylase activity, as suggested by the results of Tanaka et al . (9) . Prior to this study the shortest reported time span between 1x,25-(OH)2D 3 dosage and subsequent alteration in 25-OHD3 metabolism has been 9 hours at a 1a,25-(OH)2D3 dosage of 125 pmoles (10) . In contrast we ass able to effect an action for 1a,25-(OH)2D3 at approximately 3 hours following a dosage of 2 .4 pmoles (Table II) . This is the shortest time lag reported wherein . a diametric
Vol. 19, No . 12
Modulation of 25-0®3 Matabolien
1945
response in 25-OHD3 metabolism can be effected by either horaonal or metabolite treataent. Developaent of such an anisal aodel facilitates the in vivo vse of actinouycin D for evaluating a possible transcriptional involvement the metabolite's action . Since previous studies (10,18) have used extremely high TABLE I THYROPARATHYltOIDEC1nMY AND CHRONIC IN VIVO 1a,25-(OH)2D3 PROI~TED CHANGES IN 3H-25-(OH)D3 I~TAHOLISM AND SERi~! CALCIUM CONCENTRATIONS After seven days an a high-calcite (0 .8~) lox-phosphorus (0 .14) diet, the rats were given 30 paoles of 25-(26,27- 311)-OHD3 (10 .7 Ci/aaole) intravenously (30 u1, 954 ethanol) and killed 12 hours later with the serum analyzed as de scribed in the text . Values are the mean t the standard deviation with the number of observations given in parentheses . Group Sham Sham TPTX TPTX
(carrier) (1a,25) (carrier (1a,25)
(5) (5) (4) (5)
24,25-(OH)-3t1-2D3 (dpa/nl)
Serum Parameters 1a,25-(OH)-31t-2D3 (dpn/nl)
0 4928 t 1318 1818 t 458 4970 t 1118
3034 t 1040* 0 2142 t 842 0
Calcium (ag/dl) 8.7 8.8 6.4 9 .2
t t t t
0.5 0.4 0.61 0.2
* (p < 0 .01) carrier compared to 1a,25-(OH)2D3 in sham and TPTX groups for all serum metabolite data . (p < 0 .01) carrier compared to 1a,25-(OH)2D3 in TPTX group . TABLE
II
ACUTE 1a,25-(OH)2D3 INDUCED ALTERATIONS IN 25-OH- 3ti-D3 I~TABOLISM AND THE AFFECT OF ACTINOMYCIN D Rats xere fed a high-calcium (1 .24) low-phosphorus (0 .14) diet for 7 days before experimentation, as per Methods . Actinomycin D (1 up/gm body xeight) or carrier (1 :1 ; ethano1 :0 .15 M NaCl) was given intravenously (i .v .) 1 hour prior to an i .v . dose of 1x,25-(OH)2Dg 2 .4 nmoles) or carrier xhich was followed at 2.5 hours by an i.v . 25-OH-~-D3 injection as described in Table I . Rats xere killed 6 hours after the 25-OH-3li-D3 injection. Values are the aeon t the standard deviation with number of observations given in parentheses. Äctina~ycin D Treatment
Group Sham Shan TPTX TPTX TPT7C TPTX
(carrier) (1a,25) (carrier) (1a,25) (carrier) (1a,25)
(6) (4) (6) (7) (4) (7)
+ +
Serum (dpa/ml) 24,25-(OH)2- 3FI-D3 1a,25-(OH) 2-311-D3 0* 3648 t 1352 20o t z8o 3910 t 772 0* 800 t 210
2228 t 1322i 430 t 573 142o t 75a* 0 874 t 4365 120 t 240
* (p < 0 .01) carrier compared to 1x,25 in sham and 1YTX groups . i (p < 0 .05) carrier compared to 1a,25 in st~aa group . 5 (p < 0 .02) carrier compared to 1a,25 in TPTX/actinomycin D group.
1946
Modulation of 25-0HD3 Metabolism
Vol. 19, No . 12
levels of actinomycin D, it seemed appropriate to reevaluate the antibiotic's action at a concentration which inhibits RNA synthesis (19) but is non-toxic under the employed experimental conditions . Although use of a lower actinomycin D treatment did not completely block the 1x,25-(0~ 2D3 prompted switchover in metabolite production there was nearly an 80~ inhibition in 24,25(OH)2- 3fi-D3 induction (Table II) . In addition, 1a,25-(OH)2- 3Fl-Dg production was maintained to a limited extent in actinomycin D treated rats whereas a complete suppression was observed in control animals (minus actinomycin D) (Table II) . Incomplete blockage of the metabolites action is most likely attributable to the use of a lowered actinomycin D level . An in vitro model system which is more desirable for studying the affects of transcriptional inhibitors, is currently being developed in order to study this subject more extensively . DISCUSSION Kidney activation of 25-(l-IDg to 1a,25-(OH)2D3 controls to a significant degree the maintenance of the ionized calcium pool at a concentration which permits optimal functioning of calcium-dependent cellular processes . Excessive calcium intake in the presence of phosphorus results in the suppression of lahydroxylase and induction of 24-hydroxylase activities (6,~ , which effects a diminution in calcium similation and retention due to lowered levels of 1a,25(~)2D3 synthesis . The physiological role for 24,25-(~ 2D3 under such conditions is not presently clear although the 24-hydroxylation may signify a preparatory step for 25-OEID3 excretion similar to the formation of adrenal steroid glucuronic-acid conjugates . Modulation of the switch-over from la-hydroxylase to 24-hydroxylase activity can potentially involve alterations in levels of PTI~ CT, calcium, phosphorus and 1a,25-(OH)2D3. From this study it is now evident that the cellular modulatioy process can function independently of PTH and CT involvement. Furthermore, the ability to effect the inductive/suppressive actions of 1a,25-(OFn 2D3 within a short time span (approximately 3 hours, Table II), suggests that the metabolite may be acting directly on kidney enzyme synthetic/degradative processes . Alluding to such a possibility are the results of Chen et al . in which 1a,25-(OH)2D3 was observed to stimulate kidney nuclear-RNA synthesis (19) . A detailed description of other factors (e .g . calcium and/or phosphorus) involved in the metabolite's prompted kidney action must await further experimentation. Also suggested from this study is the requirement for a transcriptional step in the molecular mechanism for the inductive-suppressive action of 1a,25(OH)2Dg . MacIntyre et al . (10,18) have shown a similar inhibition in chicks treated with actinomycinD or a-aminitin, an RNA polymerase inhibitor . Although their results are convincing, the experimental model did not allow for stating that the action of 1a,25-(OIL 2D3 or the transcription inhibitors was effected independently of PTH and CT . Such an insight is obtainable from the present study, wherein actinomycin D was used in TPTX rats . It seems reasonable, therefore, to use as a working hypothesis the concept that 1a,25-(OH)2D3 prompts a change in kidney cell nuclear directed m-RNA synthesis which results in stimulated and suppressed assemblage of the mitochondrial 25-OHD3-24hydroxylase and la-hydroxylase enzyme systems, respectively . Although cellular and organelle concentrations of calcium and phosphorus could conceivably alter enzyme activity at the nuclear or enzyme level, it seems unlikely that the ions L se could effect such de novo protein synthesis as demonstrated in this study . It is evident theréfore, that 1a,25-(OH)2D3 occupies an important role in the renal modulation of 25-OHD3 metabolism .
Vol . 19, No . 12
Modulation of 25-0HD3 Metabolism
1947
ACKNOWLEDGMENTS The technical assistance of Vicky Aschenbrermer and Lucy Hunsaker, and manuscript assistance of B . Dershem and M . Samaha is acknavledged . This study was supported with funds from the Nutrition Foundation (8502) and the National Institutes of Health (AM-16905) . REPERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
J . L . OMDAHL, and H .F . D~ELUCA, Physiol . Rev . 53, 327-372 (1973) . M .F . HOLICK, A . KLEINER-BOSSALLER, H . K . SCHNOES, P .M . KASTEN, I .T . BOYLE, and H .F . DELUCA, J . Biol . Chem . 248, 6691-6696 (1973) . M . GARABEDIAN, M . F . HOLICK, H . F . DELUCA, and I .T . BOYLE, Proc . Nat . Acad . Sci . USA 69, 1673-1676 (1972) . b~F. ~GSÉTt, and E . KODICEK, Nature 241, 163-166 (1973) . E .B . MAfYER, J . BLACKHOUSE, L .F . HILL, G .A . LOMB, P . DBSILVA, C .M . TAYLOR, and S .W . STANBURY, Clin . Sci . Mol . Med . 48, 349-365 (1974) . I .T . BOYLE, R .W . GRAY, andd H.FDELUCA, . Proc . N_at . Acad . Sci . USA 68, 2131-2134 (1971) . J . L . OMDAHL, R .W . GRAY, I .T . BOYLE, J . KNUTSON, and H .F . DELUCA, Nature 237, 63-64 (1972) . Y.TANAKA, and H .F . DELUCA, Science ~183r 1198-1200 (1974) . Y . TANAKA, R .S . LORENC, and H .F . DELUGA, Arch . Biochem . Bio s . 171, 521-526 (1975) . I . MACINTYRE, K .W . COISTON, I .M.A . EVANS, E . IAPEZ, S .J . MACAULEY, J . PIEGNOUX-DEVILLE, E . SPANOS, and M . SZEIKE, Clin . Endo . 5 (Suppl .), 85-5 - 95-5 (1976) . R . G . LARKINS, S .J . MACAULEY, and I . MACINTYRE, Nature 252, 412-414 (1974) . P . F . BRUMBAUGH, and M .R . HAUSSLER, J . Biol . Chem . 249 1258-1262 (1974) . T . SUDA, H .F . DELUCA, and Y . TANAKA, qtr-T0,~49-1051 (1970) . J .L . OMDAHL, M . F . HOLICK, T . SUDA, Y . TANAKA, and H .F . DELUCA, Biochemistry 10, 2935-2940 (1971) . P .S . CHEN, JR ., T .Y . TORIBARA, and H . WARNER, Anal . Chem . 28, 1756-1758 (1956) . M .F . HOLICK, and H .F . DELUCA, J . ~Li i~d Res . 12, 460-465 (1971) . P .F . NEVILLE, and H .F . DELUCA, Biochemist 5, 2201-2207 (1966) . I .M.A . EVANS, K .W . COISTON, L . GA~ Î. MACINI'YRE, Clin . Sci . Mol . Med . 48, 227-230 . (1975) . . T C . CHEN, and H .F . DELUCA, Arch . Biochem . Biophys . 156, 321-327 (1973) .