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Comparison of in vivo and in vitro responses to sulfated and non-sulfated ceruletide
European Journal of Pharmacology, 47 (1978) 359--363
359
© Elsevier/North-Holland Biomedical Press
Short communication COMPARISON OF IN VIVO AND IN VITRO RESPONSES TO SULFATED AND NON,SULFATED CERULETIDE JOHN W. FARA * and STEVEN M. ERDE
Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York 11794, U.S.A. Received 15 November 1977, accepted 5 December 1977
J.W. FARA and S.M. ERDE, Comparison of in vivo and in vitro responses to sulfated and non-sulfated ceruletide, European J. Pharmacol. 47 (1978) 359--363. Responses of, guinea pig gall bladder to sulfated and non-sulfated ceruletide were compared in vitro and in vivo. Tested in vivo, sulfated ceruletide was 75 times more potent than non-sulfated. In vitro, sulfated was 150 times more potent. Thus, differences in potency are substantially less when tested in vivo, and reported differences in relative potency reflect not only chemical structure but also the method used for testing. Guinea pig gall bladder
Sulfated ceruletide
Non-sulfated ceruletide
1. Introduction The effects of sulfated versus non-sulfated peptides have been studied on gastrointestinal target organs of numerous species. Of interest has been the importance of sulfation of the tyrosine residue in location 7 (from the C-terminus) in cholecystokinin and ceruletide (= caerulein) and in the 6th position in gastrin. For CCK and ceruletide, the sulfated form is generally more potent (Amer, 1969; Anastasi et al., 1968; Chowdhury et al., 1976; Johnson et al., 1970; Vizi et al., 1974). However, species differences and dissimilar in vivo versus in vitro preparations have contributed to a wide range of reported relative potencies. For example, Johnson et al. (1970) found sulfated octapeptide of cholecystokinin to be about 160 times more potent than the nonsulfated form in contracting the in vivo cat gall bidder. On the other hand, Chowdhury et al. (1976) found a 1000-fold difference with the same peptide in cat gall bladder in vitro. * Address reprint requests to: Dr. J.W. Fara, Department of Radiology, The Johns Hopkins Hospital, Baltimore, Md. 21205, U.S.A.
Thus, the literature suggests that there are differences in the magnitude of relative potencies when determined in vitro versus in vivo, but this has never been studied per se. Therefore, the present study was done to compare in one species the in vivo and in vitro responses of guinea pig gall bladder to sulfated and non-sulfated ceruletide.
2. Materials and methods 2.1. In vivo
Either male or female guinea pigs (approx. 400 g) were fasted for 18 h and then anesthetized with urethane (3 g/kg). A tracheotomy was performed, the animals were placed on a respirator, and they were given 20 mg/kg gallamine triethiodide (Flaxedil®, Davis and Geck). The animals were then maintained at a constant respiration of 30 breaths per min. Peptides and drugs were introduced via a cannula in the external jugular vein. A laparotomy was performed and a suture tied to the apex of the intact gall bladder. The other end was connected to a Grass FT-03 strain gauge
360 transducer to m o n i t o r isometric tension. Recordings were made on a Grass Model 7C polygraph. Body temperature was maintained at 37°C by a heating pad, and reproducible results could be obtained over 2--3 h of testing. To compare responses between the two peptides, they were injected in a randomized sequence in volumes ranging from 10 to 500 pl. Doses of sulfated ceruletide tested were 0.5, 1.0, 4.0, 8.0, 16.0 and 20.0 ng/kg. For non-sulfated they were 50, 100, 400, 800, 1600 and 2000 ng/kg. The range of effective concentrations was studied in 9 guinea pigs. 2.2. I n v i t r o Gall bladders were removed from 18 h fasted guinea pigs which weighed about 400 g and had been anesthetized and treated as described above. The gall bladder was cut into longitudinal strips 10 mm X 3 mm. The strips were suspended in a Krebs solution bath (Chowdhury et al., 1975) between a stationary h o o k and an isometric strain gauge, and tension was recorded as above. The bath was maintained at 37°C and was aerated and stirred with a gas mixture of 95% 02 and 5% CO2. After a half-hour stabilization period, test doses of acetylcholine (1 pg/ml) were added to determine viability of the preparation and to determine L0 for each tissue. Dose--response curves for the muscle strips were obtained by introducing a dose of one hormone and waiting until the maximal response to that dose was reached (usually 5--15 min). The bath was then rinsed three times and 15 min allowed before a new dose was tested. Doses of sulfated ceruletide tested were 0.1, 0.2, 0.8, 4.8, 9.6 and 14.4 ng/ml. For non-sulfated they were 20, 40, 1 6 0 , 3 2 0 , 480 and 640 ng/ml. Peptides were compared in a randomized sequence on 8 tissues, and reproducible results were obtained over 6--8 h of testing. Identical responses could be obtained when similar peptide doses were repeated sequentially two or three times, and there was no evidence of tachyphylaxis when a higher dose
J.W. FARA, S.M. ERDE was given when the response to a low dose became maximal. 2.3. Hormones and drugs Sulfated (905/17) and non-sulfated (MG 1597/82) ceruletide: Pyr-Gln-Asp-Tyr-(SO3H)Thr-Gly-Trp-Met-Asp-Phe-NH2 were the kind gift of Dr. R.A. Carrano, Adria Laboratories, Wilmington, DL. Acetylcholine chloride and atropine sulfate were purchased from Sigma Chemical, St. Louis, MO. The peptides were diluted in normal saline and kept on ice during the experiments. Dosage concentrations are expressed as weight/kg body weight or weight/volume of bath. The dose required to produce half the maximal response (Ds0) and to produce the maximal response was obtained by direct observation and also by Dowd--Riggs linear transformation of Michaelis--Menten constants (Dowd and Riggs, 1965). Plotting the response/dose ratio on the abscissa and the response on the ordinate, the y-intercept gives the calculated maximal response (CMR), and the slope of the curve, with change of sign, gives the Ds0 (Chowdhury et al., 1975, 1976; Dowd and Riggs, 1965). Only the linear portion of the curves was used to calculate CMR and Ds0.
3. Results 3.1. In vitro The effective dose range for sulfated ceruletide was 0.1--10 ng/ml and for non-sulfated ceruletide 20--800 ng/ml. The latency of the response to sulfated ceruletide was the same as that to non-sulfated (30 sec to 1 rain), and this was longer than that to acetylcholine (5--10 sec). Likewise, the time to reach peak tension for a particular h o r m o n e dose was longer for the peptides than for acetylcholine (fig. 1A, B). Additionally, after one or two rinses the tension developed in response to non-sulfated ceruletide rapidly returned to
S U L F A T E D VERSUS NON-SULFATED CERULETIDE
NS
361
S
ACH
5 min
NS
FF
S
Fig. 1. (A) Isometric tension responses of guinea pig gall bladder in vitro to 40 ng/ml non-sulfated ceruletide (NS) and to 0.8 ng/mi sulfated ceruletide (S) introduced into the bath at arrows, (R) represents rinsing of the bath with Krebs, (B) Isometric tension response of guinea pig gall bladder in vitro to 1 pg/ml acetylcholine. (C) Isometric tension responses of in vivo guinea pig gall bladder to 800 ng/kg non-sulfated ceruletide (NS) and to 8.0 ng/kg sulfated ceruletide (S) given i.v. at arrows. (FF) is the response to flushing the non-sulfated ceruletide from the cannula with saline and refilling the cannula with sulfated ceruletide. A small response is observed, indicating that some o f the sulfated ceruletide entered the animal and the cannula was then full.
base line, as did the acetylcholine response. On the contrary, it generally t o o k four to five rinses to restore base line tension after exposure to sulfated ceruletide. Atropine (1 pg/ml) abolished the response to acetylcholine (1 pg/ml) b u t did n o t affect responses to either sulfated or non-sulfated ceruletide. Comparison of respective Ds0's (table 1) indicates that sulfated ceruletide is a b o u t 150 times more p o t e n t than non-sulfated when
compared in vitro. There was no significant difference in the observed or calculated maximal response to sulfated and non-sulfated ceruletide. 3.2. In vivo
The minimally effective dose of sulfated ceruletide was 0.5 ng/kg and the maximal response was obtained at 15 ng/kg. Values for non-sulfated ceruletide were 50 and 1500
TABLE 1 Comparison between the Ds0 and the calculated maximal response (CMR) for sulfated and non-sulfated ceruletide tested in vitro (n = 8) and in vivo (n = 9). Results are mean _+ S.E. Ds0 1
In vitro In vivo
CMR ~
Sulfated
Non-sulfated
Sulfated
Non-sulfated
0.55 +_ 0.09 4.66 _+ 1.14
85.11 _+ 7.67 353.39 + 16.7
2437.5 + 111.3 1385.6 _+ 86.1
2601.3 _+ 94.7 1304.1 + 104.4
1 Concentrations are ng/ml in vitro and ng/kg in vivo. 2 In mg tension.
362
ng/kg, respectively. The latency of the response to sulfated ceruletide was the same as that to non-sulfated (approx. 5--10 sec), b u t because of deleterious cardiovascular effects, gall bladder responses to acetylcholine before and after atropine could n o t be compared with the peptides. In general, it t o o k two to three times longer for the tension response to sulfated ceruletide to return to base line compared with the response to the same dose of non-sulfated (fig. 1C). Comparison o f respective Ds0's (table 1) indicates that sulfated ceruletide is a b o u t 75 times more p o t e n t than non-sulfated when compared in vivo. There was no significant difference in the observed or calculated maximal response to sulfated and non-sulfated ceruletide.
4. Discussion From these results it is apparent that there is a greater difference in p o t e n c y between sulfated and non-sulfated ceruletide when tested in vitro versus in vivo, a finding which possibly accounts for the wide range of potencies reported by individual workers using either one or the other method. The results also suggest then that either something happens to one or both of the peptides after their injection into the blood or that perhaps other circulating hormones interact with gall bladder receptor sites in vivo to minimize the presence or absence of the sulfate group in the peptides activity. Ceruletide was the only peptide available to us in both sulfated and non-sulfated form, b u t it obviously would be of interest to see whether the relationship also holds for sulfated a-o.d non-sulfated gastrin and cholecystokinin which share the same rice C-terminal amino acids and have cholecystokinetic effects (Morley, 1973). Because of this structural similarity, one might predict that the differences found here for ceruletide would hold true for these hormones as well. The n o t unexpected finding that sulfated ceruletide is more p o t e n t than non-sulfated
J.W. FARA, S.M. ERDE
agrees with all earlier ceruletide studies (Amer, 1969; Anastasi et al., 1968; Johnson et al., 1970; Vizi et al., 1974) and emphasizes the importance of sulfation of the seventh position tyrosine residue in determining this peptides potency. It has been postulated that the sulfate interacts in some way with the receptor site to produce stronger binding (Amer, 1969; Morley, 1973), a possibility which is supported in the present study, for in vivo the response to sulfated ceruletide always outlasted that to non-sulfated, and in vitro sulfated ceruletide was harder to wash off (fig. 1). In terms of the response to ceruletide compared to that of acetylcholine, the peptides consistently t o o k a much longer time to reach maximum tension (fig. 1). This may be correlated to differences in molecular weight and hence diffusion of agonist to receptor as proposed previously (Amer, 1969) or it may simply indicate a longer latency in getting contraction initiated following peptide--receptor interaction. Also, ceruletide's effect is n o t via a cholinergic receptor mechanism, for the response to the peptides was n o t blocked by atropine, whereas that to acetylcholine was.
Acknowledgements Supported in part by a research partnership grant from Adria Laboratories, Inc., Wilmington, DL. The authors gratefully acknowledge Dr. Morton I. Grossman for sharing his thoughts on in vivo versus in vitro comparisons with us at the outset of the study, and Ms. Ruth Campbell for her excellent technical assistance.
References Amer, M.S., 1969, Studies with cholecystokinin. II. Cholecystokinetic potency of porcine gastrins I and II and related peptides in three systems, Er~docrinology 84, 1277. Anastasi, A., L. Bernardi, G. Bertaccini, G. Bosisio, R. De Castiglione, V. Erspamer, O. Goffredo and M. Impicciatore, 1968, Synthetic peptides related to caerulein, Experientia 2 4 , 7 7 1 .
SULFATED VERSUS NON-SULFATED CERULETIDE Chowdhury, J.R., J.M. Berkowitz, M. Praissman and J.W. Fara, 1975, Interaction between octapeptidecholecystokinin, gastrin, and secretin on cat gall bladder in vitro, Amer. J. Physiol. 229, 1311. Chowdhury, J.R., J.M. Berkowitz, M. Praissman and J.W. Fara, 1976, Effect of sulfated and nonsulfated gastrin and octapeptide-cholecystokinin on cat gall bladder in vitro, Experientia 32, 1173. Dowd, J.E. and D.S. Riggs, 1965, A comparison of estimates of Michaelis--Menten kinetic constants from various linear transformations, J. Biol. Chem. 240,863.
363 Johnson, L.R., G.F. Stening and M.I. Grossman, 1970, Effect of sulfation on the gastrointestinal actions of caerulein, Gastroenterology 5 8 , 2 0 3 . Morley, J.S., 1973, Frontiers in gastrointestinal hormone research, in: Nobel Symposium XVI (Almquist and Wiksell, Stockholm) p. 143. Vizi, E.S., G. Bertaccini, M. Impicciatore, P. Mantovani, J. Zseli and J. Knoll, 1974, Structure--activity relationship of some analogues of gastrin and cholecystokinin on intestinal smooth muscle of the guinea pig, Naunyn--Schmiedeb. Arch. Pharmacol. 284, 233.
359
© Elsevier/North-Holland Biomedical Press
Short communication COMPARISON OF IN VIVO AND IN VITRO RESPONSES TO SULFATED AND NON,SULFATED CERULETIDE JOHN W. FARA * and STEVEN M. ERDE
Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York 11794, U.S.A. Received 15 November 1977, accepted 5 December 1977
J.W. FARA and S.M. ERDE, Comparison of in vivo and in vitro responses to sulfated and non-sulfated ceruletide, European J. Pharmacol. 47 (1978) 359--363. Responses of, guinea pig gall bladder to sulfated and non-sulfated ceruletide were compared in vitro and in vivo. Tested in vivo, sulfated ceruletide was 75 times more potent than non-sulfated. In vitro, sulfated was 150 times more potent. Thus, differences in potency are substantially less when tested in vivo, and reported differences in relative potency reflect not only chemical structure but also the method used for testing. Guinea pig gall bladder
Sulfated ceruletide
Non-sulfated ceruletide
1. Introduction The effects of sulfated versus non-sulfated peptides have been studied on gastrointestinal target organs of numerous species. Of interest has been the importance of sulfation of the tyrosine residue in location 7 (from the C-terminus) in cholecystokinin and ceruletide (= caerulein) and in the 6th position in gastrin. For CCK and ceruletide, the sulfated form is generally more potent (Amer, 1969; Anastasi et al., 1968; Chowdhury et al., 1976; Johnson et al., 1970; Vizi et al., 1974). However, species differences and dissimilar in vivo versus in vitro preparations have contributed to a wide range of reported relative potencies. For example, Johnson et al. (1970) found sulfated octapeptide of cholecystokinin to be about 160 times more potent than the nonsulfated form in contracting the in vivo cat gall bidder. On the other hand, Chowdhury et al. (1976) found a 1000-fold difference with the same peptide in cat gall bladder in vitro. * Address reprint requests to: Dr. J.W. Fara, Department of Radiology, The Johns Hopkins Hospital, Baltimore, Md. 21205, U.S.A.
Thus, the literature suggests that there are differences in the magnitude of relative potencies when determined in vitro versus in vivo, but this has never been studied per se. Therefore, the present study was done to compare in one species the in vivo and in vitro responses of guinea pig gall bladder to sulfated and non-sulfated ceruletide.
2. Materials and methods 2.1. In vivo
Either male or female guinea pigs (approx. 400 g) were fasted for 18 h and then anesthetized with urethane (3 g/kg). A tracheotomy was performed, the animals were placed on a respirator, and they were given 20 mg/kg gallamine triethiodide (Flaxedil®, Davis and Geck). The animals were then maintained at a constant respiration of 30 breaths per min. Peptides and drugs were introduced via a cannula in the external jugular vein. A laparotomy was performed and a suture tied to the apex of the intact gall bladder. The other end was connected to a Grass FT-03 strain gauge
360 transducer to m o n i t o r isometric tension. Recordings were made on a Grass Model 7C polygraph. Body temperature was maintained at 37°C by a heating pad, and reproducible results could be obtained over 2--3 h of testing. To compare responses between the two peptides, they were injected in a randomized sequence in volumes ranging from 10 to 500 pl. Doses of sulfated ceruletide tested were 0.5, 1.0, 4.0, 8.0, 16.0 and 20.0 ng/kg. For non-sulfated they were 50, 100, 400, 800, 1600 and 2000 ng/kg. The range of effective concentrations was studied in 9 guinea pigs. 2.2. I n v i t r o Gall bladders were removed from 18 h fasted guinea pigs which weighed about 400 g and had been anesthetized and treated as described above. The gall bladder was cut into longitudinal strips 10 mm X 3 mm. The strips were suspended in a Krebs solution bath (Chowdhury et al., 1975) between a stationary h o o k and an isometric strain gauge, and tension was recorded as above. The bath was maintained at 37°C and was aerated and stirred with a gas mixture of 95% 02 and 5% CO2. After a half-hour stabilization period, test doses of acetylcholine (1 pg/ml) were added to determine viability of the preparation and to determine L0 for each tissue. Dose--response curves for the muscle strips were obtained by introducing a dose of one hormone and waiting until the maximal response to that dose was reached (usually 5--15 min). The bath was then rinsed three times and 15 min allowed before a new dose was tested. Doses of sulfated ceruletide tested were 0.1, 0.2, 0.8, 4.8, 9.6 and 14.4 ng/ml. For non-sulfated they were 20, 40, 1 6 0 , 3 2 0 , 480 and 640 ng/ml. Peptides were compared in a randomized sequence on 8 tissues, and reproducible results were obtained over 6--8 h of testing. Identical responses could be obtained when similar peptide doses were repeated sequentially two or three times, and there was no evidence of tachyphylaxis when a higher dose
J.W. FARA, S.M. ERDE was given when the response to a low dose became maximal. 2.3. Hormones and drugs Sulfated (905/17) and non-sulfated (MG 1597/82) ceruletide: Pyr-Gln-Asp-Tyr-(SO3H)Thr-Gly-Trp-Met-Asp-Phe-NH2 were the kind gift of Dr. R.A. Carrano, Adria Laboratories, Wilmington, DL. Acetylcholine chloride and atropine sulfate were purchased from Sigma Chemical, St. Louis, MO. The peptides were diluted in normal saline and kept on ice during the experiments. Dosage concentrations are expressed as weight/kg body weight or weight/volume of bath. The dose required to produce half the maximal response (Ds0) and to produce the maximal response was obtained by direct observation and also by Dowd--Riggs linear transformation of Michaelis--Menten constants (Dowd and Riggs, 1965). Plotting the response/dose ratio on the abscissa and the response on the ordinate, the y-intercept gives the calculated maximal response (CMR), and the slope of the curve, with change of sign, gives the Ds0 (Chowdhury et al., 1975, 1976; Dowd and Riggs, 1965). Only the linear portion of the curves was used to calculate CMR and Ds0.
3. Results 3.1. In vitro The effective dose range for sulfated ceruletide was 0.1--10 ng/ml and for non-sulfated ceruletide 20--800 ng/ml. The latency of the response to sulfated ceruletide was the same as that to non-sulfated (30 sec to 1 rain), and this was longer than that to acetylcholine (5--10 sec). Likewise, the time to reach peak tension for a particular h o r m o n e dose was longer for the peptides than for acetylcholine (fig. 1A, B). Additionally, after one or two rinses the tension developed in response to non-sulfated ceruletide rapidly returned to
S U L F A T E D VERSUS NON-SULFATED CERULETIDE
NS
361
S
ACH
5 min
NS
FF
S
Fig. 1. (A) Isometric tension responses of guinea pig gall bladder in vitro to 40 ng/ml non-sulfated ceruletide (NS) and to 0.8 ng/mi sulfated ceruletide (S) introduced into the bath at arrows, (R) represents rinsing of the bath with Krebs, (B) Isometric tension response of guinea pig gall bladder in vitro to 1 pg/ml acetylcholine. (C) Isometric tension responses of in vivo guinea pig gall bladder to 800 ng/kg non-sulfated ceruletide (NS) and to 8.0 ng/kg sulfated ceruletide (S) given i.v. at arrows. (FF) is the response to flushing the non-sulfated ceruletide from the cannula with saline and refilling the cannula with sulfated ceruletide. A small response is observed, indicating that some o f the sulfated ceruletide entered the animal and the cannula was then full.
base line, as did the acetylcholine response. On the contrary, it generally t o o k four to five rinses to restore base line tension after exposure to sulfated ceruletide. Atropine (1 pg/ml) abolished the response to acetylcholine (1 pg/ml) b u t did n o t affect responses to either sulfated or non-sulfated ceruletide. Comparison of respective Ds0's (table 1) indicates that sulfated ceruletide is a b o u t 150 times more p o t e n t than non-sulfated when
compared in vitro. There was no significant difference in the observed or calculated maximal response to sulfated and non-sulfated ceruletide. 3.2. In vivo
The minimally effective dose of sulfated ceruletide was 0.5 ng/kg and the maximal response was obtained at 15 ng/kg. Values for non-sulfated ceruletide were 50 and 1500
TABLE 1 Comparison between the Ds0 and the calculated maximal response (CMR) for sulfated and non-sulfated ceruletide tested in vitro (n = 8) and in vivo (n = 9). Results are mean _+ S.E. Ds0 1
In vitro In vivo
CMR ~
Sulfated
Non-sulfated
Sulfated
Non-sulfated
0.55 +_ 0.09 4.66 _+ 1.14
85.11 _+ 7.67 353.39 + 16.7
2437.5 + 111.3 1385.6 _+ 86.1
2601.3 _+ 94.7 1304.1 + 104.4
1 Concentrations are ng/ml in vitro and ng/kg in vivo. 2 In mg tension.
362
ng/kg, respectively. The latency of the response to sulfated ceruletide was the same as that to non-sulfated (approx. 5--10 sec), b u t because of deleterious cardiovascular effects, gall bladder responses to acetylcholine before and after atropine could n o t be compared with the peptides. In general, it t o o k two to three times longer for the tension response to sulfated ceruletide to return to base line compared with the response to the same dose of non-sulfated (fig. 1C). Comparison o f respective Ds0's (table 1) indicates that sulfated ceruletide is a b o u t 75 times more p o t e n t than non-sulfated when compared in vivo. There was no significant difference in the observed or calculated maximal response to sulfated and non-sulfated ceruletide.
4. Discussion From these results it is apparent that there is a greater difference in p o t e n c y between sulfated and non-sulfated ceruletide when tested in vitro versus in vivo, a finding which possibly accounts for the wide range of potencies reported by individual workers using either one or the other method. The results also suggest then that either something happens to one or both of the peptides after their injection into the blood or that perhaps other circulating hormones interact with gall bladder receptor sites in vivo to minimize the presence or absence of the sulfate group in the peptides activity. Ceruletide was the only peptide available to us in both sulfated and non-sulfated form, b u t it obviously would be of interest to see whether the relationship also holds for sulfated a-o.d non-sulfated gastrin and cholecystokinin which share the same rice C-terminal amino acids and have cholecystokinetic effects (Morley, 1973). Because of this structural similarity, one might predict that the differences found here for ceruletide would hold true for these hormones as well. The n o t unexpected finding that sulfated ceruletide is more p o t e n t than non-sulfated
J.W. FARA, S.M. ERDE
agrees with all earlier ceruletide studies (Amer, 1969; Anastasi et al., 1968; Johnson et al., 1970; Vizi et al., 1974) and emphasizes the importance of sulfation of the seventh position tyrosine residue in determining this peptides potency. It has been postulated that the sulfate interacts in some way with the receptor site to produce stronger binding (Amer, 1969; Morley, 1973), a possibility which is supported in the present study, for in vivo the response to sulfated ceruletide always outlasted that to non-sulfated, and in vitro sulfated ceruletide was harder to wash off (fig. 1). In terms of the response to ceruletide compared to that of acetylcholine, the peptides consistently t o o k a much longer time to reach maximum tension (fig. 1). This may be correlated to differences in molecular weight and hence diffusion of agonist to receptor as proposed previously (Amer, 1969) or it may simply indicate a longer latency in getting contraction initiated following peptide--receptor interaction. Also, ceruletide's effect is n o t via a cholinergic receptor mechanism, for the response to the peptides was n o t blocked by atropine, whereas that to acetylcholine was.
Acknowledgements Supported in part by a research partnership grant from Adria Laboratories, Inc., Wilmington, DL. The authors gratefully acknowledge Dr. Morton I. Grossman for sharing his thoughts on in vivo versus in vitro comparisons with us at the outset of the study, and Ms. Ruth Campbell for her excellent technical assistance.
References Amer, M.S., 1969, Studies with cholecystokinin. II. Cholecystokinetic potency of porcine gastrins I and II and related peptides in three systems, Er~docrinology 84, 1277. Anastasi, A., L. Bernardi, G. Bertaccini, G. Bosisio, R. De Castiglione, V. Erspamer, O. Goffredo and M. Impicciatore, 1968, Synthetic peptides related to caerulein, Experientia 2 4 , 7 7 1 .
SULFATED VERSUS NON-SULFATED CERULETIDE Chowdhury, J.R., J.M. Berkowitz, M. Praissman and J.W. Fara, 1975, Interaction between octapeptidecholecystokinin, gastrin, and secretin on cat gall bladder in vitro, Amer. J. Physiol. 229, 1311. Chowdhury, J.R., J.M. Berkowitz, M. Praissman and J.W. Fara, 1976, Effect of sulfated and nonsulfated gastrin and octapeptide-cholecystokinin on cat gall bladder in vitro, Experientia 32, 1173. Dowd, J.E. and D.S. Riggs, 1965, A comparison of estimates of Michaelis--Menten kinetic constants from various linear transformations, J. Biol. Chem. 240,863.
363 Johnson, L.R., G.F. Stening and M.I. Grossman, 1970, Effect of sulfation on the gastrointestinal actions of caerulein, Gastroenterology 5 8 , 2 0 3 . Morley, J.S., 1973, Frontiers in gastrointestinal hormone research, in: Nobel Symposium XVI (Almquist and Wiksell, Stockholm) p. 143. Vizi, E.S., G. Bertaccini, M. Impicciatore, P. Mantovani, J. Zseli and J. Knoll, 1974, Structure--activity relationship of some analogues of gastrin and cholecystokinin on intestinal smooth muscle of the guinea pig, Naunyn--Schmiedeb. Arch. Pharmacol. 284, 233.