DIFFERENTIAL QUANTITATION OF ENDOGENOUSLY OCCUPIED AND UNOCCUPIED SITES*

The method described herein provides differential measurement of either in vivo occupied or unoccupied receptors for 1,26-dihydroxyvitamin D3 (1,26(OH)rD3) in the chick intestinal mucosa. The unfilled receptors are quantitated in the chromatin fraction by incubation with [3H]1,26(OH)2Ds at 4°C for 4 h without interference from filled sites. Fully occupied receptors can be meas- ured at 37°C for 30 min with greater than 90% recovery. However, a mixture of occupied and unoccupied recep- tors cannot be directly quantitated at 37°C because of the high degradation rate of the unfilled receptors at elevated temperatures (up to 60% degradation) which results in a falsely low total receptor estimate. Impor-tantly, preincubation at 4°C with chloromethyl ketone (TPCK, 100 the unoccupied 1,26(OH)aD3 receptor sites allowing quantitation of occupied receptors by “exchange” incubation with [3‘HJ1,26-(OH)aD3 at 30 min.

The method described herein provides differential measurement of either in vivo occupied or unoccupied receptors for 1,26-dihydroxyvitamin D3 (1,26(OH)rD3) in the chick intestinal mucosa. The unfilled receptors are quantitated in the chromatin fraction by incubation with [3H]1,26(OH)2Ds at 4°C for 4 h without interference from filled sites. Fully occupied receptors can be measured at 37°C for 30 min with greater than 90% recovery.
However, a mixture of occupied and unoccupied receptors cannot be directly quantitated at 37°C because of the high degradation rate of the unfilled receptors at elevated temperatures (up to 60% degradation) which results in a falsely low total receptor estimate. Importantly, preincubation at 4°C with ~-1-tosylamido-2phenylethyl chloromethyl ketone (TPCK, 100

PM)
blocks the unoccupied 1,26(OH)aD3 receptor sites allowing subsequent quantitation of exclusively occupied receptors by "exchange" incubation with [3'HJ1,26-(OH)aD3 at 37°C for 30 min. With this exchange of [3H]1,26(OH)2D~ in the presence of ~-1-tosylamido-2phenylethyl chloromethyl ketone, there was no significant difference between total receptor levels in intestinal mucosa of chicks with fully occupied receptors (13 nmol of l,26(OH)zD3 injected for 2 h) and the receptor levels in chicks with only unoccupied receptors (8.01 +. 0.40 uemm 7.82 f 0.66 pmol/g of tissue, respectively). Furthermore, the exchange assay gave an estimate of the level of occupied receptor identical with that obtained by direct extraction and quantitation of [3'HJ1,26(OH)aD3 present in the chromatin after in vivo [3'HJ1,26(OH)D3 injection. Therefore, this exchange assay allows quantitative measurement of occupied l,26(OH)zD3 receptors and/or unoccupied receptors in the chick intestinal mucosa. Additionally, bl-tosylamido-2-phenylethyl chloromethyl ketone may be useful to block unfilled sites in other steroid hormone systems. This 1,26(OH)aD3 receptor exchange assay w i l l be important for physiological experiments in the vitamin D This work was supported by United States Public Health Service Grant AM-09012 and Training Grant AM-07310. This is Paper XXVIII in a series "Studies on the Mode of Action of Calciferol." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. * Supported by a grant from the Swiss National Science Foundation.
8 Present address, Department of Physiology, W a n e University School of Medicine, New Orleans, LA 70112. endocrine system and may be adaptable to tissues from other species of laboratory animals or from humans. 1,25-Dihydroxyvitamin D3 (1,25(OH)zD3), an active metabolite of the secosteroid vitamin D, is now generally considered to be a steroid hormone by classical criteria (1-3). Our recent data indicate that unoccupied 1,25(OH)zD3' receptors are present in crude chromatin fractions under low salt conditions in vitro (4). Consequently, quantitation of unoccupied receptor levels in the chick intestinal mucosa is best achieved by assay of the crude chromatin fraction (5). Due to the slow dissociation rate of 1,25(OH)zD3 from its receptor ( 4 , only endogenously unoccupied 1,25(OH)&3 receptors are detected by this method (incubation with [3H]1,25(OH)zD3 at 4°C for 4 h).
The assay of endogenously occupied sites in addition to the unfilled receptors has proven very useful in other steroid hormone systems (6-9). These assays require exchange of the endogenous ligand against 3H-ligand in crude nuclear preparations in vitro. Since unoccupied 1,25(OH)ZD3 receptors of the chick intestinal mucosa are also associated with the nuclear/chromatin fraction (4), differentiation between and quantitative measurement of the unoccupied and occupied receptors is more complicated. In this paper, we report a method for differential detection and quantitative measurement of either in vivo occupied or unoccupied 1,25(OH)2D3 receptors.

Animals and Tissue Preparation-White
Leghorn cockerels obtained on the day of hatch from Pace-Setter, Alta Loma, CA were raised for 3 to 4 weeks on a standard rachitogenic diet (10). After decapitation, the small intestine was removed, stripped of contents, and washed at 4°C in 0.9% NaC1. All subsequent steps were performed at 4°C unless otherwise stated. The mucosa was scraped with a glass slide and the scraping was thoroughly homogenized in TED (10% w/ v; Ref. 5) with 10 to 12 strokes in a glass/Teflon homogenizer (motordriven pestle). After a low speed spin of the homogenate (5OOO X g, 10 min), the chromatin pellet was washed three times with vigorous blending on a Vortex mixer in the same volume of TED + 0.5% Triton X-100. Incomplete removal of cytoplasmic elements interfered with the action of TPCK in later steps of the procedure (see below).
Quantitation of Unfilled and Filled Receptors-Since both the filled and unfilled receptors were in the crude chromatin fraction under the above conditions (4), only this subcellular fraction was needed for receptor quantitation. The chromatin pellet (Triton X-100-washed) was resuspended (10% tissue w/vol) in TED buffer containing 10 m~ Naamolybdate and 500 KIU/ml of Trasylol. Molybdate stabilized the receptors at 4' and 37"C, whereas Trasylol improved receptor stability only at 37OC (data not shown). Aliquota containing the steroid as above, followed by incubation at 37°C for 30 min to determine the level of filled receptor. At the end of both incubations, 500 pl of hydroxylapatite suspension (50% v/v in TED) at 4°C with blending on a Vortex mixer. Then, 1 ml of TED-Triton was added to the tubes, and the incubation was continued for 15 min was added, and the tubes were blended on a Vortex mixer and centrifuged at 5OOO X g for 5 min, the resulting pellet was washed three times with 1 ml of TED-Triton. The radioactivity in the final pellet was extracted with 1 ml of 100% ethanol at 30°C for 30 min, the supernatant was dried under a stream of air and counted in 8 to 10 ml of toluene containing 5.25 g/liter of tertiary butylphenyl-544-biphenyl)l,3,4-oxadiazole (Amersham/Searle).  to unoccupied receptors with no effect on occupied receptors at selected concentrations (Fig. 2). The interaction of TPCK with unoccupied receptors was quite rapid at 4OC, reaching completion within 10 min (Fig. 2 4 ) . Importantly, incubation at 4OC with TPCK did not affect the ligand bound to endogenously occupied receptors. At elevated temperatures, where steroid dissociates from the occupied receptors, only 500-fold higher TPCK concentrations inhibited the [3H]1,25(OH)2D3 exchange process (Fig. 2 B ) . These data indicate that selection of the optimal concentration of TPCK is critical for the  accurate measurement of filled sites and must be carefully established in each system. Under our conditions, preincubation with 100 PM TPCK for 20 to 30 min at 4°C effectively blocks all the unoccupied 1,25(OH)2D3 receptors with only a negligible effect (only 8% underestimation in Fig. 2B) on subsequent exchange of [3H]1,25(OH)2D3 for the endogenously occupied sites. Quantitation of Unfilled and Filled Receptors for 1,25-(OH)2 D3-Application of exchange conditions to physiological/endocrinological studies requires differential quantitation of unfilled and filled 1,25(OH)zD3 receptor sites in samples containing mixtures of these sites in different ratios. To validate the TPCK assay for physiological experiments, chromatin from the intestinal mucosa of vitamin D-deficient chicks (unfilled receptors) and 1,25(OH)2D3-replete chicks (occupied receptors) were mixed in known ratios and assayed as described under "Materials and Methods." This mixing experiment indicated that unfilled and filled receptor sites could be accurately measured at 4" and at 37°C in the presence of 100 WM TPCK, respectively, whereas in the absence of TPCK the total receptor level is underestimated up to 2-fold (Fig. 3).

1,25-
(OH)2D3-replete Chicks-If both unfilled and filled receptors are measured quantitatively, the same number of total receptors should be present in vitamin D-deficient chicks and in 1,25(OH)zD3-replete chicks. Thus, unfilled and filled 1,25-(OH)zD3 receptor levels were determined in the intestinal mucosa of 1,25(0H)~D~-injected and noninjected vitamin Ddeficient chicks. Importantly, there was no difference in the total receptor levels measured in the two groups of chicks (Table 11). This result validates the receptor levels measured under nonexchange as well as under exchange conditions.

Occupied Receptor Levels Estimated by Exchange or Li-
gand Extraction-Filled receptor levels estimated by the exchange assay were compared with the level of extractable ligand after in vivo injection. For this experiment, vitamin Ddeficient chicks were injected subcutaneously with 100 units of [3H]1,25(OH)2D3 (0.1 Ci/mmol) 2 h prior to killing. Chromatin was prepared as usual and was then subjected to the exchange assay or was extracted by the method of Bligh and Dyer (16). The lipid extract was chromatographed via standard high pressure liquid chromatography methods (17) and the area under the 1,25(OH)2& peak was counted for ['HI-1,25(OH)2D3. There was no difference between the number of receptor sites detected by the exchange assay (7.73 f 0.08 pmol/g of tissue) and the amount of [3H]1,25(OH)zD3 accumulated in the chromatin fraction after injection in uiuo (7.42 +-0.31 pmol/g).
Occupied Receptor Levels in Normal Chicks-Using this assay, we evaluated the levels of occupied 1,25(OH)zD3 receptors in chicks provided a maintenance dose of vitamin D3. As shown in Table 111, the level of occupied receptor in the normal chicks remained quite low, although they were significantly elevated above the levels in the vitamin D-deficient chicks. These results indicate that, as in other steroid hormone systems (7, a), only a small fraction of the total 1,25(OH)zD3 receptor population is required for hormonal response. The difference in total receptor content in these two groups is currently under investigation.

DISCUSSION
The method described herein provides a means of quantitative detection of unoccupied and/or occupied 1,25(OH)zD3 receptors present in the chick intestinal mucosa. Unoccupied receptors can be readily measured at 4°C for 4 h with no noticeable influence from filled sites (Fig. 1). If all the receptors are occupied by ligand in vivo, they can be measured at 37°C for 30 min with about 90% recovery (Fig. 1B). Conversely, at 37°C only about 60 to 70% of the unoccupied receptors are detected due to the high rate of degradation of these sites at the elevated temperature. Occupying the unfilled 1,25(0?&D3 receptors in uitro before increasing the temperature did not increase the number of these sites detected. These results may indicate a differential stability of in vivo and in vitro filled receptor. Importantly, measurement of a mixture of filled and unfilled sites at 37°C in the absence of TPCK gave a receptor estimate that is lower than the total receptor level present in the tissue but higher than the filled receptor level (Figs. lA and 3).
In other steroid hormone systems, TPCK inhibits binding to the steroid binding site (18, 19), and protease substrates have been shown to compete for the binding of dexamethasone in HTC cells (20) and of estrone to a-fetoprotein? Interestingly, preincubation with 100 p~ TPCK at 4°C for 30 min completely blocked unfilled sites for 1,25(OH)zD3, without affecting endogenously occupied sites (Fig. 2). Surprisingly, during the subsequent temperature elevation, TPCK did not interfere with the exchange process (Fig. 2). Hence, differential quantitation of filled and d e d 1125(OH)2D3 receptors in chromatin of chick intestinal mucosa was achieved with this method (Fig. 3).
A problem associated with exchange assays is the possibility of receptor degradation at elevated temperatures which results in an underestimate of the filled receptor levels. Four independent lines of evidence show that the described exchange assay is quantitative. (a) Extrapolating the degradation rate at 37°C of in vivo filled receptor shows that 90% of the receptor is measured at 30 min (Fig. 1B). ( b ) Exchange of ligand at 4°C in presence of NaSCN as previously described for the estrogen receptor (21) gave a 30% lower estimate of the total 1,25(OH)2D3 receptor than detected by the exchange assay developed herein3 ( c ) The receptor level detected in a group of chicks dosed with 1,25(OH)&, where most of the receptor was occupied in vivo, is the same as the receptor value obtained in the vitamin D-deficient control goup (Table  11). the exchange assay. Importantly, the exchange assay is much faster and less elaborate so that large numbers of samples can be easily processed.
This assay has shown that there is only a minimal difference in occupied 1,25(OH)zD3 receptors in animals of very different physiological states (Table 111). This observation emphasizes the necessity for careful assessment of the exchange conditions in each system. Comparison of our results between different chick groups has resulted in the further conclusion that rigid controls w i l l be necessary for each series of physiological studies in order to avoid erroneous conclusions.
The described method for differential measurement of unoccupied, occupied, and total receptor sites for 1,25(OH)~D3 in the chick intestinal mucosa should be an important tool for physiological studies in the vitamin D endocrine system. The procedure can be optimized for other target tissues and different species of laboratory animals. The small amounts of tissue required for the assay will be an asset in investigating disorders in the vitamin D system in humans. Additonally, TPCK may prove useful to distinguish between unoccupied and occupied receptor sites in other steroid hormone receptor systems.