species specificity of cholecystokinin in gut and brain of several mammalian species

Species Specificity of Cholecystokinin in Gut and Brain of Several Mammalian SpeciesAuthor(s): Eugene Straus and Rosalyn S. YalowSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 75, No. 1 (Jan., 1978), pp. 486-489Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/67667 .

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Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 486-489, January 1978 Medical Sciences

Species specificity of cholecystokin mammalian species

(neuropeptides/gastrointestinal peptides/radioimmunoassay/ani

EUGENE STRAUS AND ROSALYN S. YALOW

Veterans Administration Hospital, Bronx, New York 10468; and The Mount Sinai

Contributed by Rosalyn S. Yalow, November 7, 1977

ABSTRACT Immunoreactive intact cholecystokinin and its COOH-terminal octapeptide are found in brain as well as in extracts of gut of the monkey, dog, and pig, by using an antiserum with equivalent sensitivities for detecting the octapeptide in free form or incorporated in the intact molecule. The failure to detect intact cholecystokinin in extracts from monkey or dog by using an antiserum developed by immunization with porcine cholecystokinin is presumed to be due to marked species differences in the N112-terminal portion of the molecule. Tryptic digestion converted the intact cholecystokinin from all species to a peptide resembling the COOH-terminal octapeptide. The amount of cholecystokinin in the brain is comparable to that found in the gastrointestinal tract, the traditional site for this peptide.

We have previously reported that a peptide resembling intact

cholecystokinin (CCK) in size, charge, and immunologic specificity is found in extracts of the pig cerebral cortex (1). A

peptide, first suggested to be gastrin-like (2) and later consid ered to resemble more closely the COOH-terminal octapeptide of CCK (CCK-8) (1, 3, 4), has been found in the brains of many animal species including not only mammals but also birds, fish, and amphibians (2). In this report we demonstrate that a CCK-like peptide is found in the brain as well as in the gut of the dog and monkey and that failure to detect it with an antiserum developed by immunization of a goat with porcine CCK

(pCCK) is due to the marked species specificity of this antiserum which appears not to crossreact with CCK-8.

MATERIALS AND METIIODS

Gastrointestinal Peptides. The synthetic human heptadecapeptide gastrin was a gift from J. Morley of ICI Ltd., Mac- clesfield, England; the heptadecapeptide porcine gastrin was a gift from R. Gregory through the courtesy of M. Grossman; and the sulfated synthetic CCK-8 was a gift from Squibb Re- search Institute, NJ, through the courtesy of S. J. Lucania. CCK was purchased from the Gastrointestinal Hormone Research Unit, Karolinska Institute, Stockholm, Sweden, and the COOH-terminal gastrin tetrapeptide amide was purchased from Research Plus Laboratories, Denville, NJ.

Radioimmunoassay. 125I-Labeled synthetic human gastrin I and 125I-labeled CCK were prepared by our minor modifi- cation of the chloramine-T technique (5) using about 1.2 mCi of 125I (Amersham/Searle) for 1 Mg of gastrin and 0.5 mCi of 1251 for each 1 Mg of CCK. Labeled gastrin was purified by starch gel electrophoresis and labeled CCK by adsorption to and elution from Quso G32 (5).

The production of the antisera used in this study has been described (1). One antiserum was prepared in a goat (goat 1)

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. ?1734 solely to indicate this fact.

48

in in gut and brain of several

ibody specificity/tryptic conversion)

Ichool of Medicine, City University of New York, New York, New York 1,0029

by immunization with pCCK. This antiserum does not crossreact with CCK-8 or heptadecapeptide gastrins at concentrations 1000-fold higher than the concentration of intact pCCK that decreases the ratio of antibody-bound to free labeled an- tigen to less than 10% of the ratio in the absence of unlabeled hormone. The other antiserum was prepared by immunization of a rabbit (rabbit B) with the COOH-terminal gastrin tetrapeptide amide. The antiserum used in the present study was obtained somewhat later in the course of immunization although fronm the same rabbit as reported previously (1). By using the later bleeding and 125I-labeled synthetic human gastrin as the tracer, the crossreactivities of pCCK and of CCK-8 were virtually identical on a molar basis. Heptadecapeptide gastrins reacted somewhat more strongly than the CCK peptides. Therefore, the gastrin content of all samples was also determined by using the guinea pig antiserum generally used in our gastrin radioimmunoassay, with which the CCK peptides react very poorly (6). No gastrin was detected in the brain of any of the species studied, and the gastrin content of the proximal small bowel was so low in each of the animals that it did not contribute to the CCK-8 determinations reported herein.

The incubation volume for radioimmunoassay was 2.5 ml. The standard diluent was generally 0.02 M barbital (pH 8.6) containing either 0.2 g of bovine serumn albumin per 100 ml or 2% (wt/vol) fetal bovine serum. The concentration of the labeled gastrin tracer and the labeled CCK tracer was generally <0.5 pg/ml and <10 pg/ml, respectively.

Radioimmunoassays of extracts and Sephadex fractions were performed by using methods similar to those established in our laboratory for other hormones (5, 6).

Brain and Gastrointestinal Tissue Extracts. Immediately after sacrifice of the animals, specimens for extraction were taken from the cerebral cortex and the proximal small bowel of one rhesus monkey, two dogs, and two pigs. These tissues were immediately frozen on dry ice and sectioned while still frozen; 0.1 M HCl was added to prodtuce a concentration of 0.1 g of wet tissue per ml. The tissue samples were boiled for 3 mnin and then homogenized, in their extraction solution, with a Teflon tissue grinder. The extracts from brain and gut were assayed directly with each antiserum before and after digestion with trypsin. The optimal conditions for trypsinization were determined by prior experiments on the tryptic conversion of 125I-labeled pCCK (125I-pCCK) to 125I-labeled CCK-8 (125- CCK-8). The extracts were neiutralized, buffered with 0.2 M phosphate buffer (pH 7.5) fortified with trypsin at a concentration of 10 mg/ml, and placed in a water bath at 37? for 20 hr. For some studies, a lower concentration of trypsin (1 mg/ml) and a shorter period of time (20 min) were used. At the end of

Abbreviations: GCCK, cholecystokinin; CCK-8, COOI-terminal cholecystokinin octapeptide; pCCK, porcine CCK; l25I-pCCK, 2I-- labeled pCCK; 125-CCK~8, 125-labeled CCK-8.

3



Medical Sciences: Straus and Yalow

Table 1. Immunoreactive hormon

Goat 1 assay, p,g pCCK equivalent,

Species* Organ Before trypsin Al

Pig (2) Brain 0.80 ? 0.05 Gut 1.80 0.1

Monkey (1) Brain ND Gut ND

Dog (2) Brain ND Gut ND

Data shown as mean + SEM for multiple assays. ND = not detected. * Number in parentheses = number of animals.

the reaction time, tryptic activity was destroyed by boiling for 3 min. The extracts before and after digestion were applied to

Sephadex G-50 columns. The columns were calibrated by using radioactive markers to locate the positions of the void volume and the salt peak and 25I-pCCK to mark thie region of authentic

pCCK. These methods were similar to those described from our

laboratory for the gastrin peptides (7, 8).

RESULTS

The immunoreactive contents of each of the extracts before and after tryptic digestion as determined with each antiserum are

given in Table 1. Purified pCCK was used as standard in the

assay with the goat anti-pCCK serum; synthetic CCK-8 was used as standard in the assay with rabbit B antiserum. No im-

munoreactivity was detectable in the brain or gut extracts of the dog or monkey in the pCCK assay before or after tryptic digestion. Immunoreactive pCCK averaged 0.8 and 1.8 Ag/g wet weight of tissue in the pig brain and gut extracts, respectively, before tryptic digestion and was not measurable after

digestion. Immunoreactive pCCK in the brain and gut extracts eluted in the same region as did authentic pCCK (Fig. 1).

In all animals studied, the immunoreactive content as mea- sured in the CCK-8 assay was about 5-fold greater in the gut extracts than in the brain extracts (Table 1). The former ranged from 0.40 to 0.70 ,g/g and the latter from 0.05 to 0.20 ,tg/g of wet tissue. The concentrations in the gut and brain extracts were

comparable among the different species and did not change significantly on tryptic digestion.

The conditions for tryptic conversion were determined by prior experimentation with highly purified pCCK. After treatment with 1 mg of trypsin per ml for 20 min at 37?, the intact pCCK was partially converted to two smaller forms, one

eluting in the CCK-8 region; the other, with a smaller elution volume, may'be the dodecapeptide (Fig. 2 left middle) described earlier by Jorpes anid Mutt (9) as a product of digestion of pCCK. After more prolonged digestion, with 10 mg of

trypsin per ml, there was complete conversion to CCK-8 (Fig. 2 left bottom).

Sephadex gel filtration of an extract of the monkey cerebral cortex revealed two immunoreactive peaks, one with the same elution volume as purified pCCK and the other corresponding to CCK-8 (Fig. 2 right top). Intact CCK appeared to be con-

verted by partial tryptie digestion first to an intermediate form. After more prolonged digestion there was complete conversion to CCK-8 with no change in immunoreactivity (Table 1; Fig. 2 right).

Sephadex gel filtration and assay, in the CCK-8 system, of the brain and gut extracts of the pig and dog generally revealed two peaks of comparable size in the CCK and CCK-8 regions (Fig. 3 left). A minor void volume peak was also generally ob-

Proc. Natl. Acad. Sci. USA 75 (1978) 487

content of brain and gut extracts

Rabbit B assay, /ml pig CCK-8 equivalent/ml

'ter trypsin Before trypsin After trypsin

ND 0.20 ? 0.01 0.15 + 0.01 ND 0.60 ? 0.05 0.50 + 0.03 ND 0.05 ? 0.01 0.05 + 0.02 ND 0.40 + 0.05 0.35 + 0.05 ND 0.10 + 0.01 0.10 + 0.01 ND 0.70 + 0.02 0.70 + 0.05

served. After prolonged tryptic digestion there was no change in immunoreactivity but complete conversion to CCK-8 (Table 1; Fig. 8 right).

DISCUSSION

Our antiserum prepared by immunization with pCCK fails to detect CCK in extracts of the gut or brain in species other than the pig. However, the use of the antiserum described herein, which has approximately the same sensitivity for the detection of CCK-8 whether incorporated in intact CCK or free as the octapeptide, permits the measurement of CCK in extracts of the brain or gut of dog and monkey and presumably of other species as well. When applying radioimmunoassay systems across species lines, one must appreciate that, although phylo- genetic conservation of the molecular structure of various peptides is required for preservation of biologic activity, there may be major configurational alterations that greatly affect immunochemical recognition. The total spectrum of biologic activities of CCK resides in the COOH-terminal octapeptide. In fact, CCK-8 has been estimated to be 10-fold more potent on a molar basis than intact CCK (9). The failure to detect intact CCK in dog and monkey extracts that are shown to have this hormone, by using an antiserum that reacts with the COOH- terminal portion of the molecule, predicts that there are major differences between pig and other animal CCKs in the NH2- terminal portion of the molecule. Because this portion of the

450,- PURIFIED GIH pCCK

< r ACID EXTRACT, PIG GUT

L - 300 - Lu

co - z LLJ

z 150 Li 0 lzl i . ...l -C - -C-

o 0 20 40 60 80 100 PERCENT OF EL. UTION VOLUME BETWEEN

VOID VOLUME AND SALT PEAK

FIG. 1. Immunoreactive pCCK in Sephadex G-50 gel eluates after filtration of purified GIH pCCK (Top), 0.1 N HC1 extract of mucosal tissue from the proximal small bowel of a pig (Middle), and 0.1 N HC1 extract of cerebral cortex from a pig (Bottom). Goat 1 antiserum, prepared by immunization with pCCK and GIH pCCK standards, was used for this assay.



488 Medical Sciences: Straus and Yalow

BEFORE TREA'

120 mg TRYPSIN

LuJ

z t

ne - uO - r_=

0 20 40 60 80 100

PERCENT OF ELUTION VOLUME I

FIG. 2. Immunoreactivity in eluates after Sephadex G-50 gel filtrat

identically on a molar basis with intact pCCK and its COOH-terminal

of immunoreactivity are shown for GIH pCCK (Left) and an extract of th or complete (Bottom) tryptic digestion.

molecule is not directly involved in its biologic action, it is not surprising that the amino acid sequences in this region of the molecule have diverged during the course of evolution. As yet, the amino acid sequences of CCK from other than a porcine source have not been reported.

These studies demonstrate that, like vasoactive intestinal peptide (10), the amount of CCK in the brain is comparable to that found in the gastrointestinal tract. This distribution is different from that of other gastrointestinal hormones such as gastrin (1), secretin, glucagon, and gastric inhibitory poly- peptide (11), none of which is found in the central nervous system. Still to be elucidated is the possible role in the brain of

QL

(x 400- z

400- Ii

8 fM ^G CE c t 400 a.

i 00-

0 20 40 60 80 100 PERCENT OF ELUTION VOLUME B

FIG. 3. Conditions for determining the immunoreactivity in eluates a of immunoreactivity are shown for gut and brain extracts from three i tion.

Proc. Natl. Acad. Sci. USA 75 (1978)

rMENT WITH TRYPSIN

200-

-. , , ____ ___, , ____I_______

/ml, 20 MINUTES, 37?

1501

N/ml, 20 HOURS, 37"

150-

L:s ' ''T ''--' I I ' i g T I I I ...... C- 0 20 40 60 80 100

3ETWEEN VOID VOLUME AND SALT PEAK

ion was determined by using rabbit B antiserum. This antiserum reacts octapeptide (CCK-8). CCK-8 was used as the standard. The patterns

e monkey cerebral cortex (Right) before (Top) and after partial (Middle)

these peptides previously thought to be confined to the gastrointestinal tract.

The void volume CCK-8 immunoreactivity found in Se- phadex eluates suggests a molecular form even larger than the CCK-variant, a peptide containing six additional amino acids attached to the NH2-terminal portion of CCK (12). That this immunoreactivity is due to some hormonal form of CCK and is not artifact is evidenced by its convertibility to CCK-8 on tryptic digestion. Whether this large hormonal form is a pro- hormone or simply some polymerized form of CCK has not been determined. It is of interest that the void volume immunoreactivity is measurable in pig extracts by using the CCK-8

400___

OG GUT;

400- j

4 0 0 j- _ _ _ _ _ _ _ _

REBRAL. CORTEX

300- _C

0, 20 40 60 80 100 'TWEEN VOID VOLUME AND SALT PEAK

'ter Sephadex G-50 gel filtration were as described in Fig. 2. The patterns nimal species before (Left) and after (Right) complete tryptic diges-



Medical Sciences: Straus and Yalow

but not by using the pCCK assay. If this immunoreactivity were due to a precursor "big CCK," one would have to conclude that there are configurational changes in the larger molecule that permit the COOH-terminal fragment to react in the CCK-8 assay but mask the site(s) in the CCK molecule reacting with the NH2-terminal CCK antiserum.

CCK appears to resemble gastrin (13) in that there are two biologically active forms in the tissues of origin. Which, if either, of these hormonal forms is released into the circulation in re- sponse to physiologic or pharmacologic stimuli merit further investigation. Furthermore, the possible role of the CCK-like peptides in the brain is yet to be elucidated.

1. Muller, J. E., Straus, E. & Yalow, R. S. (1977) Proc. Natl. Acad. Sci. USA 74, 3035-3037.

2. Vanderhaeghen, J. J., Signeau, J. C. & Gepts, W. (1975) Nature 257, 604-605.

3. Dockray, G. J. (1976) Nature 264, 568-570. 4. Straus, E., Muller, J. E., Choi, H., Paronetto, F. & Yalow, R. S.

(1977) Proc. Natl. Acad. Sci. USA 74, 3033-3034. 5. Berson, S. A. & Yalow, R. S. (1973) in Methods in Investigative

Proc. Natl. Acad. Sci. USA 75 (1978) 489

and Diagnostic Endocrinology, Part I-General Methodology, eds. Berson, S. A. & Yalow, R. S. (North-Holland Publishing Co., Amsterdam), pp. 84-120.

6. Yalow, R. S. & Berson, S. A. (1973) in Methods in Investigative and Diagnostic Endocrinology, Part Ill-Non-Pituitary Hor- mones, eds. Berson, S. A. & Yalow, R. S. (North-Holland Pub- lishing Co., Amsterdam), pp. 1043-1050.

7. Yalow, R. S. & Berson, S. A. (1973) in Methods in Investigative and Diagnostic Endocrinology, Part I-General Methodology, eds. Berson, S. A. & Yalow, R. S. (North-Holland Publishing Co., Amsterdam), pp. 155-167.

8. Yalow, R. S. & Berson, S. A. (1971) Gastroenterology 60, 203- 214.

9. Jorpes, J. E. & Mutt, V. (1973) in Methods in Investigative and Diagnostic Endocrinology, Part III-Non-Pituitary Hormones, eds. Berson, S. A. & Yalow, R. S. (North-Holland Publishing Co. Amsterdam), pp. 1075-1080.

10. Bryant, M. G., Polak, J. M., Modlin, I., Bloom, S. R., Albuquerque, R. H. & Pearse, A. G. E. (1976) Lancet i, 991-993.

11. Bloom, S. R., Bryant, M. G. & Polak, J. M. (1975) Gut 16, 821. 12. Mutt, V. & Jorpes, J. E. (1968) Eur. J. Biochem. 6, 156-162. 13. Berson, S. A. & Yalow, R. S. (1971) Gastroenterology 60, 215-

222.



species specificity of cholecystokinin in gut and brain of several mammalian species

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