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作者：H. J.Armbrecht, M. A.Boltz, T. L.Hodam,作者单位：1 Geriatric Research, Education, and ClinicalCenter, St. Louis Veterans Administration Medical Center, St. Louis63125; and Division of Geriatric Medicine and Department of Biochemistry and Molecular Biology,St. Louis University Health Sciences Center, St. Louis, Missouri 63104 , e. c6 l. ?1 P
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【摘要】5 e# ]. P/ R. ?- n$ k
The capacity ofparathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitaminD 3 [1,25(OH) 2 D 3 ] productiondeclines with age in the rat. The purpose of these studies was todetermine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1, the cytochrome P -450component of the 25(OH)D 3 -1 -hydroxylase. Young (2 mo)and adult (12 mo) male Fischer 344 rats were parathyroidectomized(PTX). After 72 h, PTX rats were injected with PTH or vehicle at24, 6, and 3 h before death, and renal CYP1 mRNA levels weremeasured by ribonuclease protection assay. In young rats, PTH markedlyincreased plasma 1,25(OH) 2 D 3 and renal1,25(OH) 2 D 3 production. However, in adult rats,the response to PTH was less than 30% of that seen in young rats.Renal CYP1 mRNA levels, on the other hand, were increased overfivefold by PTH in both young and adult rats. In in vitro studies,PTH/forskolin increased CYP1 mRNA levels over twofold in renalslices from both young and adult PTX rats. These studies demonstratethat the decreased capacity of PTH to increase1,25(OH) 2 D 3 production in adult rats is not dueto decreased induction of CYP1 mRNA.
- Y( K* E, V5 t7 m5 F; j 【关键词】 parathyroid hormone cytochrome P calcitriol; T. F1 `% h# s& a/ P5 g% o
INTRODUCTION9 ?5 U( U7 y4 l( t& T+ m5 V, g
! _+ [0 G5 O% ]: mPARATHYROID HORMONE (PTH) is one of the major regulators of the conversion of25-hydroxyvitamin D 3 [25(OH)D 3 ] to1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], the major biologically activeform of vitamin D, in the kidney. In young animals, a variety ofexperiments have shown that PTH markedly stimulates1,25(OH) 2 D 3 production ( 5, 10 ).PTH may accomplish this, in part, by increasing the expression of thecytochrome P -450 component of the25(OH)D 3 -1 -hydroxylase enzyme complex (CYP1 ). PTH hasbeen shown to significantly increase the mRNA levels of CYP1 inintact animals ( 13 ) and renal cell lines ( 8 ).6 J; S5 f4 y: Z9 q
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With maturation and aging, the capacity of PTH to stimulate renal1,25(OH) 2 D 3 production declines. In the rat,the capacity of the kidney to make 1,25(OH) 2 D 3 and increase plasma 1,25(OH) 2 D 3 in response toPTH declines with age ( 6, 9 ). This has also been seen inhuman clinical studies where the capacity of PTH to increaseplasma 1,25(OH) 2 D 3 decreases with age( 11, 16 ).
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The decreased capacity of PTH to increase renal1,25(OH) 2 D 3 production in adults is also seenindirectly with regard to dietary calcium adaptation. Adult rats do notadapt to feeding on a low-calcium diet by increasing renal1,25(OH) 2 D 3 production and plasma1,25(OH) 2 D 3 levels as do young rats( 4 ). This lack of adaptation occurs despite the fact thatplasma PTH levels are markedly elevated by the low-calcium diet in bothage groups.( I: ?+ g8 V7 I6 E3 z6 L
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The purpose of these studies was to determine whether the age-relateddecline in PTH-stimulated 1,25(OH) 2 D 3 production is due to decreased induction of renal CYP1 mRNA.Decreased induction of CYP1 mRNA by PTH could also account for thedecreased CYP1 mRNA levels seen in adult rats on a low-calcium diet( 4 ). To that end, we also compared the response of CYP1 mRNA levels to a low-calcium diet with the response to PTH in both age groups.
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MATERIALS AND METHODS
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Experiments were performed using male Fischer 344 rats that were2-3 mo (young) and 10-12 mo (adult) of age. Rats wereobtained from Harlan Industries (Indianapolis, IN) and were fed asemisynthetic diet containing 1.2% calcium, 0.8% phosphorus, and 3.3 IU/g of vitamin D 3 (Purina Rodent Chow, Ralston-Purina, St.Louis, MO). Rats were parathyroidectomized (PTX) under pentobarbitalsodium anesthesia. Parathyroid glands were identified and removedand/or cauterized under a dissecting microscope. On the third day after surgery, PTH was administered to half the animals and the other halfreceived vehicle only. rPTH(1-34) (3 µg/100 g bodywt) or vehicle (1 mM acetic acid, pH 4.0, 1.6% glycerol, 0.25%phenol) was injected subcutaneously at 24, 6, and 3 h beforedeath. At death, kidneys were removed for isolation of RNA or forpreparation of renal slices. Blood was collected for the measurement ofplasma 1,25(OH) 2 D 3 and PTH. A m& @9 A Z: c# Z. s$ W
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Renal slices for in vitro incubation were prepared as previouslydescribed ( 7 ). Briefly, thin cortical slices were prepared using a Stadie-Riggs microtome and incubated in plastic vials containing Krebs-Ringer bicarbonate buffer (pH 7.4) at 37°C. The stoppered vials were gassed at 1-h intervals with 95%O 2 -5% CO 2. Slices were incubated with PTH andforskolin or vehicle for the indicated length of time, and total RNAwas then isolated from the slices for determination of CYP1 mRNA levels.
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& u2 y' z, Y% W i3 m r& `6 A* \In some experiments, renal slices were used to measure renal1,25(OH) 2 D 3 production, as described previously( 6 ). Briefly, slices were incubated with 5 µM 3 H-labeled 25(OH)D 3. After 1 h, the1,25(OH) 2 D 3 product produced was quantitated byradioligand assay following partial purification by Sep-Paks.1,25(OH) 2 D 3 production was expressed aspicomoles per minute per gram of slice weight.4 c8 _) R# E/ d
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CYP1 mRNA levels were measured by ribonuclease protection assay(RPA) as previously described ( 4 ). Total RNA was isolated by RNAgents (Promega, Madison, WI). The RPA was performed using theRPAII kit from Ambion (Austin, TX). The actin probe was the -actinantisense control template from Ambion. Bands were quantitated byscanning densitometry, and CYP1 mRNA levels were normalized toeither actin mRNA or total RNA.
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Plasma 1,25(OH) 2 D 3 and PTH were measured usingcommercial kits (Nichols Institute Diagnostics, San JuanCapistrano, CA).3 ~* z1 Q$ p! H( R3 l4 Y+ C
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Data are reported as means ± SE for each treatment group.Statistical significance was determined by Student's t -test, and a confidence level greater than 95% wasconsidered significant.( g0 [( d1 E( t
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In initial studies, basal parameters related to1,25(OH) 2 D 3 production were measured in non-PTXyoung and adult rats (Fig. 1 ). Plasma1,25(OH) 2 D 3 levels and renal1,25(OH) 2 D 3 production were decreased by morethan 50% in adult rats compared with young rats. However, there was nodifference in renal CYP1 mRNA levels between young and adult rats.In addition, there was no difference in plasma PTH levels. Theseresults suggested that the adult rats did not respond to plasma PTH tothe same degree as the young animals in terms of1,25(OH) 2 D 3 production.8 p" a s9 T/ Z u
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Fig. 1. Basal parameters in young and adultnonparathyroidectomized (PTX) rats. Plasma and renal parameters relatedto 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] production (1,25D Prod.) weremeasured in non-PTX rats aged 2-3 mo (young) and 10-12 mo(adult). Plasma 1,25(OH) 2 D 3 and parathyroidhormone (PTH) were measured using commercial kits. Renal1,25(OH) 2 D 3 production was measured usingisolated renal slices. Renal 25(OH)D 3 -1 -hydroxylaseenzyme complex P -450 (CYP1 ) mRNA levels were quantitatedby ribonuclease protection assay (RPA) relative to actin. Values areexpressed relative to young rats (100%). Bars are means ± SE of4-6 animals except for CYP mRNA (9 animals). * Statisticallydifferent from young ( P t -test).
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. u: H' L( M7 a* X/ g7 FThe effect of PTH on 1,25(OH) 2 D 3 production wasstudied directly using young and adult PTX rats (Fig. 2 A ). Inyoung animals, PTH markedly increased plasma1,25(OH) 2 D 3 levels over threefold. In adultrats, PTH increased plasma 1,25(OH) 2 D 3 levelsonly slightly, and the levels attained were much less than those seenin the young. A parallel pattern was seen with regard to renal1,25(OH) 2 D 3 production. PTH markedly increasedrenal 1,25(OH) 2 D 3 production in young rats, butit only marginally increased it in adult rats.
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" t" f% C b6 S" X3 sFig. 2. Effect of PTH in young and adult PTX rats. Young(unshaded bars) and adult rats (shaded bars) were PTX and then givenPTH or vehicle only (control) subcutaneously. A : plasma1,25(OH) 2 D 3 was measured by radioligand bindingassay, and renal 1,25(OH) 2 D 3 production wasmeasured using renal slices. B : renal CYP1 mRNA levelswere measured by RPA ( C ) and are expressed as percentage ofmaximum. Bars are means ± SE of 3-6 rats, and statisticalsignificance is P t -test). a Significantly different from control of same age group. b Significantly different from young of same treatmentgroup.
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: I: o' s1 p" r9 s- C! o# qTo determine whether the differences in the action of PTH were due todifferences in CYP1 expression, CYP1 mRNA levels were measured(Fig. 2 B ). PTH markedly increased CYP1 mRNA levels in both young and adult rats. There was no difference in the magnitude ofthe stimulation. In three experiments, the average stimulation by PTHin the adult kidney was 94.8 ± 6.4% that in the young kidney (100%). This was not statistically different than 100%( P 0.46, t -test).9 [3 G" F# i- S' l& @& X# Z) h) a
; [6 N9 Y' Y n; }We showed previously that feeding a low-calcium diet also markedlyincreases CYP1 mRNA levels in young animals ( 4 ). This presumably happens in response to the high levels of plasma PTH thatoccur in response to the calcium deprivation. Therefore, it was ofinterest to compare the magnitude of the CYP1 mRNA levels inresponse to PTH (Fig. 2 B ) with the response to a low-calcium diet ( 4 ). RPA was used to compare CYP1 mRNA levels inRNA pools from both sets of experiments (Fig. 3 ). In young rats, the CYP1 mRNAlevels induced by the low-calcium diet were much higher than thoseinduced by PTH. This was also seen in the adult animals, although to alesser degree.
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Fig. 3. Comparison of the effect of low dietary calcium and PTHin young and adult rats. Pools were made of renal RNA from thePTH-treated animals (Fig. 2 B ) and from the low-calcium-dietanimals ( 4 ). The CYP1 mRNA levels in the pools werequantitated by RPA and are expressed as percent of maximum. Bars aremeans ± SE of 2 RPAs, and shaded bars indicate adult rats. a Significantly different from young of same treatment group( P t -test).
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9 y4 }: t! J; h6 P, a: D$ RFinally, the effect of PTH and forskolin on CYP1 mRNA levels wasstudied in vitro. Renal slices from young and adult PTX animals wereincubated in the presence and absence of PTH/forskolin for 8 h.Forskolin was used along with PTH since this combination has been shownto give the greatest sustained increase in renal slice1,25(OH) 2 D 3 production ( 3 ).PTH/forskolin significantly increased CYP1 mRNA levels to the samelevels in slices from both young and adult rats (Fig. 4 ). This is of interest in light ofprevious studies of 1,25(OH) 2 D 3 production inrenal slices. In similar experiments, PTH/forskolin has been shown toincrease renal 1,25(OH) 2 D 3 production in slicesfrom young rats but not in slices from adult rats ( 7 ).
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Fig. 4. Effect of PTH on CYP1 mRNA levels in renal slices.Slices from PTX young (unshaded bars) and adult rats (shaded bars) wereincubated with PTH (100 nM) and forskolin (Fsk; 10 µM) or vehicleonly (control) for 8 h. CYP1 mRNA levels were quantitated byRPA and are expressed as percent of maximum response. A :bars are means ± SE of 8 slice incubations. a Significantly different from control of same age group( P t -test). There were nosignificant differences between age groups. B :representative RPA of 8 individual slices. {" @0 u" x% W8 C, E, @
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& E' _) v: z/ x8 IThese studies provide evidence that the decreased renal1,25(OH) 2 D 3 production in the adult rat inresponse to PTH is not due to decreased levels of CYP1 mRNA. In theintact animal, the CYP1 mRNA levels in the adult kidney are the sameas those in the young (Fig. 1 ). However, plasma1,25(OH) 2 D 3 and renal1,25(OH) 2 D 3 production are decreased in theadult animals (Fig. 1 ). In the direct studies of PTH action in PTXanimals, PTH increases CYP1 mRNA to the same levels in both youngand adult animals (Fig. 2 B ). However, there is a decreasedresponse to PTH in the adult animal in terms of plasma1,25(OH) 2 D 3 and renal1,25(OH) 2 D 3 production (Fig. 2 A ).Finally, in isolated renal slices, PTH/forskolin significantly increases CYP1 mRNA to the same level in slices from both young andadult animals (Fig. 4 ). However, in similar studies reported previously, PTH/forskolin significantly increased1,25(OH) 2 D 3 production only in renal slicesfrom young animals ( 7 ).
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* i* ~) ]" O* f& f' mThere are a number of possible explanations for decreased renal1,25(OH) 2 D 3 production despite normal CYP1 mRNA levels in adult rats. First, it may be that there is decreasedtranslation of CYP1 mRNA into CYP1 protein in adult animals. Wepreviously saw this in studying the effect of1,25(OH) 2 D 3 on the expression of calbindin inthe intestine of young and adult rats ( 1 ). We found that1,25(OH) 2 D 3 increases calbindin mRNA to similar levels in both young and adult rats. However, levels of calbindin protein are significantly lower in the adult rat intestine in responseto 1,25(OH) 2 D 3.2 n9 M7 i; U$ F8 D; M9 M% I# o( p
- j$ E0 J* q8 m8 E4 Q6 LSecond, it may be that there is oxidative damage to the CYP1 proteinin the adult animal such that its activity is diminished. The CYP1 protein is located on the inner mitochondrial membrane and is,therefore, particularly vulnerable to oxidative damage. Mitochondriaare a major source of free radicals, and mitochondrial free radicalproduction increases with age ( 15 ). Mitochondrial aconitase has been shown to accumulate oxidative damage with age ( 17 )." C" ~. w$ A+ y. X3 z9 d* ^
$ F# G9 Q; q% a1 kA third possibility is that decreased renal production of1,25(OH) 2 D 3 may be due to decreasedavailability of the 25(OH)D 3 substrate. Decreased substratehas been shown to contribute to the age-related decline seen in othersteroidogenic systems. These include decreased cholesterol availabilityfor the production of adrenal steroids by adrenal cells( 14 ) and for the production of testosterone by Leydigcells ( 12 ). In our studies in the kidney, decreased renal1,25(OH) 2 D 3 production is seen even when renalslices are incubated in high concentrations of 25(OH)D 3 (Fig. 4 ). Thus any decreased availability of 25(OH)D 3 inolder rats would be due to decreased transport of 25(OH)D 3 into the kidney itself.
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6 j0 D) F+ i5 y0 D# E9 VWith regard to mechanisms, PTH and forskolin increase renal CYP1 mRNA levels via a cAMP-dependent mechanism. It has previously beenshown that the capacity of PTH to increase renal cAMP levels andstimulate protein kinase A activity does not change with age ( 2, 9 ). Because the cAMP/protein kinase A signal transduction pathways are intact, it is not surprising that the effect ofPTH/forskolin on CYP1 mRNA levels does not change with age.
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In previous studies, we showed that the capacity of a low-calcium dietto increase renal CYP1 mRNA levels is markedly decreased in adultrats ( 4 ). This occurs despite similar increases in plasmaPTH levels in response to the diet. One possible explanation for thisis that the adult kidney is refractory to the action of PTH in terms ofincreasing renal CYP1 mRNA levels. However, the present studies showthat the adult kidney is not refractory to PTH in this regard (Fig. 2 B ).% t; T5 l7 p: @. U0 z
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When one compares the response of young animals to PTH and to alow-calcium diet, the response to the diet is much greater (Fig. 3 ).This is true despite the fact that injection of PTH increases plasmaPTH to levels higher than those seen with a low-calcium diet(unpublished observations). Thus it would seem that it is the chronicelevation of PTH over several weeks that markedly increases CYP1 mRNA levels in the young. In adult animals, although the acute effectsof PTH are similar to those seen in the young, the chronic effects ofPTH are different. It may be that there are age differences in thechronic regulation of CYP1 mRNA levels by PTH and other regulatory factors.
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' X* b3 x+ j% |9 w4 E2 _7 G, c* kIn summary, PTH increases renal CYP1 mRNA levels to the same levelsin both young and adult animals. This is true whether PTH is injectedinto the intact animal or PTH is incubated with isolated renal slices.This suggests that the decreased renal 1,25(OH) 2 D 3 production seen in the adult rat isdue to age-related changes distal to the elevation of CYP1 mRNAlevels. The decreased adaptation of adult rats to a low-calcium diet interms of CYP1 is not due to decreased responsiveness to short-termadministration of PTH. It may reflect age differences in the regulationof CYP1 expression by other factors.
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' E, J4 |' ^ W' Z$ xThis work was supported by the St. Louis Geriatric Research,Education, and Clinical Center and the Medical Research Service of theDepartment of Veterans Affairs.# `1 X( ]/ F0 m- H; i
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发表于 2009-4-21 13:37
