ARCHIVES
OF BIOCHEMISTRY
Vol. 192, No. 1, January,
Embryonic
AND BIOPHYSICS
pp. 302-310,
1979
Chick Intestine in Organ and Vitamin D-Mediated ROBERT
Department
of Physical Biology/Section Cornell
Culture: Hydrocortisone Processes
A. CORRADINO
of Physiology, New York State College of VeterAary University, Ithaca, New York l&353
Received March 27, 1978; revised September
Medirin,p,
8, 1978
The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased CAMP concentration, de nova synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at This apparently levels 227.5 nM increased vitamin D-induced CaBP concentration: represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (~27.5 PM) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (~2.75 FM), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and CAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated resnonses (APA and CAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.
Glucocorticoids were found to interfere with intestinal calcium absorption by Williams and co-workers (1) using ever-ted duodenal sacs from vitamin D-replete rats injected with vitamin D and/or cortisone acetate. Cortisone depression of normal calcium transport was not reversed by excess vitamin D. In these and subsequent confirmatory experiments, calcium transport was measured either in viva or in vitro after treatment of the anima! with the steroid(s). Thus, it was difficult to determine if the glucocorticoid effect was directly on the intestinal calcium absorptive mechanism. Consequently, two possible mechanisms of glucocorticoid action have been proposed: first, alteration of the metabolism or intestinal concentration of vitamin D or its biologically active metabolites (Z-4); 0003-9861/79/010302-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of rrproduction in any form reserved.
302
second, alteration of the “biochemical reactions responsible for calcium transport” within the intestine (5). Of the biochemical reactions possibly underlying vitamin D-mediated intestinal calcium transport, there is substantial evidence for the involvement of a vitamin D-induced calcium-binding protein (CaBP)’ (6, 7). There is some evidence from intact animal studies that intestinal CaBP concentration may be decreased,2 unchanged (8), 1 Abbreviations used: CaBP, calcium-binding protein; HC, hydrocortisone; CAMP, cyclic AMP; la,25(OH)%-D,, lo,25-dihydroxy cholecalciferol; 25-OH-D,, 25.hydroxy cholecalciferol; APA, alkaline phosphatase activity. 2 J. J. Feher and R. H. Wasserman, unpublished observations.
HYDROCORTISONE
AND VITAMIN
D ACTIVITY
or increased (9) during glucocorticoid inhibition of calcium transport. This disparity may be the result of multiple actions of hydrocortisone (HC) in ‘uivo, species differences, and/or the varying doses employed. To help clarify the situation, the effects of HC on vitamin D activity were investigated in the embryonic chick duodenum maintained in organ culture. The embryonic chick duodenum contains no CaBP (lo), remains functionally and morphologically intact for up to 48 h under the culture conditions employed (ll), and responds to vitamin D, itself (12), as well as its more potent metabolites (11, 12>, by increased cyclic AMP (CAMP) concentration (13, 14), de nova synthesis of CaBP (15, 16), and enhanced uptake and transmucosal transport of calcium (‘7, 11-13, 16). The results obtained with this system closely mimic those observed in vivo. In addition, the culture technique has the obvious advantage of allowing assessment of direct effects of a substance on the intestine in the absence of detectable vitamin D metabolism (12). MATERIALS
AND METHODS
Embryonoted eggs. Day-old White Leghorn embryonated eggs (Babcock Inc., Ithaca, N. Y.) were incubated at 37.5OC and 60% relative humidity in an incubator (Petersime Incubator Co., Gettysburg, Ohio) which rotated the eggs 130” every 2 h. Viability after 20 days incubation (the day before hatching) was typically about 95%. Culture medium. The serum-free culture medium employed is described in detail elsewhere (17). It consisted of Waymouth Medium 75211 (Gibco, Grand Island, N. Y.), purchased as a dry powder with the calcium and phosphate salts omitted. After reconstitution with deionized, glass-redistilled water, the medium was supplemented (18) with 2 mgiml fatty acid-poor, crystalline bovine serum albumin (Miles Labs., Inc., Elkhart, Ind.), and 5 pglml sodium oleate (Sigma, St. Louis, MO.). To maintain buffering capacity and restore original potassium level, 2.24 mgiml NaHCO, and 0.25 mgiml KC1 were added, respectively, and the pH was adjusted to 7.2 with NaOH. After sterile filtration through a 142-mmdiameter, 0.22~pm-pore-size filter (Millipore Corp., Bedford, Mass.), the medium was stored at 4°C for no more than 2 months. On the day of an experiment, the following were added using 1000.fold concentration solutions: 100 pg/ml neomycin sulfate, 50 pgiml each
IN EMBRYONIC
CHICK
INTESTINE
303
of sodium penicillin G and streptomycin sulfate (Sigma), and 100unitslmlMycostatin (Squibb, Princeton, N. J.). Similarly, calcium and phosphate levels were adjusted to 1.25 and 0.625 mM with 1000-fold concentration solutions of CaCl, H,O and NaH,PO,, respectively. Culture technique. The culture technique is described fully elsewhere (17). Briefly, entire duodena from four 20-day chick embryos (about 400 mg tissue) were slit open and laid mucosal-side-up (except where otherwise indicated) on a specially designed stainlesssteel grid inside a petri dish. The dish contained approximately 40 ml of the medium described to which appropriate experimental additions had been made. The duodena, with only their serosal surfaces in contact with the medium, were maintained for up to 48 h in a humidified incubator (relative humidity about 95%) continuously gassed with a CO,, O,, air mixture containing 5% CO, and 50% 0,. medium. Vitamin D:, Additions to the culture (Sigma), lu,25-dihydroxyvitamin D, [lcu,25-(OH),-D,], generously provided by Dr. Milan Uskokov? (Hoffmann-LaRoche Inc., Nutley, N. J. ), hydrocortisone sodium hemisuccinate (Sigma), and actinomycin D, generously provided by Dr. Walter B. Gall (Merck and Co., Rahway, New Jersey), were added as ethanolic solutions such that the final ethanol concentration of the culture medium never exceeded 0.2%. Other steroids (all Sigma) were also added as ethanolic solutions. In every experiment the final ethanol concentrations of control and treatment media were identical. Assay procedures. After the appropriate incubation period (usually 48 h), some of the duodena were homogenized prior to CaBP radial immunodiffusion assay (ll), total protein assay (19), and alkaline phosphatase assay (Technicon AutoAnalyzer II Method No. SE4-0006FC4). Other duodena were frozen in liquid N,, lyophilized, and extracted with 1 N HClO,; DNA assay was performed on the residues (14, 20). Cyclic AMP assays of the K,CO,neutralized extracts were then performed (21-23) using a commercial kit (Schwartz/Mann, Orangeburg, N. Y.). Radiocalcium uptake (and mucosal-to-serosal transport where indicated) was measured on other cultured duodena during a 3@min incubation in a 45Ca-containing, low Na+, low Ca2+ buffer (24), and calculations were performed as previously detailed (11). Uptake of radiolabeled nutrients other than calcium (“‘P-labeled phosphate, D-[‘4C]glu~ose, or L-[‘~Clalanine; New England Nuclear, Boston) from the same buffer was also determined. Counting was done in a liquid scintillation spectrometer (Beckman LS 355) using techniques described earlier (11). RESULTS
In the normal course of development in the chick, there is no detectable CaBP in
ROBERT A. CORRADINO
304
TABLE I EFFECT
OF HYDROCORTISONE
ON VITAMIN
D,
ACTIVITY: CONCENTRATIONDEPENDENCE” ““Ca uptake
HC in medium (PM)
D, in medium (26 PM)
0 0 0.275’ 0.275 2.75 2.75 27.5 27.5 275 275
+ + f + +
CaBP (cLd100 mg duodenum) 0 15.0 ? 0 23.4 + 0 22.9 2 0 21.6 2 0 22.0 i-
0.7 0.6” 0.8y 0.8y 0.6”
Cal. 1 (% dose per 100 mg duodenum)* 3.71 + 6.08 t 2.29 t 5.88 f 2.33 -t 6.15 k 2.26 k 4.73 + 2.35 + 4.56 t
0.19 0.13 0.11’ 0.10 0.08’ 0.12 0.09/ 0.09h 0.07f 0.11”
Cal. 2 (% of -HC, -D, control)’
Cal. 3 (% of -D, control)”
100 164 62 159 63 166 61 128 63 123
100 164 100 257 100 264 100 209 100 194
a Values are Z ? SE; five or six duodena per treatment. Slit-open duodena cultured 48 h. b Values are the percentage of total *Wa counts available in the buffer solution taken up per 100mg duodenal tissue. In this and all other tables, statistical evaluations were performed on the “raw” uptake data (expressed as a percentage of total isotope counts available in the buffer solution taken up per 100 mg duodenal tissue) by Analysis of Variance and Duncan’s New Multiple Range Test at the 1% significance level. Differences between treatment means were determined on this data. The values were then converted to “percentage of control” for clarity of presentation. c Derived by dividing each Wa uptake value by the mean value obtained in the absence of both D, and HC. d Data expressed as percentage of minus D, control at each HC level: a measure of the net 45Cauptake stimulated by vitamin D,. p Equivalent to 0.1 pg HCiml medium. ’ Significantly plus D, control at zero HC. h Significantly minus D,, minus HC control. r Significantly plus D,, minus HC control. c Significantly minus D,, plus HC control.
100 135” 1896,” 2gqvJdJ Slit-open
r+4C]glucose
L-[YJalanine
100 126b 120” 1636.6.1
100 100 130h.d 131b.d
duodena cultured
48 h. Statistical
HYDROCORTISONE
AND VITAMIN
D ACTIVITY
D, either alone or in combination with HC. However, there was a stimulatory effect of HC alone. HC and vitamin D3 activity: Effects on CAMP and alkaline phosphatase. HC also
affected two other indicators of vitamin D activity in the organ-cultured duodenum, namely, increased alkaline phosphatase activity (11) and cyclic AMP concentration (13, 14). At 2.75 PM, HC alone stimulated alkaline phosphatase activity and increased cyclic AMP concentration. The effects of vitamin D, and HC were essentially additive (Table IV). HC effect on vitamin D, activity: Specilcity. The effects of other steroid
hormones (estradiol, progesterone, or testosterone) on vitamin D, activity were assessed. At 27.5 j..&M in the medium only HC both increased vitamin D,-induced CaBP concentration and reduced vitamin D,-dependent 45Cauptake (data not shown). HC and vitamin D, activity: Effect of D. To determine if new
actinomycin
protein synthesis was required for the HC effects on calcium uptake and CaBP concentration, duodena were cultured in the presence of an inhibitor of DNA-directed RNA synthesis, actinomycin D. The data of TABLE
IN EMBRYONIC
CHICK
307
INTESTINE
Table V indicate that the action of HC (2.75 PM) on basal calcium uptake was unaffected by actinomycin D. Vitamin D,dependent calcium uptake, as well as CaBP synthesis, was greatly inhibited by actinomycin D as expected (13, 15, 16). The HC stimulation of CaBP concentration was abolished by the inhibitor. DISCUSSION
The literature records some evidence for glucocorticoid-altered distribution or metabolism of vitamin D (2-4). Alternatively, other reports have shown that in glucocorticoid-treated rats: first, there was no alteration of vitamin D, concentration in the intestinal mucosa or in the metabolism of vitamin D, to 25-OH-D, (9); second, there was no alteration of 25-OH-D, metabolism to Lx,~~-(OH)~-D, or in the concentration of this putative active form of vitamin D, in intestinal cells (5, ZS), or intestinal cell nuclei (5); and, third, there was no reversal of cortisone depression of intestinal calcium transport by administration of excess vitamin D, (1,2) or 25-OH-D, (5, 9) or by administration of the potent metabolite, lcu,25-(OH)z-D,. There is no IV
EFFECT OF HYDROCORTISONE ON VITAMIN D, ACTIVITY: ALKALINE PHOSPHATASE AND CYCLIC AMP”
HC in medium (PM)
D, in medium (26 Wf)
CaBP (cLg/lOO mg duodenum)
“5Ca uptake
0 0 2.75 2.75
+ -
0 12.1 * 0.8 0 20.7 e 1.1’
100 176 73 181
+
Alkaline phosphatase activity” (% of -HC, -D, control) 100 160” 181” 231”,’
Cyclic AMP’
100 157” 150” 222”,’
a Values are 2 + SE; 8 duodena per treatment for Wa uptake, 12 duodena per treatment for all other measurements. Slit-open duodena cultured 48 h. Statistical analyses were performed on the “raw” data as given in Table I. Differences between treatment means were determined on this data. The values were converted to “% of -HC, -D, control” for clarity of presentation. b Initially expressed as micromolesp-nitrophenylphosphate hydrolyzed per hour per milligram total protein in the whole homogenate sample. The value in the absence of both vitamin D, and HC was 93 2 6. r Initially expressed as picomoles cyclic AMP per milligram DNA in the whole duodenum. The value in the absence of both vitamin D, and hydrocortisone was 104 + 3. d Significantly >minus D,, minus HC control. e Significantly >plus D,, minus HC control. f Significantly >minus D,, plus HC control.
308
ROBERT
A. CORRADINO TABLE
V
EFFECT OF ACTINOMYCIND ON VITAMIN D-HYDROCORTISONEINTERACTION” HC in medium (2.75 /.LM) + + + +
D, in medium
Actinomycin D in medium
(26 FM)
(0.8 PM)
bYlO mg duodenum)
+
-
0 8.9 2 1.1
-
-
0 14.4 t- 1.3 0 3.8 + 0.6” 0 3.8 k 0.4”
+ + +
+ + + +
CaBP
%a
uptake
(8 of -HC, -D, control) 100 208 72 212 102 1346 74 138h
ClValues are 2 + SE; five or six duodena per treatment. Slit-open duodena cultured 48 h. Statistical analyses as given in Table I. b Significantly
OF BIOCHEMISTRY
Vol. 192, No. 1, January,
Embryonic
AND BIOPHYSICS
pp. 302-310,
1979
Chick Intestine in Organ and Vitamin D-Mediated ROBERT
Department
of Physical Biology/Section Cornell
Culture: Hydrocortisone Processes
A. CORRADINO
of Physiology, New York State College of VeterAary University, Ithaca, New York l&353
Received March 27, 1978; revised September
Medirin,p,
8, 1978
The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased CAMP concentration, de nova synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at This apparently levels 227.5 nM increased vitamin D-induced CaBP concentration: represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (~27.5 PM) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (~2.75 FM), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and CAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated resnonses (APA and CAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.
Glucocorticoids were found to interfere with intestinal calcium absorption by Williams and co-workers (1) using ever-ted duodenal sacs from vitamin D-replete rats injected with vitamin D and/or cortisone acetate. Cortisone depression of normal calcium transport was not reversed by excess vitamin D. In these and subsequent confirmatory experiments, calcium transport was measured either in viva or in vitro after treatment of the anima! with the steroid(s). Thus, it was difficult to determine if the glucocorticoid effect was directly on the intestinal calcium absorptive mechanism. Consequently, two possible mechanisms of glucocorticoid action have been proposed: first, alteration of the metabolism or intestinal concentration of vitamin D or its biologically active metabolites (Z-4); 0003-9861/79/010302-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of rrproduction in any form reserved.
302
second, alteration of the “biochemical reactions responsible for calcium transport” within the intestine (5). Of the biochemical reactions possibly underlying vitamin D-mediated intestinal calcium transport, there is substantial evidence for the involvement of a vitamin D-induced calcium-binding protein (CaBP)’ (6, 7). There is some evidence from intact animal studies that intestinal CaBP concentration may be decreased,2 unchanged (8), 1 Abbreviations used: CaBP, calcium-binding protein; HC, hydrocortisone; CAMP, cyclic AMP; la,25(OH)%-D,, lo,25-dihydroxy cholecalciferol; 25-OH-D,, 25.hydroxy cholecalciferol; APA, alkaline phosphatase activity. 2 J. J. Feher and R. H. Wasserman, unpublished observations.
HYDROCORTISONE
AND VITAMIN
D ACTIVITY
or increased (9) during glucocorticoid inhibition of calcium transport. This disparity may be the result of multiple actions of hydrocortisone (HC) in ‘uivo, species differences, and/or the varying doses employed. To help clarify the situation, the effects of HC on vitamin D activity were investigated in the embryonic chick duodenum maintained in organ culture. The embryonic chick duodenum contains no CaBP (lo), remains functionally and morphologically intact for up to 48 h under the culture conditions employed (ll), and responds to vitamin D, itself (12), as well as its more potent metabolites (11, 12>, by increased cyclic AMP (CAMP) concentration (13, 14), de nova synthesis of CaBP (15, 16), and enhanced uptake and transmucosal transport of calcium (‘7, 11-13, 16). The results obtained with this system closely mimic those observed in vivo. In addition, the culture technique has the obvious advantage of allowing assessment of direct effects of a substance on the intestine in the absence of detectable vitamin D metabolism (12). MATERIALS
AND METHODS
Embryonoted eggs. Day-old White Leghorn embryonated eggs (Babcock Inc., Ithaca, N. Y.) were incubated at 37.5OC and 60% relative humidity in an incubator (Petersime Incubator Co., Gettysburg, Ohio) which rotated the eggs 130” every 2 h. Viability after 20 days incubation (the day before hatching) was typically about 95%. Culture medium. The serum-free culture medium employed is described in detail elsewhere (17). It consisted of Waymouth Medium 75211 (Gibco, Grand Island, N. Y.), purchased as a dry powder with the calcium and phosphate salts omitted. After reconstitution with deionized, glass-redistilled water, the medium was supplemented (18) with 2 mgiml fatty acid-poor, crystalline bovine serum albumin (Miles Labs., Inc., Elkhart, Ind.), and 5 pglml sodium oleate (Sigma, St. Louis, MO.). To maintain buffering capacity and restore original potassium level, 2.24 mgiml NaHCO, and 0.25 mgiml KC1 were added, respectively, and the pH was adjusted to 7.2 with NaOH. After sterile filtration through a 142-mmdiameter, 0.22~pm-pore-size filter (Millipore Corp., Bedford, Mass.), the medium was stored at 4°C for no more than 2 months. On the day of an experiment, the following were added using 1000.fold concentration solutions: 100 pg/ml neomycin sulfate, 50 pgiml each
IN EMBRYONIC
CHICK
INTESTINE
303
of sodium penicillin G and streptomycin sulfate (Sigma), and 100unitslmlMycostatin (Squibb, Princeton, N. J.). Similarly, calcium and phosphate levels were adjusted to 1.25 and 0.625 mM with 1000-fold concentration solutions of CaCl, H,O and NaH,PO,, respectively. Culture technique. The culture technique is described fully elsewhere (17). Briefly, entire duodena from four 20-day chick embryos (about 400 mg tissue) were slit open and laid mucosal-side-up (except where otherwise indicated) on a specially designed stainlesssteel grid inside a petri dish. The dish contained approximately 40 ml of the medium described to which appropriate experimental additions had been made. The duodena, with only their serosal surfaces in contact with the medium, were maintained for up to 48 h in a humidified incubator (relative humidity about 95%) continuously gassed with a CO,, O,, air mixture containing 5% CO, and 50% 0,. medium. Vitamin D:, Additions to the culture (Sigma), lu,25-dihydroxyvitamin D, [lcu,25-(OH),-D,], generously provided by Dr. Milan Uskokov? (Hoffmann-LaRoche Inc., Nutley, N. J. ), hydrocortisone sodium hemisuccinate (Sigma), and actinomycin D, generously provided by Dr. Walter B. Gall (Merck and Co., Rahway, New Jersey), were added as ethanolic solutions such that the final ethanol concentration of the culture medium never exceeded 0.2%. Other steroids (all Sigma) were also added as ethanolic solutions. In every experiment the final ethanol concentrations of control and treatment media were identical. Assay procedures. After the appropriate incubation period (usually 48 h), some of the duodena were homogenized prior to CaBP radial immunodiffusion assay (ll), total protein assay (19), and alkaline phosphatase assay (Technicon AutoAnalyzer II Method No. SE4-0006FC4). Other duodena were frozen in liquid N,, lyophilized, and extracted with 1 N HClO,; DNA assay was performed on the residues (14, 20). Cyclic AMP assays of the K,CO,neutralized extracts were then performed (21-23) using a commercial kit (Schwartz/Mann, Orangeburg, N. Y.). Radiocalcium uptake (and mucosal-to-serosal transport where indicated) was measured on other cultured duodena during a 3@min incubation in a 45Ca-containing, low Na+, low Ca2+ buffer (24), and calculations were performed as previously detailed (11). Uptake of radiolabeled nutrients other than calcium (“‘P-labeled phosphate, D-[‘4C]glu~ose, or L-[‘~Clalanine; New England Nuclear, Boston) from the same buffer was also determined. Counting was done in a liquid scintillation spectrometer (Beckman LS 355) using techniques described earlier (11). RESULTS
In the normal course of development in the chick, there is no detectable CaBP in
ROBERT A. CORRADINO
304
TABLE I EFFECT
OF HYDROCORTISONE
ON VITAMIN
D,
ACTIVITY: CONCENTRATIONDEPENDENCE” ““Ca uptake
HC in medium (PM)
D, in medium (26 PM)
0 0 0.275’ 0.275 2.75 2.75 27.5 27.5 275 275
+ + f + +
CaBP (cLd100 mg duodenum) 0 15.0 ? 0 23.4 + 0 22.9 2 0 21.6 2 0 22.0 i-
0.7 0.6” 0.8y 0.8y 0.6”
Cal. 1 (% dose per 100 mg duodenum)* 3.71 + 6.08 t 2.29 t 5.88 f 2.33 -t 6.15 k 2.26 k 4.73 + 2.35 + 4.56 t
0.19 0.13 0.11’ 0.10 0.08’ 0.12 0.09/ 0.09h 0.07f 0.11”
Cal. 2 (% of -HC, -D, control)’
Cal. 3 (% of -D, control)”
100 164 62 159 63 166 61 128 63 123
100 164 100 257 100 264 100 209 100 194
a Values are Z ? SE; five or six duodena per treatment. Slit-open duodena cultured 48 h. b Values are the percentage of total *Wa counts available in the buffer solution taken up per 100mg duodenal tissue. In this and all other tables, statistical evaluations were performed on the “raw” uptake data (expressed as a percentage of total isotope counts available in the buffer solution taken up per 100 mg duodenal tissue) by Analysis of Variance and Duncan’s New Multiple Range Test at the 1% significance level. Differences between treatment means were determined on this data. The values were then converted to “percentage of control” for clarity of presentation. c Derived by dividing each Wa uptake value by the mean value obtained in the absence of both D, and HC. d Data expressed as percentage of minus D, control at each HC level: a measure of the net 45Cauptake stimulated by vitamin D,. p Equivalent to 0.1 pg HCiml medium. ’ Significantly plus D, control at zero HC. h Significantly minus D,, minus HC control. r Significantly plus D,, minus HC control. c Significantly minus D,, plus HC control.
100 135” 1896,” 2gqvJdJ Slit-open
r+4C]glucose
L-[YJalanine
100 126b 120” 1636.6.1
100 100 130h.d 131b.d
duodena cultured
48 h. Statistical
HYDROCORTISONE
AND VITAMIN
D ACTIVITY
D, either alone or in combination with HC. However, there was a stimulatory effect of HC alone. HC and vitamin D3 activity: Effects on CAMP and alkaline phosphatase. HC also
affected two other indicators of vitamin D activity in the organ-cultured duodenum, namely, increased alkaline phosphatase activity (11) and cyclic AMP concentration (13, 14). At 2.75 PM, HC alone stimulated alkaline phosphatase activity and increased cyclic AMP concentration. The effects of vitamin D, and HC were essentially additive (Table IV). HC effect on vitamin D, activity: Specilcity. The effects of other steroid
hormones (estradiol, progesterone, or testosterone) on vitamin D, activity were assessed. At 27.5 j..&M in the medium only HC both increased vitamin D,-induced CaBP concentration and reduced vitamin D,-dependent 45Cauptake (data not shown). HC and vitamin D, activity: Effect of D. To determine if new
actinomycin
protein synthesis was required for the HC effects on calcium uptake and CaBP concentration, duodena were cultured in the presence of an inhibitor of DNA-directed RNA synthesis, actinomycin D. The data of TABLE
IN EMBRYONIC
CHICK
307
INTESTINE
Table V indicate that the action of HC (2.75 PM) on basal calcium uptake was unaffected by actinomycin D. Vitamin D,dependent calcium uptake, as well as CaBP synthesis, was greatly inhibited by actinomycin D as expected (13, 15, 16). The HC stimulation of CaBP concentration was abolished by the inhibitor. DISCUSSION
The literature records some evidence for glucocorticoid-altered distribution or metabolism of vitamin D (2-4). Alternatively, other reports have shown that in glucocorticoid-treated rats: first, there was no alteration of vitamin D, concentration in the intestinal mucosa or in the metabolism of vitamin D, to 25-OH-D, (9); second, there was no alteration of 25-OH-D, metabolism to Lx,~~-(OH)~-D, or in the concentration of this putative active form of vitamin D, in intestinal cells (5, ZS), or intestinal cell nuclei (5); and, third, there was no reversal of cortisone depression of intestinal calcium transport by administration of excess vitamin D, (1,2) or 25-OH-D, (5, 9) or by administration of the potent metabolite, lcu,25-(OH)z-D,. There is no IV
EFFECT OF HYDROCORTISONE ON VITAMIN D, ACTIVITY: ALKALINE PHOSPHATASE AND CYCLIC AMP”
HC in medium (PM)
D, in medium (26 Wf)
CaBP (cLg/lOO mg duodenum)
“5Ca uptake
0 0 2.75 2.75
+ -
0 12.1 * 0.8 0 20.7 e 1.1’
100 176 73 181
+
Alkaline phosphatase activity” (% of -HC, -D, control) 100 160” 181” 231”,’
Cyclic AMP’
100 157” 150” 222”,’
a Values are 2 + SE; 8 duodena per treatment for Wa uptake, 12 duodena per treatment for all other measurements. Slit-open duodena cultured 48 h. Statistical analyses were performed on the “raw” data as given in Table I. Differences between treatment means were determined on this data. The values were converted to “% of -HC, -D, control” for clarity of presentation. b Initially expressed as micromolesp-nitrophenylphosphate hydrolyzed per hour per milligram total protein in the whole homogenate sample. The value in the absence of both vitamin D, and HC was 93 2 6. r Initially expressed as picomoles cyclic AMP per milligram DNA in the whole duodenum. The value in the absence of both vitamin D, and hydrocortisone was 104 + 3. d Significantly >minus D,, minus HC control. e Significantly >plus D,, minus HC control. f Significantly >minus D,, plus HC control.
308
ROBERT
A. CORRADINO TABLE
V
EFFECT OF ACTINOMYCIND ON VITAMIN D-HYDROCORTISONEINTERACTION” HC in medium (2.75 /.LM) + + + +
D, in medium
Actinomycin D in medium
(26 FM)
(0.8 PM)
bYlO mg duodenum)
+
-
0 8.9 2 1.1
-
-
0 14.4 t- 1.3 0 3.8 + 0.6” 0 3.8 k 0.4”
+ + +
+ + + +
CaBP
%a
uptake
(8 of -HC, -D, control) 100 208 72 212 102 1346 74 138h
ClValues are 2 + SE; five or six duodena per treatment. Slit-open duodena cultured 48 h. Statistical analyses as given in Table I. b Significantly
