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Cyclic nucleotide activity in gastrointestinal tissues and fluids
ANATYT!CA!<
RJOCHEMJSTRY
78,
?95405
(:977)
Cyclic Nucleotide Activity in Gastrointestinal Tissues and Fluids EDMUND H. SCHWARTZEL, JR.,S.
BACHMAN,ANDROBERT
A. LEVINE
Department of Medicine, Upstate Medical Center, 750 E. Adams Street, Syracuse, New York 13210 Received June 21, 1976; accepted November 15, 1976 Published values of adenosine 3’,5’-monophosphate (CAMP) and guanosine 3’,5’-monophosphate (cCiMP) in gastrointestinal tissues and fluids have varied depending upon extraction and purification methods and assay procedures. Cyclic nucleotide levels in a variety of gastrointestinal tissues and fluids from several animal species were measured by the Gilman protein kinase assay for CAMP, and by a radioimmunoassay for CAMP and cGMP. The neutral alumina/Dowex-lformate column was the most accurate for the preparation of bile and gastric juice samples for the measurement of CAMP and cGMP, as verified by the use of column blanks, spiked samples, phosphodiesterase treatment, and serial sample dilution. Tissue samples gave consistent results regardless of the various column procedures employed.
The cyclic nucleotides, adenosine 3’,5’-monophosphate (CAMP) and guanosine 3’,5’-monophosphate (cGMP), have been implicated as “second messengers” in the regulation of a number of gastrointestinal functions. These include secretion in the stomach (1) and intestine (2); absorption in the small intestine (3); cellular growth (3) and the regulation of liver metabolism (4). Levels of CAMP in tissues and fluids reported in the literature have shown wide variation depending on the method of sample preparation (5,6). Reports of gastrointestinal cGMP levels have been few. In order to establish baseline values of cyclic nucleotide levels for their possible physiologic involvement in various gastrointestinal functions, we compared cyclic nucleotide levels in a number of gastrointestinal tissues and fluids. Samples were prepared for cyclic nucleotide assay using four different commonly reported column procedures (7- 11). The reliability of each column procedure was evaluated on the basis of recovery of radioimmunoassayable CAMP and cGMP from spiked samples; recovery of H3 - CAMP and H3. cGMP from spiked samples; levels of cyclic nucleotides measured in column blanks, the susceptibility of samples to hydrolysis by cyclic nucleotide phosphodiesterase, and values measured in serially diluted samples. 395 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction m any form reserved.
ISSN
0003-2697
396
SCHWARTZEL,
MATERIALS
BACHMAN,
AND
LEVINE
AND METHODS
I. Tissue Biopsy (A) Animal tissue. Fasted male Sprague- Dawley rats (200-400 g) were killed by decapitation; the body cavity was rapidly opened, and samples of liver, gastric antrum or fundus, and small intestine were removed with scissors and forceps. All tissue samples were rinsed thoroughly with cold saline. Small tissue samples (less than 50 mg) were frozen directly in liquid nitrogen, whereas larger tissue samples (50-300 mg) were frozen using Wollenberger clamps (12) precooled in liquid nitrogen. All tissue samples were stored in liquid nitrogen. Samples of dog antrum and fundus mucosa were obtained from fasted mongrel dogs (lo- 15 kg) in acute experiments under pentobarbital anesthesia. After opening the body cavity, stab incisions were made in the area of the antrum and fundus, and mucosa (100-200 mg) was easily separated with forceps from underlying muscle along one edge of the incision. Full-thickness samples (200-400 mg) were similarly rapidly dissected from along the other edge of the incision, and all samples were rinsed with cold saline and frozen with liquid nitrogen precooled Wollenberger clamps. (B) Human tissue. Human biopsies were obtained under three sets of conditions. In one series, patients undergoing endoscopic examination for a number of upper gastrointestinal conditions were biopsied through the fiberoptic endoscope. Biopsy forceps were placed in either the antrum or fundus under direct visual control, and a biopsy (2-5 mg) was obtained. The time from the cutting of the biopsy to freezing directly in liquid nitrogen averaged 20 set (100 biopsies). Three to five biopsies were obtained from each anatomical area and were pooled to give a total biopsy weight of 15-25 mg. In a second series of experiments, patients and normal medical student volunteers undergoing routine betazole or pentagastrin gastric analysis were biopsied with a Woods tube-suction biopsy instrument. Multiple biopsies of each area were frozen directly in liquid nitrogen and were pooled to give a total sample weight of 15-25 mg. The time from removal of the biopsy to freezing in liquid nitrogen averaged 20 set (200 biopsies). In a third series of patients, samples were obtained at surgery. These included stomach (antrum and fundus, full thickness), pancreas, and small and large intestines. Samples (0.5- 1.O g) were removed from the sterile operating field on gauze pads, rinsed with cold saline, and frozen in liquid nitrogen. The time from removal of the sample to freezing in liquid nitrogen averaged 30 set (25 samples). Written informed consent was obtained from patients and volunteers prior to any procedure.
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
397
II. Tissue Homogenization Tissues were weighed in a frozen state and added directly to ice-cold 6% trichloracetic acid (TCA) in a Tenbroeck (7 ml) tissue grinder. Biopsies were divided into IOO-mg portions before homogenization. Small samples (~50 mg) were homogenized with 2 ml of 6% TCA; larger samples (>50 mg) were homogenized with 4 ml of 6% TCA. Tissue extracts were centrifuged (SOOOg) in the cold, and the supernatant was transferred to 50-m! glass-stoppered centrifuge tubes. The supernatant was extracted 3X with 10 vol of water-saturated diethyl ether. The last traces of ether were removed from the aqueous phase in a 60°C water bath under a stream of air. The extract was then applied directly to the column. III.
Biological
Fluids:
Collection
(A) Gastric juice, bile, and pancreatic juice. Basal gastric juice was collected by a nasogastric tube from fasted patients and normal student volunteers for four consecutive 15-min periods. Either betazole- or pentagastrin-stimulated samples were collected in ice and maintained at 2-5°C until frozen. Samples containing gross contamination with either bile or blood were not used for cyclic nucleotide determination. Samples containing large quantities of mucous, usually basal samples, were filtered through a l.O-cm layer of Pyrex glass wool packed into a 1.5 x lo-cm glass column. Only the clear filtrate or clear poststimulation gastric juice was used for column chromatography. Human bile was collected over ice through T-tube drainage from patients following choledocholithotomy. Pancreatic juice was collected from one patient following cannulation of the pancreatic duct during an endoscopic retrograde cannulation procedure. (B) Human serum. Blood was drawn by venipuncture and allowed to clot at room temperature. After centrifugation, the serum was decanted and frozen. IV. Biological
Fluids: Preparation
for Chromatography
Gastric juice, pancreatic juice, bile or serum (0.5 ml), and 0.5 ml of cold 0.6 N perchloric acid (0.5 ml) were mixed and allowed to stand in an ice bath for 10 min. After centrifugation at 5OOOg for 10 min, the clear supernatant was removed with a Pasteur pipet and used in the Dowex 50H+/water chromatography system. Gastric and pancreatic juices were applied directly to Dowex 50 H+/HCl and Dowex- l-formateiformic acid columns, The sample volumes applied to columns ranged from 2 to 4 ml of juice. pH Neutralization prior to
398
SCHWARTZEL,
BACHMAN,
AND
LEVINE
column chromatography did not effect separation, recovery, or the level of cyclic nucleotides as measured in blanks or spiked samples. Cold 6% TCA (2 ml) was added to 2 ml of bile or serum. The contents were mixed and allowed to stand in an ice bath for 10 min. After centrifugation at 5OOOg for 10 min, the supernatant was extracted with water-saturated ether as described for tissue extracts. The sample was then applied to a Dowex 50 H+/HCl or Dowex-1-formate/formic acid column. V. Column
Chromatography
(A) Dowex 50 H+lwater and HCI elution. Blanks for all columns consisted of a volume of 0.1 N HCl equal to a given sample volume. Standards were added to fluids at levels higher than that usually found in those fluids. Dowex 50 H+ columns (AG 5OW-X8, 100-200 mesh, Bio-Rad Laboratories) to be eluted with water were prepared by the method of Krishna et al. (7) in disposable Pasteur pipets (5.5 x 50 mm) plugged with Pyrex glass wool. Columns were first rinsed with 10 ml of distilled water, and the sample was (1.0 ml from the perchloric acid procedure) applied. The column was then eluted with 10 ml of distilled water. The elutant from this and other column procedures was taken to dryness at 60°C under a stream of air. Dried samples were dissolved in 0.5 ml of 0.05 M sodium acetate buffer (pH 6.2) and frozen until cyclic nucleotide assay. For the Dowex 50 H+/HCl column procedure (8), the same resin was packed in a glass column (7.0 x 150 mm) plugged with Pyrex glass wool. The resin bed was washed with five bed-volumes of 0.1 N HCl, and the sample (up to 4 ml) was applied. The column was eluted with 100 ml of 0.1 N HCl. cGMP was eluted in the first 20 ml, and CAMP in the 20-80-ml fraction. (B) Dowex-I-formate
and aluminalDowex-I-formate
chromatography.
Dowex-1-formate resin was generated from Dowex-l-chloride (1 x 8 100-200 mesh, Bio-Rad Laboratories) by washing with 1.0 N NaOH followed by 2.0 N formic acid. The resin was stored in 0.5 N formic acid at 4°C until used. A 7 x 30-mm resin bed was packed into a glass column plugged with Pyrex glass wool and equipped with a 50-ml reservoir. The resin bed was washed with distilled water until the pH of the elutant was > 4.0. In the Dowex-1-formate column procedure without a neutral alumina precolumn (9), the sample was applied directly to the resin bed. When an alumina (AG-7, 100-200 mesh, Bio-Rad Laboratories) precolumn was used (10,l l), the barrel of a ~-CC disposable plastic syringe containing a 1.4-cm filter paper disk was placed in the column reservoir over the prepacked water-washed Dowex-1-formate resin. Dry neutral alumina was added to the syringe barrel to the l-ml mark, and the sample was applied directly to the dry alumina. After the sample had run through the alumina into the resin bed, the cyclic nucleotides were eluted from the alumina and into the resin bed with 10 ml of 25 mM Tris-formate (pH 7.4).
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
399
The Dowex-I-formate resin was then washed with 10 ml of distilled water. CAMP was then eluted with 10 ml of 2 N formic acid, and cGMP was eluted with 20 ml of 4 N formic acid. Samples were concentrated and prepared for analysis as described earlier. VI. Cyclic Nucleotide
Assay
(A) Gilman assay. CAMP was measured in a number of samples via the protein kinase binding assay of Gilman (13). 3,5’-Cyclic AMP-dependent protein kinase (from beef heart; Sigma, St. Louis, MO.) was reconstituted at pH 6.5 in 0.0005 M acetate buffer. Aliquots of column fractions (50- 100 ~1) were added to 100 ~1 of 0.1 M sodium acetate/acetic acid buffer (pH 4.0) in tubes in an ice bath. Two picomoles of H3*cAMP in 10 ~1 of the pH 4.0 acetate buffer was added to each tube, and the reaction was initiated by the addition of binding protein (5 $/tube). All tubes were incubated at 0°C for 30 min. Binding protein recovery (Millipore filter) and scintillation counting procedures were carried out as described by Gilman (13). (B) Radioimmunoassay. CAMP and cGMP were measured using the radioimmunoassay kits (Catalog Nos. 201618 and 201812) produced by Schwarz/Mann (Orangeburg, New York, 10962) according to the procedures of Steiner et a/. (14). VII. Cyclic Nucleotide
Verijication
Procedures
(A) Phosphodiesterase treatment. Phosphodiesterase (from beef heart) was purchased from Boehringer Mannheim. The ammonium sulfate suspension was diluted with glycylglycine buffer, 0.1 M pH 7.5, to give 0.01 unit/IO ~1. One-hundred microliter aliquots of column fractions (pH adjusted to 7.5) were mixed with 10 ~1 of MgSO,*7H,O (1 mg/ml) and 10 ~1 of enzyme suspension. Samples were incubated at 25°C for 60 min. Samples were heated for 2 min in a boiling-water bath and were used directly in the Gilman and radioimmunoassay procedures. (B) Serial sample dilution. Selected samples were serially diluted (up to l/100) and 100 pl-fractions were run in the radioimmunoassay for both CAMP and cGMP. RESULTS
Tables 1 and 2 show the results of column blanks. Of the four columns tested, the Dowex 50 H+ eluted with water gave the highest blank values for both cyclic nucleotides. The neutral alumina/Dowex-l-formate combination was the only column to give a zero blank for both cyclic nucleotides. While blank values for the Dowex 50 H+/O. 1 N HCI and the Dowex-I-formate columns were lower than the blank values in the Dowex 50 H+/water system, the material responsible for these values was not hydrolyzed by cyclic nucleotide phosphodiesterase.
400
SCHWARTZEL,
BACHMAN, TABLE
CAMP IN GASTROINTESTINAL
AND LEVINE
I TISSUES AND FLUIDS
Column Dowex 50 H+/ Blank CAMP (RIA)” CAMP (G)b
Dowex-1-formate
Water
0.1 N HCl
Without alumina
With alumina
4.6 10.6
1.1 4.5
0.8 4.0
0.0 2.6
9.3 k 3.5 5.9 ‘-’ 2.1 12.7 zt 2.2 105 4 11.0 90 ” 30 1.95’
12.3 4 1.4 8.5 f 1.1 11.4 ” 3.7 84 ” 12.1 105 ” 25 1.75’
16.6 ? 1.7
14.4 t 2.3
Sample Human Gastric juice Basal (RIA) Post-stim. (RIA) (G) Bile (RIA) (G) Pancreatic (RIA) Dog Gastric juice (RIA)
Fluid (pm/ml)
26 2 5.6 21 2 4.3 471 c 68 250 zt 30 1000 -
12.5 k 6.3 ‘19.0 + 145 k 131 f -
0.5 1.5 2.6 15 23
10.6 -r- 2.1
Tissue (pm/g) No column Human Gastric antrum (G) Gastric fundus (G) Dog Gastric antrum Gastric fundus Rat Liver (RIA) ((3 Small intestine
546 zk 52 1750 ” 175 807?106 1450 ‘- 225
534 + 27 650 k 25 550 ” 50
490 2 35 730 ? 30 940 2 21
(RIA) (RIA)
-
-
3%’ -
525 k 53 580 + 33
(RIA)
1040 + 125 425 2 40
510 2 30 -
410 k 40 360 2 30 -
380 2 20 420 2 40 725 2 71
(RIA) (RIA)
597 841 784 876
-c 43 t 56 5 112 -c 34
a (RIA), Radioimmunoassay. b (G), Gilman assay. r One sample only.
Addition of radioimmunoassayable CAMP to human gastric juice resulted in recoveries in the range of 65-80% for the Dowex 50 H+ column when eluted with either water or 0.1 N HCl. Recovery of radioimmunoassayable CAMP from the Dowex-I-formate column alone was in the range of 70-N%. Recovery of radioimmunoassayable cGMP
GASTROINTESTINAL
TABLE cGMP
401
CYCLIC NUCLEOTIDES
IN GASTROINTESTINAL
2 TISSUES
AND
(RIA)
FLUIDS
Column Dowex 50 H+/ Blank cGMP
Water
0.1 N HCI
Without alumina
With alumina
8.8
1.3
0.6
0.0
4.1 ” 0.6 2.8 -c 0.7 9.6 2 I.2 0.95”
2.2 k 0.2 0.6 -c 0.1 6.0 -+ 0.5 1.15”
Fluid (pm/ml)
Sample Human Gastric Juice Basal Post-stimulation Bile Pancreatic
Dowex-1-formate
14.9 2 2.9 15.9 k 2.5 19.1 t 5.8 -
2.1 2 0.3 1.4 -+ 0.4 16.9 2 2.3 -
Tissue (pm/g) No column
Human Gastric antrum Gastric fundus Liver Rat Chicken
17.5 * 3.0 22.6 t 0.5
17.4 ” 4.4 15.7 * 3.1
38.4 ” 6.1 22.0 IL 1.1
27.1 ? 3.5 34.3 k I.5
14.6 r 2.1 18.7 2 0.6
-
17.1 ” 2.4 29.7 2 2.1
13.8 d 6.6 -
0 One sample only.
was 10% lower than CAMP for all four columns, whereas the recovery of both tritiated CAMP and cGMP was lo- 15% higher than that observed for radioimmunoassayable cyclic nucleotides. Values calculated (picomole/ tube) from serially diluted fluid samples showed a random + 10% variation when plotted against the standard CAMP or cGMP curve, only when the fluid sample had been prepared by the neutral alumina/Dowex-1-formate column system. Diluted tissue samples showed a r 10% variation with the standard curves regardless of the column procedure. CAMP levels in gastric juice and bile were highest when measured by the Gilman assay after Dowex 50 H+/water chromatography (Table 1). Tissue samples, when measured directly without columns, also gave high values. CAMP levels measured by the Gilman method after Dowex 50 H+/O. 1 N HCI or Dowex-I-formate formic acid chromatography were more like the values obtained using radioimmunoassay. However, only samples prepared with the alumina/Dowex- I-formate combination gave values that were the same by either the Gilman or RIA procedures.
402
SCHWARTZEL,
BACHMAN,
AND
LEVINE
While the RIA procedure for CAMP was less influenced than the Gilman assay by interfering compounds in gastrointestinal fluids prepared by Dowex 50 H+/water chromatography, measured values were still 3-5 times higher than those obtained by alumina/Dowex-1-formate chromatography. Gastric juice gave similar values for RIA when prepared by Dowex 50 H+/HCl or either of the Dowex- 1-formate procedures. Human bile, unlike gastric juice, gave decreasing values as samples were prepared, respectively, by the Dowex 50 H+/water, Dowex 50 H+/HCl, and both Dowex-1-formate column procedures (Table 1). Only fluid samples prepared by the alumina/Dowex-1-formate procedure gave values as low as column blanks when treated with phosphodiesterase or gave proportional amounts per tube after serial sample dilution. Tissue samples gave consistent results regardless of the method of column preparation. Samples assayed without prior chromatography with phosphodiesterase gave values higher than any tissue sample prepared by any of the three column procedures employed. Tissue samples prepared by any of the previously mentioned column procedures gave values after phosphodiesterase treatment equal to column blanks. Serial dilution of tissue samples prepared by any of the three column procedures confirmed the measured material as CAMP. cGMP levels (Table 2) in gastrointestinal fluids were at least 6 times less than corresponding CAMP levels. Fluid samples prepared by Dowex 50 H+/water chromatography gave the highest levels of cGMP, but less than half of this material was hydrolyzed by phosphodiesterase. cGMP levels in tissues were at least 20 times lower than the corresponding CAMP levels. Individual tissue samples gave identical to slightly higher values of cGMP, depending on the chromatographic procedure employed (Table 2). Treatment of samples with phosphodiesterase prepared by any of the other column procedures gave values similar to column blanks when measured by the RIA procedure. cGMP in gastrointestinal tissues and fluids showed chromatographic separation and serial sample dilution patterns similar to that seen for CAMP. Table 3 presents a summary CAMP and cGMP values for representative gastrointestinal tissues and fluids determined in our laboratory using the neutral alumina/Dowex-1-formate columns and the RIA procedure. Note the higher CAMP compared to cGMP levels for all samples. DISCUSSION
The alumina/Dowex- 1-formate chromatographic combination is the system of choice for the preparation of gastrointestinal fluids for either CAMP or cGMP determinations. Of the other three columns tested, the Dowex 50 H+/O. 1 N HCl and the Dowex-1-formatelformic acid columns are suitable for fluids such as bile where the cyclic nucleotide concentration is relatively high. These columns are not suited for either gastric or
GASTROINTESTINAL
TABLE MEAN
GASTROINTESTINAL
403
CYCLIC NUCLEOTIDES
CYCLIC
3 NUCLEOTIDE
Source
CAMP
Fluid (pm/ml) Human serum Human gastric juice Canine gastric juice Human pancreatic juice Human bile Baboon bile Canine bile Rat bile
5 12 14 2 125 5,833 10.300 100
Tissue (pm/g) Human stomach Canine antrum mucosa Canine fundus mucosa Rat stomach Human jejunum Rat jejunum Human ileum Rat ileum Rat colon Human pancreas Baboon liver Chicken liver Rat liver
847 600 650 600 l.ooo 950 83.5 950 980 959 950 380
’ All samples processed by alumina/Dowex-I-formate
CONCENTFUTION~
cGMP
-
1 2 3 1 6
23 17 20 13 90 45 15 45 17 37 12 8
and measured by RIA.
pancreatic juices since the column blanks are equal to the levels of cGMP found in these fluids. The Dowex 50 H+/O. 1 N HCl column has the added disadvantage of having a relatively long running time and yielding large-volume column fractions. The higher recoveries observed with tritium-labeled, as opposed to radioimmunoassayable, cyclic nucleotides may reflect additional losses from the concentration/recovery step necessary to prepare samples for the Gilman assay or RIA. We also found that tritium-labeled cyclic nucleotides contain varying amounts of unidentified radioactive contaminants or breakdown products which may affect recoveries. We have recovered up to 20% of the counts applied to a Dowex-I-formatelformic acid column in the water-wash fraction prior to the elution of CAMP with 2 N formic acid. We have, therefore, routinely purified all tritiated cyclic nucleotides on Dowex-1-formate columns prior to use, either in samples as an indicator of recovery or, in the case of H3-CAMP, for use in the Gilman assay. The low recovery of cyclic nucleotides, particularly cGMP in the alumina/ Dowex- I-formate system, may be a function of the alumina. White (15) has
404
SCHWARTZEL,
BACHMAN,
AND LEVINE
reported markedly different yields of cyclic nucleotides from alumina obtained from different suppliers. Selection of a neutral or basic alumina on a trial and error basis to give relatively high yields of cGMP is advisable in order to detect changes in concentration in fluids such as gastric and pancreatic juices which have low basal levels of cGMP. The failure of the Gilman assay to yield results in both tissues and fluids comparable to those obtained with the RIA with all samples, except for those prepared by the alumina/Dowex-l-formate system, suggests that an interfering compound(s) is most effectively removed by the latter column combination. It is also possible that some of the problem is due to the preparation of binding protein. J. D. Gardner (personal communication) has reported that binding protein, prepared in his laboratory from beef muscle, is less sensitive to interfering substances than binding protein obtained from commercial sources. The values for CAMP and cGMP in gastrointestinal fluids reported here are lower than those that we (16) have previously reported. It is not surprising that measurement of biological fluids of such diverse composition as gastric juice, bile, and pancreatic juice would involve complex technical problems. Nevertheless, we feel that the values reported here are reliable on the basis of low-column blanks, hydrolysis by phosphodiesterase, and the results of serial sample dilution, as recommended by G. Brooker (personal communication). The time period from isolation to freezing seems to be critical in tissues with the highest rates of metabolic activity, such as cardiac muscle and brain tissue (17). Preliminary acute experiments in our laboratory with in vivo dog stomach muscosa and rat intestinal muscosa showed that cyclic nucleotide levels were unchanged in biopsy specimens that were removed and kept at room temperature for as long as 1 min before freezing with precooled Wollenberger clamps. Comparison of cyclic nucleotide levels in specimens that were frozen with Wollenberger clamps versus direct freezing in liquid nitrogen showed no significant difference. These results are in agreement with the report of Stull and Mayer (18) who found no significant difference in rabbit muscle CAMP when tissue was frozen by either of the above techniques. These observations suggest that cyclic nucleotide levels in gastrointestinal tissues are relatively stable. Our results suggest that, under the usual conditions of gastrointestinal tissue and fluid sampling, reliable estimates of cyclic nucleotide content can be obtained if care is taken to select the most sensitive column procedure and if adequate recovery and verification procedures are employed. ACKNOWLEDGMENTS This research was supported, in part, by Grant AM 17192 USPHS.
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
405
REFERENCES 1. Bieck, P. R., Gates, J. A., Robison, G. A., and Adkins, R. B. (197 l)Amer, J. Physiol. 224, 158- 164. 2. Kimberg, D. V., Field, M., Johnson, J., Henderson, A., and Gershon, E. (1971)3. C/in. Invest. 50, 1218- 1230. 3. Kimberg. D. V. (1974) Gasfroenterolog~ 67, 1023- 1064. 4. Pilkis. S. J., Claus, T. H., Johnson, R. A., and Park, C. R. (1975)J. Bid/. Chem. 250, 6328-6336.
5. Mao, C. C., Jacobson, E. D., and Shanbour, L. L. (1973)Amer. J. Physiol. 225893-896. 6. Ruoff, Hans -J.. and Sewing, K. (1975) Eur. J. Pharmacol. 32, 227-232. 7. Krishna, G., Weiss. B.. and Brodie, B. B. (1968) J. Phartnncol. and Exp. Ther. 163, 379-385. 8.
Murad, F., Manganiello,
V., and Vaughan, M. (1971) Proc.
Nat.
Acad.
Sci.
USA
68,
736-739. 9. Brooker.G..Thomas, L. J., Jr.,andAppleman, M. M.(l%8)Biochemistv 7,4177-4181. 10. White, A. A., and Zenser, T. V. (197l)Anal. Biochem. 41, 372-396. 11. O’Dea, R. F., Bodley, J. W., Lin, L., Haddox. M. K., and Goldberg, N. D. (1974) in Methods in Enzymology (Hardman, J. G., and O’Malley. B. W., eds.) Vol. 38C, pp. 85-95. Academic Press, New York. 12. Wollenberger, A., Ristau. O., and Schoffa. G. (1960) PJIuegers Arch. Gesamfe Physiol. Menschen
Tiere
270, 399.
13. Gilman, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305-312. 14. Steiner, A. L., Kipnis, D. M., Utiger, R., and Parker, C. (1969) Proc. USA
Nat.
Acud.
Sci.
64, 367-372.
15. White, A. A. (1974)in Methods in Enzymology (Hardman, J. G.. and O’Malley. B. W.. eds.) Vol. 38C, pp. 41-46. Academic Press, New York. 16. Levine, R. A., and Schwartzel, E. H., Jr. (1974) Gasfroenferology 66, 730. 17. Mayer, S. E., Stul!, J. T., and Wastila. W. B. (1974) in Methods in Enzymology (Hardman, J. G. and O’MaUey. B. W.. eds.) Vol. 38C, pp. 3-9, Academic Press, New York. 18. Stull. J. T., and Mayer. S. E. (197l)J. Biol. Chem. 246, 5716-5723.
RJOCHEMJSTRY
78,
?95405
(:977)
Cyclic Nucleotide Activity in Gastrointestinal Tissues and Fluids EDMUND H. SCHWARTZEL, JR.,S.
BACHMAN,ANDROBERT
A. LEVINE
Department of Medicine, Upstate Medical Center, 750 E. Adams Street, Syracuse, New York 13210 Received June 21, 1976; accepted November 15, 1976 Published values of adenosine 3’,5’-monophosphate (CAMP) and guanosine 3’,5’-monophosphate (cCiMP) in gastrointestinal tissues and fluids have varied depending upon extraction and purification methods and assay procedures. Cyclic nucleotide levels in a variety of gastrointestinal tissues and fluids from several animal species were measured by the Gilman protein kinase assay for CAMP, and by a radioimmunoassay for CAMP and cGMP. The neutral alumina/Dowex-lformate column was the most accurate for the preparation of bile and gastric juice samples for the measurement of CAMP and cGMP, as verified by the use of column blanks, spiked samples, phosphodiesterase treatment, and serial sample dilution. Tissue samples gave consistent results regardless of the various column procedures employed.
The cyclic nucleotides, adenosine 3’,5’-monophosphate (CAMP) and guanosine 3’,5’-monophosphate (cGMP), have been implicated as “second messengers” in the regulation of a number of gastrointestinal functions. These include secretion in the stomach (1) and intestine (2); absorption in the small intestine (3); cellular growth (3) and the regulation of liver metabolism (4). Levels of CAMP in tissues and fluids reported in the literature have shown wide variation depending on the method of sample preparation (5,6). Reports of gastrointestinal cGMP levels have been few. In order to establish baseline values of cyclic nucleotide levels for their possible physiologic involvement in various gastrointestinal functions, we compared cyclic nucleotide levels in a number of gastrointestinal tissues and fluids. Samples were prepared for cyclic nucleotide assay using four different commonly reported column procedures (7- 11). The reliability of each column procedure was evaluated on the basis of recovery of radioimmunoassayable CAMP and cGMP from spiked samples; recovery of H3 - CAMP and H3. cGMP from spiked samples; levels of cyclic nucleotides measured in column blanks, the susceptibility of samples to hydrolysis by cyclic nucleotide phosphodiesterase, and values measured in serially diluted samples. 395 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction m any form reserved.
ISSN
0003-2697
396
SCHWARTZEL,
MATERIALS
BACHMAN,
AND
LEVINE
AND METHODS
I. Tissue Biopsy (A) Animal tissue. Fasted male Sprague- Dawley rats (200-400 g) were killed by decapitation; the body cavity was rapidly opened, and samples of liver, gastric antrum or fundus, and small intestine were removed with scissors and forceps. All tissue samples were rinsed thoroughly with cold saline. Small tissue samples (less than 50 mg) were frozen directly in liquid nitrogen, whereas larger tissue samples (50-300 mg) were frozen using Wollenberger clamps (12) precooled in liquid nitrogen. All tissue samples were stored in liquid nitrogen. Samples of dog antrum and fundus mucosa were obtained from fasted mongrel dogs (lo- 15 kg) in acute experiments under pentobarbital anesthesia. After opening the body cavity, stab incisions were made in the area of the antrum and fundus, and mucosa (100-200 mg) was easily separated with forceps from underlying muscle along one edge of the incision. Full-thickness samples (200-400 mg) were similarly rapidly dissected from along the other edge of the incision, and all samples were rinsed with cold saline and frozen with liquid nitrogen precooled Wollenberger clamps. (B) Human tissue. Human biopsies were obtained under three sets of conditions. In one series, patients undergoing endoscopic examination for a number of upper gastrointestinal conditions were biopsied through the fiberoptic endoscope. Biopsy forceps were placed in either the antrum or fundus under direct visual control, and a biopsy (2-5 mg) was obtained. The time from the cutting of the biopsy to freezing directly in liquid nitrogen averaged 20 set (100 biopsies). Three to five biopsies were obtained from each anatomical area and were pooled to give a total biopsy weight of 15-25 mg. In a second series of experiments, patients and normal medical student volunteers undergoing routine betazole or pentagastrin gastric analysis were biopsied with a Woods tube-suction biopsy instrument. Multiple biopsies of each area were frozen directly in liquid nitrogen and were pooled to give a total sample weight of 15-25 mg. The time from removal of the biopsy to freezing in liquid nitrogen averaged 20 set (200 biopsies). In a third series of patients, samples were obtained at surgery. These included stomach (antrum and fundus, full thickness), pancreas, and small and large intestines. Samples (0.5- 1.O g) were removed from the sterile operating field on gauze pads, rinsed with cold saline, and frozen in liquid nitrogen. The time from removal of the sample to freezing in liquid nitrogen averaged 30 set (25 samples). Written informed consent was obtained from patients and volunteers prior to any procedure.
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
397
II. Tissue Homogenization Tissues were weighed in a frozen state and added directly to ice-cold 6% trichloracetic acid (TCA) in a Tenbroeck (7 ml) tissue grinder. Biopsies were divided into IOO-mg portions before homogenization. Small samples (~50 mg) were homogenized with 2 ml of 6% TCA; larger samples (>50 mg) were homogenized with 4 ml of 6% TCA. Tissue extracts were centrifuged (SOOOg) in the cold, and the supernatant was transferred to 50-m! glass-stoppered centrifuge tubes. The supernatant was extracted 3X with 10 vol of water-saturated diethyl ether. The last traces of ether were removed from the aqueous phase in a 60°C water bath under a stream of air. The extract was then applied directly to the column. III.
Biological
Fluids:
Collection
(A) Gastric juice, bile, and pancreatic juice. Basal gastric juice was collected by a nasogastric tube from fasted patients and normal student volunteers for four consecutive 15-min periods. Either betazole- or pentagastrin-stimulated samples were collected in ice and maintained at 2-5°C until frozen. Samples containing gross contamination with either bile or blood were not used for cyclic nucleotide determination. Samples containing large quantities of mucous, usually basal samples, were filtered through a l.O-cm layer of Pyrex glass wool packed into a 1.5 x lo-cm glass column. Only the clear filtrate or clear poststimulation gastric juice was used for column chromatography. Human bile was collected over ice through T-tube drainage from patients following choledocholithotomy. Pancreatic juice was collected from one patient following cannulation of the pancreatic duct during an endoscopic retrograde cannulation procedure. (B) Human serum. Blood was drawn by venipuncture and allowed to clot at room temperature. After centrifugation, the serum was decanted and frozen. IV. Biological
Fluids: Preparation
for Chromatography
Gastric juice, pancreatic juice, bile or serum (0.5 ml), and 0.5 ml of cold 0.6 N perchloric acid (0.5 ml) were mixed and allowed to stand in an ice bath for 10 min. After centrifugation at 5OOOg for 10 min, the clear supernatant was removed with a Pasteur pipet and used in the Dowex 50H+/water chromatography system. Gastric and pancreatic juices were applied directly to Dowex 50 H+/HCl and Dowex- l-formateiformic acid columns, The sample volumes applied to columns ranged from 2 to 4 ml of juice. pH Neutralization prior to
398
SCHWARTZEL,
BACHMAN,
AND
LEVINE
column chromatography did not effect separation, recovery, or the level of cyclic nucleotides as measured in blanks or spiked samples. Cold 6% TCA (2 ml) was added to 2 ml of bile or serum. The contents were mixed and allowed to stand in an ice bath for 10 min. After centrifugation at 5OOOg for 10 min, the supernatant was extracted with water-saturated ether as described for tissue extracts. The sample was then applied to a Dowex 50 H+/HCl or Dowex-1-formate/formic acid column. V. Column
Chromatography
(A) Dowex 50 H+lwater and HCI elution. Blanks for all columns consisted of a volume of 0.1 N HCl equal to a given sample volume. Standards were added to fluids at levels higher than that usually found in those fluids. Dowex 50 H+ columns (AG 5OW-X8, 100-200 mesh, Bio-Rad Laboratories) to be eluted with water were prepared by the method of Krishna et al. (7) in disposable Pasteur pipets (5.5 x 50 mm) plugged with Pyrex glass wool. Columns were first rinsed with 10 ml of distilled water, and the sample was (1.0 ml from the perchloric acid procedure) applied. The column was then eluted with 10 ml of distilled water. The elutant from this and other column procedures was taken to dryness at 60°C under a stream of air. Dried samples were dissolved in 0.5 ml of 0.05 M sodium acetate buffer (pH 6.2) and frozen until cyclic nucleotide assay. For the Dowex 50 H+/HCl column procedure (8), the same resin was packed in a glass column (7.0 x 150 mm) plugged with Pyrex glass wool. The resin bed was washed with five bed-volumes of 0.1 N HCl, and the sample (up to 4 ml) was applied. The column was eluted with 100 ml of 0.1 N HCl. cGMP was eluted in the first 20 ml, and CAMP in the 20-80-ml fraction. (B) Dowex-I-formate
and aluminalDowex-I-formate
chromatography.
Dowex-1-formate resin was generated from Dowex-l-chloride (1 x 8 100-200 mesh, Bio-Rad Laboratories) by washing with 1.0 N NaOH followed by 2.0 N formic acid. The resin was stored in 0.5 N formic acid at 4°C until used. A 7 x 30-mm resin bed was packed into a glass column plugged with Pyrex glass wool and equipped with a 50-ml reservoir. The resin bed was washed with distilled water until the pH of the elutant was > 4.0. In the Dowex-1-formate column procedure without a neutral alumina precolumn (9), the sample was applied directly to the resin bed. When an alumina (AG-7, 100-200 mesh, Bio-Rad Laboratories) precolumn was used (10,l l), the barrel of a ~-CC disposable plastic syringe containing a 1.4-cm filter paper disk was placed in the column reservoir over the prepacked water-washed Dowex-1-formate resin. Dry neutral alumina was added to the syringe barrel to the l-ml mark, and the sample was applied directly to the dry alumina. After the sample had run through the alumina into the resin bed, the cyclic nucleotides were eluted from the alumina and into the resin bed with 10 ml of 25 mM Tris-formate (pH 7.4).
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
399
The Dowex-I-formate resin was then washed with 10 ml of distilled water. CAMP was then eluted with 10 ml of 2 N formic acid, and cGMP was eluted with 20 ml of 4 N formic acid. Samples were concentrated and prepared for analysis as described earlier. VI. Cyclic Nucleotide
Assay
(A) Gilman assay. CAMP was measured in a number of samples via the protein kinase binding assay of Gilman (13). 3,5’-Cyclic AMP-dependent protein kinase (from beef heart; Sigma, St. Louis, MO.) was reconstituted at pH 6.5 in 0.0005 M acetate buffer. Aliquots of column fractions (50- 100 ~1) were added to 100 ~1 of 0.1 M sodium acetate/acetic acid buffer (pH 4.0) in tubes in an ice bath. Two picomoles of H3*cAMP in 10 ~1 of the pH 4.0 acetate buffer was added to each tube, and the reaction was initiated by the addition of binding protein (5 $/tube). All tubes were incubated at 0°C for 30 min. Binding protein recovery (Millipore filter) and scintillation counting procedures were carried out as described by Gilman (13). (B) Radioimmunoassay. CAMP and cGMP were measured using the radioimmunoassay kits (Catalog Nos. 201618 and 201812) produced by Schwarz/Mann (Orangeburg, New York, 10962) according to the procedures of Steiner et a/. (14). VII. Cyclic Nucleotide
Verijication
Procedures
(A) Phosphodiesterase treatment. Phosphodiesterase (from beef heart) was purchased from Boehringer Mannheim. The ammonium sulfate suspension was diluted with glycylglycine buffer, 0.1 M pH 7.5, to give 0.01 unit/IO ~1. One-hundred microliter aliquots of column fractions (pH adjusted to 7.5) were mixed with 10 ~1 of MgSO,*7H,O (1 mg/ml) and 10 ~1 of enzyme suspension. Samples were incubated at 25°C for 60 min. Samples were heated for 2 min in a boiling-water bath and were used directly in the Gilman and radioimmunoassay procedures. (B) Serial sample dilution. Selected samples were serially diluted (up to l/100) and 100 pl-fractions were run in the radioimmunoassay for both CAMP and cGMP. RESULTS
Tables 1 and 2 show the results of column blanks. Of the four columns tested, the Dowex 50 H+ eluted with water gave the highest blank values for both cyclic nucleotides. The neutral alumina/Dowex-l-formate combination was the only column to give a zero blank for both cyclic nucleotides. While blank values for the Dowex 50 H+/O. 1 N HCI and the Dowex-I-formate columns were lower than the blank values in the Dowex 50 H+/water system, the material responsible for these values was not hydrolyzed by cyclic nucleotide phosphodiesterase.
400
SCHWARTZEL,
BACHMAN, TABLE
CAMP IN GASTROINTESTINAL
AND LEVINE
I TISSUES AND FLUIDS
Column Dowex 50 H+/ Blank CAMP (RIA)” CAMP (G)b
Dowex-1-formate
Water
0.1 N HCl
Without alumina
With alumina
4.6 10.6
1.1 4.5
0.8 4.0
0.0 2.6
9.3 k 3.5 5.9 ‘-’ 2.1 12.7 zt 2.2 105 4 11.0 90 ” 30 1.95’
12.3 4 1.4 8.5 f 1.1 11.4 ” 3.7 84 ” 12.1 105 ” 25 1.75’
16.6 ? 1.7
14.4 t 2.3
Sample Human Gastric juice Basal (RIA) Post-stim. (RIA) (G) Bile (RIA) (G) Pancreatic (RIA) Dog Gastric juice (RIA)
Fluid (pm/ml)
26 2 5.6 21 2 4.3 471 c 68 250 zt 30 1000 -
12.5 k 6.3 ‘19.0 + 145 k 131 f -
0.5 1.5 2.6 15 23
10.6 -r- 2.1
Tissue (pm/g) No column Human Gastric antrum (G) Gastric fundus (G) Dog Gastric antrum Gastric fundus Rat Liver (RIA) ((3 Small intestine
546 zk 52 1750 ” 175 807?106 1450 ‘- 225
534 + 27 650 k 25 550 ” 50
490 2 35 730 ? 30 940 2 21
(RIA) (RIA)
-
-
3%’ -
525 k 53 580 + 33
(RIA)
1040 + 125 425 2 40
510 2 30 -
410 k 40 360 2 30 -
380 2 20 420 2 40 725 2 71
(RIA) (RIA)
597 841 784 876
-c 43 t 56 5 112 -c 34
a (RIA), Radioimmunoassay. b (G), Gilman assay. r One sample only.
Addition of radioimmunoassayable CAMP to human gastric juice resulted in recoveries in the range of 65-80% for the Dowex 50 H+ column when eluted with either water or 0.1 N HCl. Recovery of radioimmunoassayable CAMP from the Dowex-I-formate column alone was in the range of 70-N%. Recovery of radioimmunoassayable cGMP
GASTROINTESTINAL
TABLE cGMP
401
CYCLIC NUCLEOTIDES
IN GASTROINTESTINAL
2 TISSUES
AND
(RIA)
FLUIDS
Column Dowex 50 H+/ Blank cGMP
Water
0.1 N HCI
Without alumina
With alumina
8.8
1.3
0.6
0.0
4.1 ” 0.6 2.8 -c 0.7 9.6 2 I.2 0.95”
2.2 k 0.2 0.6 -c 0.1 6.0 -+ 0.5 1.15”
Fluid (pm/ml)
Sample Human Gastric Juice Basal Post-stimulation Bile Pancreatic
Dowex-1-formate
14.9 2 2.9 15.9 k 2.5 19.1 t 5.8 -
2.1 2 0.3 1.4 -+ 0.4 16.9 2 2.3 -
Tissue (pm/g) No column
Human Gastric antrum Gastric fundus Liver Rat Chicken
17.5 * 3.0 22.6 t 0.5
17.4 ” 4.4 15.7 * 3.1
38.4 ” 6.1 22.0 IL 1.1
27.1 ? 3.5 34.3 k I.5
14.6 r 2.1 18.7 2 0.6
-
17.1 ” 2.4 29.7 2 2.1
13.8 d 6.6 -
0 One sample only.
was 10% lower than CAMP for all four columns, whereas the recovery of both tritiated CAMP and cGMP was lo- 15% higher than that observed for radioimmunoassayable cyclic nucleotides. Values calculated (picomole/ tube) from serially diluted fluid samples showed a random + 10% variation when plotted against the standard CAMP or cGMP curve, only when the fluid sample had been prepared by the neutral alumina/Dowex-1-formate column system. Diluted tissue samples showed a r 10% variation with the standard curves regardless of the column procedure. CAMP levels in gastric juice and bile were highest when measured by the Gilman assay after Dowex 50 H+/water chromatography (Table 1). Tissue samples, when measured directly without columns, also gave high values. CAMP levels measured by the Gilman method after Dowex 50 H+/O. 1 N HCI or Dowex-I-formate formic acid chromatography were more like the values obtained using radioimmunoassay. However, only samples prepared with the alumina/Dowex- I-formate combination gave values that were the same by either the Gilman or RIA procedures.
402
SCHWARTZEL,
BACHMAN,
AND
LEVINE
While the RIA procedure for CAMP was less influenced than the Gilman assay by interfering compounds in gastrointestinal fluids prepared by Dowex 50 H+/water chromatography, measured values were still 3-5 times higher than those obtained by alumina/Dowex-1-formate chromatography. Gastric juice gave similar values for RIA when prepared by Dowex 50 H+/HCl or either of the Dowex- 1-formate procedures. Human bile, unlike gastric juice, gave decreasing values as samples were prepared, respectively, by the Dowex 50 H+/water, Dowex 50 H+/HCl, and both Dowex-1-formate column procedures (Table 1). Only fluid samples prepared by the alumina/Dowex-1-formate procedure gave values as low as column blanks when treated with phosphodiesterase or gave proportional amounts per tube after serial sample dilution. Tissue samples gave consistent results regardless of the method of column preparation. Samples assayed without prior chromatography with phosphodiesterase gave values higher than any tissue sample prepared by any of the three column procedures employed. Tissue samples prepared by any of the previously mentioned column procedures gave values after phosphodiesterase treatment equal to column blanks. Serial dilution of tissue samples prepared by any of the three column procedures confirmed the measured material as CAMP. cGMP levels (Table 2) in gastrointestinal fluids were at least 6 times less than corresponding CAMP levels. Fluid samples prepared by Dowex 50 H+/water chromatography gave the highest levels of cGMP, but less than half of this material was hydrolyzed by phosphodiesterase. cGMP levels in tissues were at least 20 times lower than the corresponding CAMP levels. Individual tissue samples gave identical to slightly higher values of cGMP, depending on the chromatographic procedure employed (Table 2). Treatment of samples with phosphodiesterase prepared by any of the other column procedures gave values similar to column blanks when measured by the RIA procedure. cGMP in gastrointestinal tissues and fluids showed chromatographic separation and serial sample dilution patterns similar to that seen for CAMP. Table 3 presents a summary CAMP and cGMP values for representative gastrointestinal tissues and fluids determined in our laboratory using the neutral alumina/Dowex-1-formate columns and the RIA procedure. Note the higher CAMP compared to cGMP levels for all samples. DISCUSSION
The alumina/Dowex- 1-formate chromatographic combination is the system of choice for the preparation of gastrointestinal fluids for either CAMP or cGMP determinations. Of the other three columns tested, the Dowex 50 H+/O. 1 N HCl and the Dowex-1-formatelformic acid columns are suitable for fluids such as bile where the cyclic nucleotide concentration is relatively high. These columns are not suited for either gastric or
GASTROINTESTINAL
TABLE MEAN
GASTROINTESTINAL
403
CYCLIC NUCLEOTIDES
CYCLIC
3 NUCLEOTIDE
Source
CAMP
Fluid (pm/ml) Human serum Human gastric juice Canine gastric juice Human pancreatic juice Human bile Baboon bile Canine bile Rat bile
5 12 14 2 125 5,833 10.300 100
Tissue (pm/g) Human stomach Canine antrum mucosa Canine fundus mucosa Rat stomach Human jejunum Rat jejunum Human ileum Rat ileum Rat colon Human pancreas Baboon liver Chicken liver Rat liver
847 600 650 600 l.ooo 950 83.5 950 980 959 950 380
’ All samples processed by alumina/Dowex-I-formate
CONCENTFUTION~
cGMP
-
1 2 3 1 6
23 17 20 13 90 45 15 45 17 37 12 8
and measured by RIA.
pancreatic juices since the column blanks are equal to the levels of cGMP found in these fluids. The Dowex 50 H+/O. 1 N HCl column has the added disadvantage of having a relatively long running time and yielding large-volume column fractions. The higher recoveries observed with tritium-labeled, as opposed to radioimmunoassayable, cyclic nucleotides may reflect additional losses from the concentration/recovery step necessary to prepare samples for the Gilman assay or RIA. We also found that tritium-labeled cyclic nucleotides contain varying amounts of unidentified radioactive contaminants or breakdown products which may affect recoveries. We have recovered up to 20% of the counts applied to a Dowex-I-formatelformic acid column in the water-wash fraction prior to the elution of CAMP with 2 N formic acid. We have, therefore, routinely purified all tritiated cyclic nucleotides on Dowex-1-formate columns prior to use, either in samples as an indicator of recovery or, in the case of H3-CAMP, for use in the Gilman assay. The low recovery of cyclic nucleotides, particularly cGMP in the alumina/ Dowex- I-formate system, may be a function of the alumina. White (15) has
404
SCHWARTZEL,
BACHMAN,
AND LEVINE
reported markedly different yields of cyclic nucleotides from alumina obtained from different suppliers. Selection of a neutral or basic alumina on a trial and error basis to give relatively high yields of cGMP is advisable in order to detect changes in concentration in fluids such as gastric and pancreatic juices which have low basal levels of cGMP. The failure of the Gilman assay to yield results in both tissues and fluids comparable to those obtained with the RIA with all samples, except for those prepared by the alumina/Dowex-l-formate system, suggests that an interfering compound(s) is most effectively removed by the latter column combination. It is also possible that some of the problem is due to the preparation of binding protein. J. D. Gardner (personal communication) has reported that binding protein, prepared in his laboratory from beef muscle, is less sensitive to interfering substances than binding protein obtained from commercial sources. The values for CAMP and cGMP in gastrointestinal fluids reported here are lower than those that we (16) have previously reported. It is not surprising that measurement of biological fluids of such diverse composition as gastric juice, bile, and pancreatic juice would involve complex technical problems. Nevertheless, we feel that the values reported here are reliable on the basis of low-column blanks, hydrolysis by phosphodiesterase, and the results of serial sample dilution, as recommended by G. Brooker (personal communication). The time period from isolation to freezing seems to be critical in tissues with the highest rates of metabolic activity, such as cardiac muscle and brain tissue (17). Preliminary acute experiments in our laboratory with in vivo dog stomach muscosa and rat intestinal muscosa showed that cyclic nucleotide levels were unchanged in biopsy specimens that were removed and kept at room temperature for as long as 1 min before freezing with precooled Wollenberger clamps. Comparison of cyclic nucleotide levels in specimens that were frozen with Wollenberger clamps versus direct freezing in liquid nitrogen showed no significant difference. These results are in agreement with the report of Stull and Mayer (18) who found no significant difference in rabbit muscle CAMP when tissue was frozen by either of the above techniques. These observations suggest that cyclic nucleotide levels in gastrointestinal tissues are relatively stable. Our results suggest that, under the usual conditions of gastrointestinal tissue and fluid sampling, reliable estimates of cyclic nucleotide content can be obtained if care is taken to select the most sensitive column procedure and if adequate recovery and verification procedures are employed. ACKNOWLEDGMENTS This research was supported, in part, by Grant AM 17192 USPHS.
GASTROINTESTINAL
CYCLIC
NUCLEOTIDES
405
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15. White, A. A. (1974)in Methods in Enzymology (Hardman, J. G.. and O’Malley. B. W.. eds.) Vol. 38C, pp. 41-46. Academic Press, New York. 16. Levine, R. A., and Schwartzel, E. H., Jr. (1974) Gasfroenferology 66, 730. 17. Mayer, S. E., Stul!, J. T., and Wastila. W. B. (1974) in Methods in Enzymology (Hardman, J. G. and O’MaUey. B. W.. eds.) Vol. 38C, pp. 3-9, Academic Press, New York. 18. Stull. J. T., and Mayer. S. E. (197l)J. Biol. Chem. 246, 5716-5723.