A simplified procedure for cyclic nucleotide radioimmunoassay and its application to human blood leukocytes

Journal of Immunological Methods, 19 (1978) 301--308 © Elsevier/North-Holland Biomedical Press

301

A SIMPLIFIED PROCEDURE FOR CYCLIC NUCLEOTIDE RADIOI M M U N O A S S A Y AND ITS A P P L I C A T I O N TO H U M A N BLOOD LEUKOCYTES *

GARY E. HATCH, WILLIAM K. NICHOLS and HARRY R. HILL ** Division of Clinical Immunology, Department of Pediatrics, and Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, UT 84132, 5\S.A. (Received 10 June J977, accepted 30 July 1977)

Rapid treatment of leukocyte suspensions was found to be an effective alternative to acid treatment in the preparation of these cells for radioimmunoassay of cyclic GMP and cyclic AMP. A 2-sec heating to boiling temperature, followed by sonication and micropore filtration, was employed. This procedure adequately inactivated or removed enzymes and binding proteins that can alter cyclic nucleotide concentrations or otherwise interfere with the radioimmunoassay. Moreover, heating in this manner did not appear to affect the stability of cyclic nucleotides or cause significant formation of cyclic GMP from endogenous GTP. Recovery of cyclic nucleotides after heating and filtration was high (89--96%), making possible the measurement of both cyclic GMP and cyclic AMP in small cellular samples. Variation in cyclic nucleotide recovery was small (+2--4% SD), therefore individual recovery determinations were unnecessary.

INTRODUCTION Studies of cellular cyclic nucleotides have traditionally been difficult because of the laborious procedures required for preparing radioimmunoassay s a m p l e s . T h e s e p r o c e d u r e s r e q u i r e t h e d e n a t u r a t i o n o f m a c r o m o l e c u l e s i n v o l v e d in t h e s y n t h e s i s , d e g r a d a t i o n , a n d b i n d i n g o f t h e c y c l i c n u c l e o t i d e s . P r o t e i n p r e c i p i t a n t s , such as t r i c h l o r o a c e t i c a n d p e r c h l o r i c acids, are e m p l o y e d f o r this p u r p o s e . T h e s e p r e c i p i t a n t s m u s t l a t e r be r e m o v e d f r o m samples because they interfere with the radioimmunoassay. Ether extraction o f t r i c h l o r o a c e t i c acid ( B r o o k e r , 1 9 7 4 ) , p r e c i p i t a t i o n o f p e r c h l o r i c acid w i t h K O H ( S a n d l e r et al., 1 9 7 5 ) , or its e l i m i n a t i o n b y use o f a l u m i n a c o l u m n s ( H a d d o x et al., 1 9 7 6 ) , are p r o c e d u r e s c o m m o n l y e m p l o y e d t o r e m o v e t h e s e agents. T h e u n p r e d i c t a b l e losses o f c y c l i c n u c l e o t i d e t h a t o c c u r d u r i n g t h e s e extraction steps often necessitate a separate d e t e r m i n a t i o n of cyclic nucleo-

* Supported by U.S. Public Health Service Grants AI13150, AM18354, AM16330 and GM00153. ** H.R. Hill is an Investigator of the Howard Hughes Medical Institute.

302 tide recovery for each sample. Much of the sample must be used for recovery determinations and, in addition, the radiolabeled cyclic nucleotide which is added for assessing recovery may itself interfere with the radioimmunoassay. The present studies were designed to determine if a rapid heat-inactivation could be employed to simplify the preparation of immunoassay samples. Previous studies have shown that rat brain cyclic nucleotide concentrations obtained by 2-sec microwave heating were comparable to those obtained by rapid freezing followed by perchloric acid treatment (Guidotti et al., 1974). We report that a 2-sec heat treatment of purified human blood neutrophil suspensions simplifies the procedure for preparing these cells for radioimmunoassay and, in addition, increases overall recovery of cellular cyclic nucleotides. MATERIALS AND METHODS Materials

Iodinated (12sI) cyclic nucleotides, cyclic nucleotide standards, and antibodies to cyclic GMP and cyclic AMP were obtained from Collaborative Research, Waltham, MA; Ficoll--Hypaque (Ficol--Paque) from Pharmacia, Piscataway, NJ; micropore filters (No. HAWP02500) from Millipore Corp., Bedford, MA; Medium 199 with Hank's salts from Microbiological Associates Inc., Bethesda, MD; GTP, sodium azide, and Hepes from Sigma, St. Louis, MO; and beef heart phosphodiesterase from Boehringer Mannheim, San Francisco, CA. Methods

Leukocyte-rich plasma was obtained by sedimentation (1 h, 37°C) of heparinized venous blood (10 U/ml) from normal adult donors. Suspensions of polymorphonuclear leukocytes (PMNs) were separated from leukocyte rich plasma b y density gradient centrifugation (½ h, 400g) on Ficoll--Hypaque (Boyum, 1967). Cells were suspended at a final concentration of 5 × 106 per ml in medium 199 to which Hepes buffer had been added to a concentration of 15 mM. The final pH of this solution was 7.4. Fieoll--Hypaque centrifugation yielded PMN suspensions containing less than 2% mononuclear cells. Cellular incubations were carried out in 15 × 100 mm glass culture tubes containing 0.5 ml of cell suspension. Siliconization of the culture tubes was found to have no noticeable effect on cyclic nucleotide concentrations and was therefore omitted. Incubations were terminated by holding the tubes containing cells in a large bunsen burner flame until boiling began (2 sec). If necessary, samples heated in this manner were stored a t - - 2 0 ° C . Heated cellular suspensions were sonicated and then filtered (0.45 p pore size) to remove cellular debris. Filtrate cyclic nucleotides were acetylated {Harper and Brooker, 1975) by adding 30 pl of a freshly prepared solution of triethyl-

303 amine and acetic anhydride (2 : 1 vol/vol) per ml of sample. Cyclic nucleotide standards used in the assay were prepared in Medium 199--Hepes. Sodium azide (final conc. of 0.5%) added to the media was found to be effective in inhibiting microbial growth. This agent did not interfere with acetylation, antibody binding, or subsequent phosphodiesterase treatments of cyclic nucleotide samples and was therefore added to cyclic nucleotide standards stored at 4°C for up to 1 month. Immunoassays were performed by adding 100 pl of the cyclic nucleotide standard or of the filtered, heat-inactivated cell suspension to each assay tube. Antibody and iodinated cyclic nucleotides were prepared in 50 mM sodium acetate buffer, pH 4.75, containing 20 mM calcium chloride. Immunoassay tubes containing a total volume of 300 pl were incubated at 4°C overnight and separation of antibody bound cyclic nucleotide was performed by micropore filtration (Millipore application manual AM304). The radioactivity of each filter disc was determined from the time required to generate 4000 counts, in order to eliminate the necessity of adjusting the concentration of iodinated cyclic nucleotide during radioactive decay of these compounds. Time values obtained were analyzed using a Hewlett Packard Model 10 calculator (Loveland, CO, program No. 9810A). Phosphodiesterase treatment was performed by adding beef heart phosphodiesterase (PD, final concentration of 0.3 mg/ml) to samples containing cyclic nucleotides and incubating at room temperature for 2 h, after which the enzyme was inactivated by rapid heat treatment. RESULTS

E[fects o f heat treatment and micropore filtration Removal o f enzymes and binding molecules Heating of cell suspensions until boiling occurred required 2 sec and resulted in a volume loss of about 2% of the sample (table 1). Experiments were performed to determine whether this rapid heating procedure inactivated the cellular macromolecules that may interfere with the radioimmunoassay. Table 2 shows the effect of adding known amounts of cyclic nucleotide to medium 199 alone and to medium 199 containing heat-killed neutrophils. Concentrations of cyclic nucleotides determined after addition of standards were comparable with the expected values. The fact that heat-killed cells did not affect the radioimmunoassay of added cyclic nucleotides indicates that PD, cyclases, and binding proteins were inactivated or removed by heating and filtration. Cyclic nucleotides are undetectable in PD-treated samples, indicating that 'blank' materials which mimic the cyclic nucleotides in the immunoassay are not present in heated PMNs as previously reported in other tissue extracts (Steiner et al., 1972). bYJrmation of cyclic GMP from GTP Kimura and Murad (1974) demonstrated conversion of GTP to cyclic

304 TABLE 1 Loss o f s a m p l e v o l u m e due t o e v a p o r a t i o n during h e a t t r e a t m e n t . Culture t u b e s (15 x 100 ram) c o n t a i n i n g 0.5 ml o f cellular s u s p e n s i o n in M e d i u m 199 were w e i g h e d , h e a t - t r e a t e d , and a l l o w e d t o s t a n d at r o o m t e m p e r a t u r e until c o n d e n s e d w a t e r w h i c h c o l l e c t e d o n t h e inside walls o f the t u b e s during h e a t i n g had e v a p o r a t e d (1 h). T u b e s were t h e n w e i g h e d again. P e r c e n t w e i g h t loss is calculated to r e p r e s e n t the p e r c e n t of 0.5 ml o f s o l u t i o n t h a t was lost due t o t h e h e a t t r e a t m e n t . The p e r c e n t v o l u m e loss due t o t h e 1 h o u r r o o m t e m p e r a t u r e period w i t h o u t h e a t t r e a t m e n t was 0.9%. Sample

Time to boiling (sec)

P e r c e n t loss of volume

1 2 3 4 5 6 7 8

1.8 2.2 1.8 2.0 1.8 2.2 2.2 1.8

2.16 2.68 1.78 2.88 1.70 2.75 2.12 2.34

Mean ± SD

1.98 ± 0.19

2.30 ± 0.44

GMP during heat t r eat m e nt . We have sought to assess the c o n t r i b u t i o n of this reaction to PMN cyclic GMP concent r a t i ons as det erm i ned by the heat treatm e n t described here. Table 3 shows the effect of 2-sec heating on cyclic GMP f o r m a t i o n f r om various concentrations of added GTP. Less than TABLE 2 T h e m e a s u r e m e n t o f cyclic n u c l e o t i d e s t a n d a r d s a d d e d t o heat-killed cellular samples and the effect of phosphodiesterase treatment. Equal a m o u n t s o f cyclic GMP and cyclic AMP s t a n d a r d s were a d d e d to cell-free m e d i a and t o heat-killed PMNs. E x p e r i m e n t s 1 and 2 s h o w t h e cyclic GMP and cyclic AMP levels, as d e t e r m i n e d b y r a d i o i m m u n o a s s a y , o f m e d i u m plus 100 ±moles o f t h e s e agents. Exp e r i m e n t 3 d e p i c t s the levels o f t h e s e cyclic n u c l e o t i d e s in heat-killed PMNs; e x p e r i m e n t s 4 and 5 s h o w t h e levels in t h e same PMN p r e p a r a t i o n s t o w h i c h 100 ±moles o f cyclic AMP or cyclic GMP were a d d e d . E x p e r i m e n t 6 s h o w s t h e e f f e c t o f p h o s p h o d i e s t e r a s e treatm e n t o f cyclic n u c l e o t i d e c o n c e n t r a t i o n s . Values r e p r e s e n t m e a n ÷ SE o f 3--5 i n c u b a t i o n s with 3 assay t u b e s per i n c u b a t i o n . Test

Sample

1. 2. 3. 4.

100 ±moles cAMP 100 ±moles cGMP Heat-killed PMNs Heat-killed PMNs ±moles cAMP Heat-killed PMNs ±moles cGMP Heat-killed PMNs

5. 6.

+ buffer + buffer

±moles cGMP

±moles cAMP

<5 102 + 3 36 ± 2

106 +_ 5 <5 178 _+ 5

--

280 _+ 11

+ 100 + 100 + PD

138 ± 4 <5

-<5

305 TABLE 3 Heat treatment of guanosine 5'-triphosphate (GTP) and its effect on cyclic GMP concentrations. Guanosine 5'-triphosphate was added to Medium 199 and to heat-killed PMN suspensions. Part of each sample was then subjected to a 2-sec heat treatment. Differences in cyclic GMP concentrations between heated and unheated samples, as determined by radioimmunoassay, were used to calculate the percent of added GTP that was converted to cyclic GMP by the heat treatment. Radioligand displacement in the immunoassay due to 10 mM GTP was equivalent to the displacement seen with 1 nM cyclic GMP. Values represent the mean ± SE for 3 incubations with 2 assay tubes per incubation. Experiment

Percent conversion of GTP to cyclic GMP

1. Medium 199 + 0.01 mM GTP Heat-killed PMNs + 0.01 mM GTP

0.0019 0.0011

2. Medium 199 + 0.1 mM GTP Heat-killed PMNs + 0.1 mM GTP

0.0009 0.0022

3. Medium 199 + 1.0 mM GTP Heat-killed PMNs + 1.0 mM GTP

0.00023 0.00019

4. Medium 199 + 10.0 mM GTP Heat-killed PMNs + 10.0 mM GTP

0.00001 0.00001

0 . 0 0 2 2 % o f the a d d e d GTP was c o n v e r t e d t o cyclic GMP u n d e r these conditions. This p e r c e n t a g e was n o t increased at l o w e r G T P c o n c e n t r a t i o n s and was decreased at higher GTP levels.

Recovery of cyclic nucleotides after filtration T h e r e m o v a l o f h e a t - c o a g u l a t e d cellular debris was f o u n d t o be necessary a f t e r heating, because its presence interfered with later s e p a r a t i o n o f free and a n t i b o d y - b o u n d cyclic n u c l e o t i d e s . M i c r o p o r e filtration o f each cellular sample was f o u n d to be the m o s t rapid and effective m e t h o d o f r e m o v i n g h e a t - c o a g u l a t e d debris. Table 4 shows the r e c o v e r y o f cyclic n u c l e o t i d e s a f t e r m i c r o p o r e filtration. B o t h n e u t r o p h i l and m o n o n u c l e a r cell suspensions y i e l d e d high r e c o v e r y values f o r b o t h cyclic n u c l e o t i d e s ( 8 9 - - 9 6 % ) . Plasma samples, w h i c h c o n t a i n e d larger a m o u n t s o f p r o t e i n , s h o w e d l o w e r recoveries ( 6 0 - - 7 7 % ) . T h e variability in recoveries, h o w e v e r , was always low (_+1.4-4.0% S.D.). T h u s , individual r e c o v e r y d e t e r m i n a t i o n s f o r each sample are unnecessary w h e n h e a t i n g and m i c r o p o r e filtration are e m p l o y e d .

Effects of high protein concentrations U n d e r m o s t c o n d i t i o n s , h e a t t r e a t m e n t f o l l o w e d b y s o n i c a t i o n and microp o r e filtration p r o v i d e d a c o n v e n i e n t m e t h o d o f rapidly p r e p a r i n g radioimm u n o a s s a y samples. O c c a s i o n a l l y , h o w e v e r , large a m o u n t s o f p r o t e i n are required in cellular i n c u b a t i o n s , the presence o f w h i c h slows d o w n the filt r a t i o n step and interferes with later s e p a r a t i o n o f b o u n d and free radio-

306 TABLE 4 R e c o v e r y o f cyclic n u c l e o t i d e s after h e a t inactivation, s o n i c a t i o n , and m i c r o p o r e filtration. Radioactive (12sI) cyclic n u c l e o t i d e s were a d d e d to 0.6 ml o f h e a t - i n a c t i v a t e d cell susp e n s i o n s or plasma samples (30 c p m / p l final c o n c e n t r a t i o n ) . Samples were t h e n sonicated and filtered. C o u n t s o b t a i n e d f r o m 100 pl o f t h e cell s u s p e n s i o n b e f o r e filtering were c o m p a r e d w i t h t h e c o u n t s o b t a i n e d o n 100 pl o f s o l u t i o n after filtration to o b t a i n the p e r c e n t r e c o v e r y value. N e u t r o p h i l and m o n o n u c l e a r cell samples were s u s p e n s i o n s c o n t a i n i n g 5 x 106 cells/ml in m e d i u m 199. Plasma samples were 1 : 10 dilutions o f fresh plasma in p h o s p h a t e b u f f e r e d saline. T h e n u m b e r o f separate samples is in p a r e n t h e s e s . Exp, H e a t - t r e a t e d sample

1. 2. 3. 4. 5,

Polymorphonuclear leukocytes Polymorphonuclear leukocytes Mononuclear leukocytes Mononuclear leukocytes B l o o d plasma

P e r c e n t recovery ± S.D. Cyclic GMP

Cyclic AMP

92.5 95.0 96.0 94.1 76.8

94.3 93.3 89.1 60.3

+ 2.9 + 2.3 +_ 2.3 + 2.0 ± 4.0

(8) (10) (9) (7) (10)

± ± ± ±

2.0 1.7 1.4 2.6

(10) (10) (8) (10)

labeled cyclic nucleotide. Table 5 shows the effect of protein in cellular samples on the no-antibody control of a cyclic GMP radioimmunoassay. Similar results were seen in cyclic AMP assays. Proteins normally present in heated suspensions of PMNs only slightly increased the no-ant i body control over th at seen with m e di um 199 alone. However, the presence of serum (9% TABLE 5 The e f f e c t o f h e a t - t r e a t e d PMNs, e r y t h r o c y t e s , and s e r u m o n the n o - a n t i b o d y c o n t r o l o f a cyclic GMP r a d i o i m m u n o a s s a y . A typical r a d i o i m m u n o a s s a y was p e r f o r m e d as d e s c r i b e d in M e t h o d s using m i c r o p o r e filt r a t i o n for the final s e p a r a t i o n o f b o u n d and free radiolabel. Values given in this table r e p r e s e n t t h e m a g n i t u d e o f t h e c o n t r o l t o w h i c h n o a n t i b o d y was a d d e d e x p r e s s e d as a p e r c e n t a g e o f t h e t o t a l c o u n t s per m i n u t e o f i o d i n a t e d cyclic GMP a d d e d . H e a t e d n e u t r o phils slightly increased t h e blank and e r y t h r o c y t e s significantly increased t h e blank. Each value is calculated f r o m the m e a n o f duplicate assay tubes. Agents a d d e d

Medium Medium Medium Medium Medium Medium Medium Medium

199 199 199 199 199 199 199 199

No a n t i b o d y c o n t r o l ( p e r c e n t o f t o t a l c o u n t s a d d e d *)

+ + + + + + +

PMNs (5 X 106/ml) PMNs + 1,0% s e r u m PMNs + 2,1% s e r u m PMNs + 3.8% s e r u m PMNs + 5.7% s e r u m PMNs + 9.0% s e r u m e r y t h r o c y t e s (1.5 × 109/ml)

* C o u n t s a d d e d were 6800 c p m per assay t u b e .

3.0% 3.2% 3.6% 3.8% 4.4% 7.0% 8.4% 5.7%

307 v/v) increased this value almost 3-fold (3.0% increased to 8.4%). Concentrations of heated serum higher than 10% are very difficult to filter. Similar increases in the no-antibody control of high protein samples were seen when a m m o n i u m sulphate precipitation was employed in the final separation of bound and free radiolabel. The increased no-antibody control is therefore due to proteins or other macromolecules that are not removed by the heat treatment and initial micropore filtration. It can be concluded that the heatkill procedure is most useful when cell suspensions contain relatively little protein. For this reason all of our assays are carried out on washed, purified leukocyte preparations to which no serum, plasma or other proteins have been added. DISCUSSION We have shown (table 1) that heat treatment of neutrophil suspensions can be performed rapidly (2 sec) with little loss of sample volume due to evaporation. This procedure appears to inactivate inhibitors of cyclic AMP and cyclic GMP binding that have been reported to be present in neutrophils (Goldstein et al., 1973; Tsung and Weissmann, 1973; Zurier et al., 1974). Phosphodiesterase treatment of cellular samples (table 2) indicates that the nucleotides measured are cyclic GMP and cyclic AM['. Experiments in which known amounts of the cyclic nucleotides were added to heat-killed cells indicate that the cyclic nucleotides are not degraded by the heat treatment or by remaining cellular phosphodiesterases. The effectiveness of heat treatment in inactivating cellular enzymes was also indicated by Guidotti et al. (1974), who showed that rat brain adenylate cyclase and phosphodiesterase were inactivated by a 2-sec exposure to microwave heating. The possible formation of cyclic GMP from GTP during the 2-see heat treatment has also been investigated. We have found (table 3) that less than 0.002% of the GT[' added to medium 199 or to heat-killed ['MNs is converted to cyclic GMP during the 2 sec heat treatment. Assuming intracellular GTP concentrations are about 0.3 to 0.5 mM (Goldberg et al., 1973), a 0.002% conversion of endogenous GTP to cyclic GMP would cause an increase in cellular cyclic GM[' of less than 1 fmole/106 cells (or 10 -8 moles/ 10 ~3 cells, assuming 10 .3 wet cells per liter). Kimura and Murad (1974) indicate that 0.05% of the added GT[' can be converted to cyclic GMP after 3--5 min of heating at 100°C, however, their data also show that less prolonged heating times result in less formation of cyclic GMP. We conclude that at the probable intracellular GTP concentrations (0.5 mM or lower), and using the 2-sec heat treatment, the contribution of this reaction to cyclic GMP measurements would be negligible. Similar conversion of ATP to cyclic AMP can apparently occur under conditions of prolonged heating (30 min, 100°C) in 0.4 N barium hydroxide (Cook et al., 1957), however, this effect is not observed in physiologic buffers during shorter heating times (Kimura and Murad, 1974).

308 A d i s a d v a n t a g e o f t h e h e a t killing m e t h o d is t h a t very large a m o u n t s o f p r o t e i n or o t h e r m a c r o m o l e c u l e s can r e d u c e cyclic n u c l e o t i d e r e c o v e r y , m a k e m i c r o p o r e f i l t r a t i o n difficult, and i n t e r f e r e w i t h the final s e p a r a t i o n o f b o u n d and free radiolabel. M o d e r a t e a m o u n t s o f p r o t e i n (up t o 5% plasma} usually p r e s e n t few p r o b l e m s if t h e y are p r e s e n t in all i n c u b a t i o n s or if adeq u a t e c o n t r o l s are p e r f o r m e d . We h a v e successfully m e a s u r e d p l a s m a cyclic n u c l e o t i d e c o n c e n t r a t i o n s b y initially diluting p l a s m a 1 : 10 in saline p r i o r to h e a t i n g and filtering. P l a s m a cyclic n u c l e o t i d e c o n c e n t r a t i o n s o b t a i n e d b y this m e t h o d agreed w i t h p r e v i o u s l y r e p o r t e d values. In assessing cellular cyclic n u c l e o t i d e s we have used s e p a r a t e d , w a s h e d l e u k o c y t e p r e p a r a t i o n s to w h i c h n o s e r u m , p l a s m a or o t h e r p r o t e i n s have b e e n a d d e d , h o w e v e r . A d v a n t a g e s o f the h e a t t r e a t m e n t o v e r t h e t r a d i t i o n a l acid kill p r o c e d u r e s are t h a t no e x t r a c t i o n o f p r o t e i n p r e c i p i t a n t s is r e q u i r e d and r e c o v e r y o f cyclic n u c l e o t i d e s is high and s h o w s little variation. This m a k e s it possible to a c c u r a t e l y assay cyclic AMP and cyclic GMP levels in s a m p l e s c o n t a i n i n g f e w e r cells. T h e m e t h o d a p p e a r s t o be sensitive in d e t e c t i n g small changes in l e u k o c y t e cyclic n u c l e o t i d e levels i n d u c e d b y p h a r m a c o l o g i c agents ( H a t c h et al., 1 9 7 7 ) . ACKNOWLEDGEMENTS T h e a u t h o r s wish to t h a n k N.A. H o g a n , M.R. Portas, and W.K. R a w l i n s o n f o r t e c h n i c a l assistance, and Val Callanan f o r secretarial aid. REFERENCES Boyum, A., 1967, Scand. J. Clin. Lab. Invest. 21, 77. Brooker, G., 1974, Methods Biochem. Anal. 22, 95. Cook, W.H., D. Lipkin and R. Markham, 1957, J. Am. Chem. Soc. 79, 3607. Goldberg, N.D., R.F. O'Dea and M.K. Haddox, 1973, in: Advances in Cyclic Nucleotide Research (P. Greengard and G.A. Robinson, eds.) Vol. 3, (Raven Press, New York) pp. 155. Goldstein, I., S. Hoffstein, J. Gallin and G. Weissmann, 1973, Proc. Natl. Acad. Sci. U.S.A. 71, 2916. Guidotti, A., D.L. Cheney, M. Trabucchi, C. Wang and R.A. Hawkins, 1974, Neuropharmacology 13, 1115. Haddox, M.K., L.T. Furcht, S.R. Gentry, J.H. Stephcnson and N.D. Goldgerg, 1976, Nature (London) 262, 146. Harper, J.F. and G. Brooker, 1975, J. Cyclic Nucleotide Res. 1, 207. Hatch, G.E., W.K. Nichols and H.R. Hill, 1977, J. Immunol. 119, 450. Kimura, H. and F. Murad, 1974, J. Biol. Chem. 249,329. Sandier, J.A., R.I. Clyman, V.C. Manganiello and M. Vaughan, 1975, J. Clin. Invest. 55, 431. Steiner, A.L., A.S. Pagliara, L.R. Chase and D.M. Kipnis, 1972, J. Biol. Chem. 247, 1114. Tsung, P.K. and G. Weissmann, 1973, Biochem. Biophys. Res. Commun, 51, 836. Zurier, R.B., F. Weissmann, S. Hoffstein, S. Kammerman and H.H. Tai, 1974, J. Clin. Invest. 53. 297.