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Histamine release induced by neurotensin from rat peritoneal mast cells
European Journal of Pharmacology, 76 (1981) 129-136 Elsevier/North-Holland Biomedical Press
129
H I S T A M I N E RELEASE I N D U C E D BY N E U R O T E N S I N F R O M RAT P E R I T O N E A L M A S T CELLS M A S A O K U R O S E and K I Y O M I SAEKI
*
Department of Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Received 10 April 1981, revised MS received 7 August 1981, accepted 24 August 1981
M. K U R O S E and K. SAEKI, Histamine release induced by neurotensin from rat peritoneal mast cells, European J. Pharmacol. 76 ( 1981) 129-136. Neurotensin induced the release of histamine from both purified and non-purified rat peritoneal mast cells by a non-cytotoxic mechanism. It was effective at a concentration as low as 10 - 8 M. The dose-response curve for the neurotensin effect was triphasic: an initial gentle rise, a plateau (2.5X 1 0 - 7 - 2 . 5 X l0 6M) and a second rise. Disodium cromoglycate markedly, but benzalkonium chloride only slightly, inhibited the neurotensin-induced release. The effect of neurotensin was markedly different from that of substance P, bradykinin and compound 4 8 / 8 0 with respect to both the dose-response curve and the sensitivity to benzalkonium chloride. These inducers of histamine release showed somewhat different effects with changes in pH and temperature. Their actions did not require extracellular Ca 2+ . The results indicate that neurotensin is a potent histamine releaser acting directly on mast cells. Its mode of action may be different from those of the other substances tested. Neurotensin
Mast cell
Substance P
Disodium cromoglycate
I. Introduction
Neurotensin (NT) is a tridecapeptide found in the brain (Carraway and Leeman, 1976a) and the digestive tract (Carraway and Leeman, 1976b; Kitabgi et al., 1976; Orci et al., 1976; Polak et al., 1977) of various animals. It has a wide variety of biological activities such as inducing hypotension, increasing vascular permeability, contraction or relaxation of the gut (Carraway and Leeman, 1973), hyperglycemia (Carraway et al., 1973) associated with hyperglucagonemia (Brown and Vale, 1976; Nagai and Frohman, 1976) and hypothermia (Bissette et al., 1976) and stimulation of the atrial muscle (Quirion et al., 1978). It has been shown that the NT-induced hyperglucagonemia (Brown et al., 1976; Nagai and Frohman, 1978) and the increase in vascular permeability (Chahl, 1979) as well as the stimulatory effect of NT on the atrial muscle (Quirion et al., 1980) may be entirely or partially mediated by the histamine released from tissues. Furthermore, ra* To whom all correspondence should be addressed.
Compound 48/80
Histamine release
diolabelling has shown that NT binds to rat peritoneal mast cells (Lazarus et al., 1977). Considering these findings, it seemed of critical importance to determine if NT can elicit a histamine release reaction in mast cells in order to better understand the mechanism of action of this polypeptide. Compound 48/80 is a mixture of linear copolymers synthesized from p-methoxyphenethylmethylamine and formaldehyde. It is a potent inducer of histamine release from mast cells, acting by a non-cytotoxic mechanism (Johnson and Moran, 1969). Substance P and bradykinin also cause histamine release from mast cells (Johnson and Erdrs, 1973). The mode of action of these two peptides as histamine releasers appears to be similar to that of compound 48/80 (Read and Kiefer, 1979). In this study, we found that NT does in fact have a potent histamine-releasing effect on rat peritoneal mast cells. We subsequently compared the characteristics of NT action with those of compound 48/80, substance P and bradykinin action.
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
130
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats aged 9-12 weeks and weighing 300-350g (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were used. 2.2. Chemicals and drugs Neurotensin triacetate, substance P triacetate and bradykinin triacetate were obtained from Protein Research Foundation (Minoh, Japan); compound 4 8 / 8 0 from Burroughs Wellcome and Co. (Tuckahoe, NY); disodium cromoglycate (DSCG) from Fujisawa Pharmaceutical Co. (Osaka, Japan); benzalkonium chloride (BAC, 95% pure powder) and antimycin A from Nakarai Chemicals (Kyoto, Japan); EDTA (disodium salt) from Wako Pure Chemical Industries (Osaka); and bovine serum albumin (fraction V) from Armour Pharmaceutical Co. (Kankakee, IL).
leasers and drugs to be tested were dissolved in PBS to 10 times their final concentrations and 0.1 ml of each was added to the cell suspension. After incubation, each tube was immediately~ transferred to an ice bath and centrifuged at 650 X g for 10 min at 0°C. Both the supernatant and the precipitate were assayed for histamine content. Siliconized glassware was used throughout. Histamine release was determined in duplicate samples. When the rate of histamine release was studied, 4 m l of ice-cold PBS was added to the reaction mixture at various times after the addition of a histamine releaser, and then each tube was transferred immediately to an ice bath. To study the effect of pH, suspending media with various pH values were prepared by using phosphate buffer of different pH. For determining the effect of temperature, cell suspensions were preincubated at various temperatures for 5 min and the histamine release reaction was allowed to proceed for 5 min at the same temperature. 2.4. Isolation of mast cells
2.3. Histamine release from rat peritoneal mast cells In most experiments, phosphate-buffered saline (PBS) of the following composition was used: NaC1 154 mM; KC1 2.7 mM; CaC12 0.9 mM; N a 2 H P O 4 - K H 2 P O 4 buffer pH 7.0, 6.7 mM; glucose 5.55 mM; bovine serum albumin 0.05%. The rat peritoneal cells containing 4-8% mast cells were obtained by peritoneal lavage with icecold PBS not containing bovine serum albumin. Usually the cells collected from 3 - 4 rats were pooled and used for one set of experiments. Unless otherwise indicated, further purification of mast cells was not performed. The cells were washed once with a large volume of ice-cold PBS. Amounts of cell suspension such that each tube contained 1.0-1.5 × l0 s mast cells were put into glass centrifuge tubes. After centrifuging at 350 X g for 5 min at 4°C, the precipitated cells were resuspended in fresh PBS. They were usually preincubated for 5 min at 37°C. The peptide to be tested or compound 48/80 was added to the cell suspension and the incubation was continued for a further 5 min at the same temperature. The final reaction volume was 1.0 ml. The histamine re-
When necessary, mast cells were isolated by centrifuging (110 X g for 20 min at 4°C) the peritoneal cell suspensions on a concentrated solution (37% w / w ) of bovine serum albumin (Saeki, 1964). This procedure yielded 80-95% mast cells. 2.5. Determination of h&tarnine The histamine content of each sample was determined fluorometrically by the method of Shore et al. (1959). After fluorofore formation, 2 M citric acid instead of 3 N HC1 was used as the acidifying agent (Anton and Sayre, 1969). The extraction procedures with organic solvents were omitted as described by Loeffler et al. (1971). None of the histamine releasers and drugs used interfered with the histamine assay to any significant extent. The percentage of histamine release was calculated using the following equation: histamine release (%) -- (histamine content of the supernatant) X 100/[(histamine content of the supernatant)+ (histamine content of the precipitate)]. Results were expressed as the mean of duplicate determinations and corrected for 2-4% spontaneous release. Per-
131 cent inhibition of histamine release was calculated as follows: 96 inhibition= [(% release from the non-treated cells)-(% release from the treated cells)] X 100/(% release from the non-treated cells).
pound 48/80, substance P and bradykinin are also shown in fig. 1. Although compound 4 8 / 8 0 is a mixture of polymers of different size, the molecular weight of active constituents has been estimated to be 1300 (Read and Lenney, 1972). This value was adopted in our study. The lowest effective concentrations of substance P, bradykinin and compound 48/80 were about 10-6, 5 X 10-6 and 4 X 10 -8 M, respectively. The dose-response curves for these 3 histamine releasers had steep slopes and ran nearly parallel with each other. When N T and the other substances were tested on purified mast cells, almost the same degree of histamine release reaction was observed as in the mixed peritoneal cells: N T (10 -6 M), N T ( 1 0 - 4 M ) , substance P ( 1 0 - S M ) and compound 48,/80 (7.7X 1 0 - 7 M ) induced 21.0±3.9, 50.1 ± 2.3, 59.1---2.4 and 77.7-4-1.0% (mean ±S.E.M., n - - 3 ) histamine release from purified mast cells, respectively. Degranulation of the mast cells treated with these substances could be observed
3. Results
3.1. Dose-response relationship for the histaminereleasing effect of peptides and compound 48/80 N T induced histamine release from nonpurified rat peritoneal mast cells. It was effective at a concentration as low as l0 -8 M. The dose-response curve for the N T effect was triphasic: an initial gentle rise, a plateau (2.5 X 10-7-2.5 X 1 0 - 6 M ) and a second rise (fig. 1). N T did not cause as high a degree of histamine release as was induced by the other releasers tested; the histamine released by 10-4 M N T was only about 40% of the total. The dose-response curves for the effect of com-
% HISTAMINE RELEASE 8 O"
,~1~-
/ 60-
,
~/
-8
-7
-6
-
--4
CONCENTRATION (IogM)
Fig. 1. Dose-responsecurves for histamine release from rat peritoneal mast cells induced by NT (©), substance P (Q), bradykinin(A) and compound48/80 (A). Each point is the mean of 3 or 4 experimentson different pools of cells and the verticalbar represents the S.E.M.
132
under the phase-contrast microscope (Nikon S-type microscope, × 600).
% HISTAMINE RELEASE 50"
3.2. Time course of histamine release 40-
The histamine release induced by NT (10 _6 and 10-4M), substance P (10-SM) and compound 48/80 (1/~g/ml) was completed within 30 sec after the addition of these substances.
30- / / 20-
3.3. Effect of temperature and p H The temperature optimum for NT-induced histamine release was in the range of 30-37°C, whereas the temperature optima for the substance P- and the compound 48/80-induced release were around 30 and 37°C, respectively (fig. 2). These reactions were almost completely abolished at l0 and 45°C. The NT-induced histamine release had a broad range of optimal pH peaking around 6.6. The substance P-induced release had a sharp peak around pH 6.6. Compound 48/80 showed its maximal effect around pH 7.4 (fig. 3). The effects of substance P and compound 48/80 were almost completely suppressed at pH 5.5, whereas NT still
p/
,/
..e
/ ~7
0
o
7'.o
o--O-
[]
715
a'.o
pH Fig. 3. Effect of pH on the histamine release induced by NT, substance P and compound 4 8 / 8 0 from rat peritoneal mast cells. The concentrations of releasers and the symbols used are the same as in fig. 2. The values have not been corrected for spontaneous release. Results from a single experiment.
induced a considerable degree of histamine release at the same pH. 3.4. Effect of antimycin A In glucose-free medium, antimycin A (10 -6 M), an inhibitor of electron transport, completely blocked the histamine-releasing effect of NT, substance P and compound 48,/80 (fig. 4). Glucose (5.55 mM) partially counteracted the inhibitory effect of antimycin A on the NT-, substance Pand compound 48/80 (0.1 #g/ml)-induced release. Histamine release with high concentrations of compound 48/80, though inhibited by antimycin A without glucose, was hardly inhibited by antimycin A in the presence of glucose. In the absence of antimycin A, glucose (5.55 mM) slightly inhibited the effect of NT.
% HISTAMINE RELEASE
1 20-
0 --
, I0
20
30 40 TEMPERATURE
5'0 (*C)
Fig. 2. Effect of temperature on the histamine release induced by NT, substance P and compound 4 8 / 8 0 from rat peritoneal mast cells. The symbols used are as follows: NT, 10 4 M (O) and 10 - 6 M (0); substance P, 10 -5 M (A); compound 48/80, 0.25 # g / m l (A); buffer alone (D). The values in the presence of histamine releasers have not been corrected for spontaneous release. Results from a single experiment.
3.5. Effect of Ca 2+ in the medium The histamine release induced by 10 - 6 M NT, 10-SM substance P or 0.1 #g/ml compound 48/80 in Ca2+-containing medium was significantly less marked than in Ca 2+ -free medium (table 1). However, the degree of the histamine re-
133 TABLE I Effect of Ca 2+ on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were washed twice with a large volume of ice-cold Ca2+-free PBS (PBS without added Ca 2÷) which contained no or 0.5 mM EDTA. They were resuspended in the same fresh media as used for washing and preincubated at 37°C for 15 min before the addition of each releaser. Ca 2+ was added I min before the addition of each releaser to give a final concentration of 0.9 mM. All values are the means ±S.E.M. of 4 experiments on different pools of cells. Inducers of histamine release (concentration)
Medium
N T (10 -6 M)
N T (10 --4 M)
Substance P (10 -5 M)
Compound 48/80 (0. I # g / m l )
Compound 48/80 (I # g / m l )
EDTA
Ca 2+
0 0.5 0 0 0.5 0 0 0.5 0 0 0.5 0 0 0.5 0
0 0 0.9 mM 0 0 0.9 mM 0 0 0.9 mM 0 0 0.9mM 0 0 0.9mM
mM
mM
mM
mM
mM
% Histamine release
% Inhibition
16.7±0.7 15.2±1.1 11.8±2.0 46.7±4.9 46.7± 1.5 36.1 ± 1.9 50.4 -+ 5.4 47.7±3.4 39.6±6.1 37.0± 8.4 38.3--+6.0 29.2-+8.8 60.2 -4-5.2 57.5±4.4 63.7_+7.1
8.9± 6.1 30.2± 9.5 l -3.0_+ 10.1 20.2_+ 8.8 4.1± 4.1 21.7± 6.6 i - 10.0-+ 10.6 25.9± 8.1 I 4.2± 1.4 - - 5 . 1 ± 3.5
i P<0.05 compared with the corresponding values in the Ca2+-free PBS without EDTA.
TABLE 2
TABLE 3
Effect of DSCG on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. DSCG was added to cell suspensions 30 sec before the addition of each releaser. All values are the means ±S.E.M. of 4 experiments on different pools of cells.
Effect of BAC on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were preincubatdd with BAC at 37°C for 10 min before the addition of each releaser. All values are the means ± S.E.M. of 4 experiments on different pools of cells.
Inducers of histamine release (concentration)
DSCG (#M)
%Histamine release
% Inhibition
Inducers of histamine release (concentration)
BAC (#g/ml)
% Histamine release
% Inhibition
NT (10-6 M)
0 10 100 0 10 100 0 10 100 0 10 100 0 10 100
15.7±1.1 0.9±0.8 0.7±0.1 36.3±0.8 7.2±0.7 6.7±0.6 43.5±4.5 10.6-+3.7 8.3±2.2 19.9_+ 5.1 4.2_+ 1.3 4.3±1.1 60.3 _+4.6 51.4-+6.0 49.8±7.4
93.4±5.1 95.8±0.8 80.1±2.0 81.6±1.9 77.4_+6.3 81.7_+3.3 75.2±7.3 77.9±1.6 15.4_+4.9 18.3±7.7
N T ( 1 0 - 6 M)
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
23.2---5.0 19.3---3.7 17.1±4.0 40.6±5.7 31.7±4.3 23.6±4.6 51.4 ± 3.0 26.1±3.6 9 . 8+- 1.8 21.8±5.6 5.2± 1.4 0.9-+0.4 68.8±7.1 61.0-+5.6 38.7±7.3
14.9 + 28.1± 21.4± 41.5± 50.0± 81.1± 75.9± 95.9± 10.9± 45.0±
NT ( 1 0 - 4 M)
Substance P (10-5 M) Compound 48/80 (0.1 # g / m l ) Compound 4 8 / 8 0 (1/~ g / m l )
2 2 NT ( 1 0 - 4 M) 2 2 i 2 i 2
P<0.005, 2 P<0.001 compared with the corresponding control values.
Substance P ( 1 0 - s M) Compound 48/80 (0.1/~g/ml) Compound 48/80 (I p g / m l )
4.3 i 6.5 I 5.91 10.0 J 4.0 4 3.35 2.6 5 2.5 s 2.4 z 6.3 3
I P<0.05, : P<0.02, 3 P<0.01, 4 P<0.005, 5 P<0.001 compared with the corresponding control values.
134
% HISTAMINE RELEASE
t
80T
t t t ~ t
60t f t
t
40-
'"'l
:.::1 :2:1
~t
0
* o
20t
*
0 10-6M
10-4M
I
I
NT
IO-SM I
O.l,~g/ml I
Subsl. P
Ipg/ml l
I
Comp. 48/80
Fig. 4. Effect of antimycin A on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were washed twice with one of the following media: glucose-free PBS without antimycin A (D); glucose-free PBS with 1 #M antimycin A ( n , the second from the left of each group of columns); ordinary PBS without antimycin A (l~); ordinary PBS with 1 #M antimycin A ([]). They were resuspended in the same fresh medium as used for washing and preincubated at 37°C for 5 rain before the addition of each releaser. Results are the means of 4 experiments on different pools of cells and vertical bars represent the S.E.M. Significantly different from the corresponding values in glucose-free PBS without antimycin A by paired t-test: * P<0.05, • * P<0.01, *** P<0.005, **** P<0.001. Significantly different from the corresponding values in glucose-free PBS with I # M antimycin A: t P<0.05, tt P<0.02, tit P<0.01, t t t t P<0.001. Significantly different from the corresponding values in ordinary PBS without antimycin A: ~ P<0.05, O ~ P<0.01.
lease induced by 10-4M NT or 1/~g/ml compound 48/80 was not significantly affected by the presence of Ca 2+. In CaZ+-free medium, EDTA (0.5 raM) had no significant effect on the release reaction induced by any of the substances tested.
3.6. Effect of disodium cromoglycate (DSCG) The histamine release reactions induced by NT and substance P were markedly inhibited by DSCG, an antiallergic drug (table 2). DSCG also had a marked inhibitory effect on the release caused by 0.1 /~g/ml compound 48/80, but was almost ineffective when a higher concentration (1 #g/ml) of this histamine releaser was used.
3. 7. Effect of benzalkonium chloride (BA C) BAC is an inhibitor of the histamine release caused by releasers such as compound 48/80 (Read and Kiefer, 1979). The histamine-releasing action of NT (10 -6 and 10-4M) was only slightly or moderately inhibited by BAC (table 3). In concentrations higher than 5/~g/ml, BAC itself caused histamine release. On the other hand, BAC produced a marked dose-dependent inhibition of the histamine-releasing effects of substance P and low concentrations (0.1 #g/ml) of compound 48/80, and also of bradykinin (10 -5 and 2.5 × 10 -5 M, data not shown). However, the releasing reaction induced by high concentrations of compound 48/80 was less sensitive to the BAC effect.
135
4. Discussion In the present experiments, it was shown that N T induces the release of histamine from both purified and non-purified peritoneal mast cells of the rat and that this release is associated with the degranulation of the mast cells. The release reaction is dependent on temperature and pH of the medium and requires an energy supply by the metabolic processes of the cells as evidenced by the effect of antimycin A. These facts indicate that N T acts directly on rat mast cells and causes the secretion of histamine by a non-cytotoxic mechanism. N T is effective at a concentration as low as 10 -8 M. The high potency of N T is comparable to that of compound 4 8 / 8 0 which is one of the most potent histamine liberators. Hence, N T may be the most potent of the known substances with histamine-releasing activity which are contained in mammalian tissues. N T can elicit histamine release at far lower concentrations than substance P or bradykinin. Interestingly, N T is more potent than these 2 polypeptides in increasing vascular permeability (Chahl, 1979). The present results are consistent with the view that histamine alone mediates the vascular permeability increase induced by N T (Chahl, 1979). Probably, histamine also mediates to at least some degree the vasodilatation and hypotension induced by NT. The histamine-containing cells in the gut and pancreas are different from typical mast cells such as peritoneal mast cells (Lorenz et al., 1969). It has been shown that these cells are resistant to the histamine-releasing action of compound 48/80 (Mongar and Schild, 1952; Enerb~ick, 1966). Although the hyperglucagonemia-inducing effect of N T has been suggested to be mediated by histamine (Brown et al., 1976; Nagai and Frohman, 1978), the origin of such histamine remains to be determined. The histamine-releasing properties of the substances tested are similar with respect to the following: the release reactions do not depend on extracellular Ca 2÷ , they require an energy supply by the metabolic processes of the cells and are rapidly completed. However, the N T effect is strikingly different from that of the other histamine releasers
both as to shape of the dose-response curve and sensitivity to BAC. NT, substance P and compound 4 8 / 8 0 show somewhat different effects with changes in temperature and pH. These results suggest that there are considerable differences in the mode of action of these histamine releasers. The histamine-releasing action of high concentrations of compound 48/80 is less sensitive to the inhibitory effect of BAC than are low concentrations of this compound. This is compatible with the findings of Read and Kiefer (1979) that BAC is a competitive inhibitor of compound 48/80. In the present experiments, the degree of inhibition by DSCG of the compound 48/80'induced release varied markedly with changes in the concentration of compound 48/80. Similar results have also been reported by Orr et al. (1971) and Ennis et al. (1980). Since extracellular Ca 2+ is not required for the compound 48/80-induced histamine release from rat peritoneal mast cells, Ennis et al. (1980) have questioned the view (Foreman and Garland, 1976; Foreman et al., 1977) that the inhibitory effect of DSCG on the histamine release is based on interference with the calcium-gating mechanism in mast cellsl The opinion of Ennis et al. (1980) may be compatible with the present findings that DSCG markedly inhibited the NTand substance P-induced release reactions which likewise do not require extracellular Ca 2+ .
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136 Carraway, R. and S.E. Leeman, 1973, The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem. 248, 6854. Carraway, R. and S.E. Leeman, 1976a, Radioimmunoassay for neurotensin, a hypothalamic peptide, J. Biol. Chem. 25 I, 7035. Carraway, R. and S.E. Leeman, 1976b, Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem. 25 I, 7045. Chahl, L.A., 1979, The effect of putative peptide neurotransmitters on cutaneous vascular permeability in the rat, Naunyn-Schmiedeb. Arch. Pharmacol. 309, 159. Enerb~ck, L., 1966, Mast cells in rat gastrointestinal mucosa. 3. Reactivity towards compounds 48/80, Acta Pathol. Microbiol. Scand. 66, 313. Ennis, M., G. Atkinson and F.L. Pearce, 1980, Inhibition of histamine release induced by compound 48/80 and peptide 401 in the presence and absence of calcium. Implications for the mode of action of antiallergic compounds, Agents Actions 10, 222. Foreman, J.C. and L.G. Garland, 1976, Cromoglycate and other antiallergic drugs: a possible mechanism of action, Br. Med. J. 1,820. Foreman, J.C., M.B. Hallette and J.L. Mongar, 1977, Site of action of the antiallergic drugs cromoglycate and doxantrazole, Br. J. Pharmacol. 59, 473P. Johnson, A.R. and E.G. ErdiSs, 1973, Release of histamine from mast cells by vasoactive peptides, Proc. Soc. Exp. Biol. Med. 142, 1252. Johnson, A.R. and N.C. Moran, 1969, Selective release of histamine from rat mast cells by compound 48/80 and antigen, Am. J. Physiol. 216, 453. Kitabgi, P., R. Carraway and S.E. Leeman, 1976, Isolation of a tridecapeptide from bovine intestinal tissue and its partial characterization as neurotensin, J. Biol. Chem. 251, 7053. Lazarus, L.H., M.H. Perrin, M.R. Brown and J.E. Rivier, 1977, Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells, Biochem. Biophys. Res. Commun. 76, 1079. Loeffler, L.J., W. Lovenberg and A. Sjoerdsma, 1971, Effects of dibutyryl-3',5'-cyclic adenosine monophosphate, phosphodiesterase inhibitors and prostaglandin E t on compound 48/80-induced histamine release from rat peritoneal mast cells in vitro, Biochem. Pharmacol. 20, 2287. Lorenz, W., A. Schauer, St. Heitland, R. Calvoer and E. Werle,
1969, Biochemical and histochemical studies on the distribution of histamine in the digestive tract of man, dog and other mammals, Naunyn-Schmiedeb. Arch. Pharmakol. 265, 81. Mongar, J.L. and H.O. Schild, 1952, A comparison of the effects of anaphylactic shock and of chemical histamine releasers, J. Physiol. (London) 118, 461. Nagai, K. and L.A. Frohman, 1976, Hyperglycemia and hyperglucagonemia following neurotensin administration, Life Sci. 19, 273. Nagai, K. and L.A. Frohman, 1978, Neurotensin hyperglycemia: evidence for histamine mediation and the assessment of a possible physiologic role, Diabetes 27, 577. Orci, L., O. Beatens, C. Rufener, M. Brown, W. Vale and R. Guillemin, 1976, Evidence for immunoreactive neurotensin in dog intestinal mucosa, Life Sci. 19, 559. Orr, T.S.C., D.E. Hall, J.M. Gwilliam and J.S.G. Cox, 1971, The effect of disodium cromoglycate on the release of histamine and degranulation of rat mast cells induced by compound 48/80, Life Sci. 10 (Part I), 805. Polak, J.M., S.N. Sullivan, S.R. Bloom, A.M.J. Buchan, P. Facer, M.R. Brown and A.G.E. Pearse, 1977, Specific localization of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry, Nature (London) 270, 183. Quirion, R., D. Regoli, F. Rioux and S. St-Pierre, 1980, The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea-pig atria, Br. J. Pharmacol. 68, 83. Quirion, R., F. Rioux and D. Regoli, 1978, Chronotropic and inotropic effects of neurotensin on spontaneously beating auricles, Can. J. Physiol. Pharmacol. 56, 671. Read, G.W. and E.F. Kiefer, 1979, Benzalkonium chloride: selective inhibitor of histamine release induced by compound 48/80 and other polyamines, J. Pharmacol. Exp. Ther. 211, 711. Read, G.W. and J.F. Lenney, 1972, Molecular weight studies on the active constituents of compound 48/80, J. Med. Chem. 15, 320. Saeki, K., 1964, Effects of compound 48/80, chymotrypsin and antiserum on isolated mast cells under aerobic and anaerobic conditions, Jap. J. Pharmacol. 14, 375. Shore, P.A., A. Burkhalter and V.H. Cohn, Jr., 1959, A method for the fluorometric assay of histamine in tissues, J. Pharmacol. Exp. Ther. 127, 182.
129
H I S T A M I N E RELEASE I N D U C E D BY N E U R O T E N S I N F R O M RAT P E R I T O N E A L M A S T CELLS M A S A O K U R O S E and K I Y O M I SAEKI
*
Department of Pharmacology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan Received 10 April 1981, revised MS received 7 August 1981, accepted 24 August 1981
M. K U R O S E and K. SAEKI, Histamine release induced by neurotensin from rat peritoneal mast cells, European J. Pharmacol. 76 ( 1981) 129-136. Neurotensin induced the release of histamine from both purified and non-purified rat peritoneal mast cells by a non-cytotoxic mechanism. It was effective at a concentration as low as 10 - 8 M. The dose-response curve for the neurotensin effect was triphasic: an initial gentle rise, a plateau (2.5X 1 0 - 7 - 2 . 5 X l0 6M) and a second rise. Disodium cromoglycate markedly, but benzalkonium chloride only slightly, inhibited the neurotensin-induced release. The effect of neurotensin was markedly different from that of substance P, bradykinin and compound 4 8 / 8 0 with respect to both the dose-response curve and the sensitivity to benzalkonium chloride. These inducers of histamine release showed somewhat different effects with changes in pH and temperature. Their actions did not require extracellular Ca 2+ . The results indicate that neurotensin is a potent histamine releaser acting directly on mast cells. Its mode of action may be different from those of the other substances tested. Neurotensin
Mast cell
Substance P
Disodium cromoglycate
I. Introduction
Neurotensin (NT) is a tridecapeptide found in the brain (Carraway and Leeman, 1976a) and the digestive tract (Carraway and Leeman, 1976b; Kitabgi et al., 1976; Orci et al., 1976; Polak et al., 1977) of various animals. It has a wide variety of biological activities such as inducing hypotension, increasing vascular permeability, contraction or relaxation of the gut (Carraway and Leeman, 1973), hyperglycemia (Carraway et al., 1973) associated with hyperglucagonemia (Brown and Vale, 1976; Nagai and Frohman, 1976) and hypothermia (Bissette et al., 1976) and stimulation of the atrial muscle (Quirion et al., 1978). It has been shown that the NT-induced hyperglucagonemia (Brown et al., 1976; Nagai and Frohman, 1978) and the increase in vascular permeability (Chahl, 1979) as well as the stimulatory effect of NT on the atrial muscle (Quirion et al., 1980) may be entirely or partially mediated by the histamine released from tissues. Furthermore, ra* To whom all correspondence should be addressed.
Compound 48/80
Histamine release
diolabelling has shown that NT binds to rat peritoneal mast cells (Lazarus et al., 1977). Considering these findings, it seemed of critical importance to determine if NT can elicit a histamine release reaction in mast cells in order to better understand the mechanism of action of this polypeptide. Compound 48/80 is a mixture of linear copolymers synthesized from p-methoxyphenethylmethylamine and formaldehyde. It is a potent inducer of histamine release from mast cells, acting by a non-cytotoxic mechanism (Johnson and Moran, 1969). Substance P and bradykinin also cause histamine release from mast cells (Johnson and Erdrs, 1973). The mode of action of these two peptides as histamine releasers appears to be similar to that of compound 48/80 (Read and Kiefer, 1979). In this study, we found that NT does in fact have a potent histamine-releasing effect on rat peritoneal mast cells. We subsequently compared the characteristics of NT action with those of compound 48/80, substance P and bradykinin action.
0014-2999/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
130
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats aged 9-12 weeks and weighing 300-350g (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were used. 2.2. Chemicals and drugs Neurotensin triacetate, substance P triacetate and bradykinin triacetate were obtained from Protein Research Foundation (Minoh, Japan); compound 4 8 / 8 0 from Burroughs Wellcome and Co. (Tuckahoe, NY); disodium cromoglycate (DSCG) from Fujisawa Pharmaceutical Co. (Osaka, Japan); benzalkonium chloride (BAC, 95% pure powder) and antimycin A from Nakarai Chemicals (Kyoto, Japan); EDTA (disodium salt) from Wako Pure Chemical Industries (Osaka); and bovine serum albumin (fraction V) from Armour Pharmaceutical Co. (Kankakee, IL).
leasers and drugs to be tested were dissolved in PBS to 10 times their final concentrations and 0.1 ml of each was added to the cell suspension. After incubation, each tube was immediately~ transferred to an ice bath and centrifuged at 650 X g for 10 min at 0°C. Both the supernatant and the precipitate were assayed for histamine content. Siliconized glassware was used throughout. Histamine release was determined in duplicate samples. When the rate of histamine release was studied, 4 m l of ice-cold PBS was added to the reaction mixture at various times after the addition of a histamine releaser, and then each tube was transferred immediately to an ice bath. To study the effect of pH, suspending media with various pH values were prepared by using phosphate buffer of different pH. For determining the effect of temperature, cell suspensions were preincubated at various temperatures for 5 min and the histamine release reaction was allowed to proceed for 5 min at the same temperature. 2.4. Isolation of mast cells
2.3. Histamine release from rat peritoneal mast cells In most experiments, phosphate-buffered saline (PBS) of the following composition was used: NaC1 154 mM; KC1 2.7 mM; CaC12 0.9 mM; N a 2 H P O 4 - K H 2 P O 4 buffer pH 7.0, 6.7 mM; glucose 5.55 mM; bovine serum albumin 0.05%. The rat peritoneal cells containing 4-8% mast cells were obtained by peritoneal lavage with icecold PBS not containing bovine serum albumin. Usually the cells collected from 3 - 4 rats were pooled and used for one set of experiments. Unless otherwise indicated, further purification of mast cells was not performed. The cells were washed once with a large volume of ice-cold PBS. Amounts of cell suspension such that each tube contained 1.0-1.5 × l0 s mast cells were put into glass centrifuge tubes. After centrifuging at 350 X g for 5 min at 4°C, the precipitated cells were resuspended in fresh PBS. They were usually preincubated for 5 min at 37°C. The peptide to be tested or compound 48/80 was added to the cell suspension and the incubation was continued for a further 5 min at the same temperature. The final reaction volume was 1.0 ml. The histamine re-
When necessary, mast cells were isolated by centrifuging (110 X g for 20 min at 4°C) the peritoneal cell suspensions on a concentrated solution (37% w / w ) of bovine serum albumin (Saeki, 1964). This procedure yielded 80-95% mast cells. 2.5. Determination of h&tarnine The histamine content of each sample was determined fluorometrically by the method of Shore et al. (1959). After fluorofore formation, 2 M citric acid instead of 3 N HC1 was used as the acidifying agent (Anton and Sayre, 1969). The extraction procedures with organic solvents were omitted as described by Loeffler et al. (1971). None of the histamine releasers and drugs used interfered with the histamine assay to any significant extent. The percentage of histamine release was calculated using the following equation: histamine release (%) -- (histamine content of the supernatant) X 100/[(histamine content of the supernatant)+ (histamine content of the precipitate)]. Results were expressed as the mean of duplicate determinations and corrected for 2-4% spontaneous release. Per-
131 cent inhibition of histamine release was calculated as follows: 96 inhibition= [(% release from the non-treated cells)-(% release from the treated cells)] X 100/(% release from the non-treated cells).
pound 48/80, substance P and bradykinin are also shown in fig. 1. Although compound 4 8 / 8 0 is a mixture of polymers of different size, the molecular weight of active constituents has been estimated to be 1300 (Read and Lenney, 1972). This value was adopted in our study. The lowest effective concentrations of substance P, bradykinin and compound 48/80 were about 10-6, 5 X 10-6 and 4 X 10 -8 M, respectively. The dose-response curves for these 3 histamine releasers had steep slopes and ran nearly parallel with each other. When N T and the other substances were tested on purified mast cells, almost the same degree of histamine release reaction was observed as in the mixed peritoneal cells: N T (10 -6 M), N T ( 1 0 - 4 M ) , substance P ( 1 0 - S M ) and compound 48,/80 (7.7X 1 0 - 7 M ) induced 21.0±3.9, 50.1 ± 2.3, 59.1---2.4 and 77.7-4-1.0% (mean ±S.E.M., n - - 3 ) histamine release from purified mast cells, respectively. Degranulation of the mast cells treated with these substances could be observed
3. Results
3.1. Dose-response relationship for the histaminereleasing effect of peptides and compound 48/80 N T induced histamine release from nonpurified rat peritoneal mast cells. It was effective at a concentration as low as l0 -8 M. The dose-response curve for the N T effect was triphasic: an initial gentle rise, a plateau (2.5 X 10-7-2.5 X 1 0 - 6 M ) and a second rise (fig. 1). N T did not cause as high a degree of histamine release as was induced by the other releasers tested; the histamine released by 10-4 M N T was only about 40% of the total. The dose-response curves for the effect of com-
% HISTAMINE RELEASE 8 O"
,~1~-
/ 60-
,
~/
-8
-7
-6
-
--4
CONCENTRATION (IogM)
Fig. 1. Dose-responsecurves for histamine release from rat peritoneal mast cells induced by NT (©), substance P (Q), bradykinin(A) and compound48/80 (A). Each point is the mean of 3 or 4 experimentson different pools of cells and the verticalbar represents the S.E.M.
132
under the phase-contrast microscope (Nikon S-type microscope, × 600).
% HISTAMINE RELEASE 50"
3.2. Time course of histamine release 40-
The histamine release induced by NT (10 _6 and 10-4M), substance P (10-SM) and compound 48/80 (1/~g/ml) was completed within 30 sec after the addition of these substances.
30- / / 20-
3.3. Effect of temperature and p H The temperature optimum for NT-induced histamine release was in the range of 30-37°C, whereas the temperature optima for the substance P- and the compound 48/80-induced release were around 30 and 37°C, respectively (fig. 2). These reactions were almost completely abolished at l0 and 45°C. The NT-induced histamine release had a broad range of optimal pH peaking around 6.6. The substance P-induced release had a sharp peak around pH 6.6. Compound 48/80 showed its maximal effect around pH 7.4 (fig. 3). The effects of substance P and compound 48/80 were almost completely suppressed at pH 5.5, whereas NT still
p/
,/
..e
/ ~7
0
o
7'.o
o--O-
[]
715
a'.o
pH Fig. 3. Effect of pH on the histamine release induced by NT, substance P and compound 4 8 / 8 0 from rat peritoneal mast cells. The concentrations of releasers and the symbols used are the same as in fig. 2. The values have not been corrected for spontaneous release. Results from a single experiment.
induced a considerable degree of histamine release at the same pH. 3.4. Effect of antimycin A In glucose-free medium, antimycin A (10 -6 M), an inhibitor of electron transport, completely blocked the histamine-releasing effect of NT, substance P and compound 48,/80 (fig. 4). Glucose (5.55 mM) partially counteracted the inhibitory effect of antimycin A on the NT-, substance Pand compound 48/80 (0.1 #g/ml)-induced release. Histamine release with high concentrations of compound 48/80, though inhibited by antimycin A without glucose, was hardly inhibited by antimycin A in the presence of glucose. In the absence of antimycin A, glucose (5.55 mM) slightly inhibited the effect of NT.
% HISTAMINE RELEASE
1 20-
0 --
, I0
20
30 40 TEMPERATURE
5'0 (*C)
Fig. 2. Effect of temperature on the histamine release induced by NT, substance P and compound 4 8 / 8 0 from rat peritoneal mast cells. The symbols used are as follows: NT, 10 4 M (O) and 10 - 6 M (0); substance P, 10 -5 M (A); compound 48/80, 0.25 # g / m l (A); buffer alone (D). The values in the presence of histamine releasers have not been corrected for spontaneous release. Results from a single experiment.
3.5. Effect of Ca 2+ in the medium The histamine release induced by 10 - 6 M NT, 10-SM substance P or 0.1 #g/ml compound 48/80 in Ca2+-containing medium was significantly less marked than in Ca 2+ -free medium (table 1). However, the degree of the histamine re-
133 TABLE I Effect of Ca 2+ on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were washed twice with a large volume of ice-cold Ca2+-free PBS (PBS without added Ca 2÷) which contained no or 0.5 mM EDTA. They were resuspended in the same fresh media as used for washing and preincubated at 37°C for 15 min before the addition of each releaser. Ca 2+ was added I min before the addition of each releaser to give a final concentration of 0.9 mM. All values are the means ±S.E.M. of 4 experiments on different pools of cells. Inducers of histamine release (concentration)
Medium
N T (10 -6 M)
N T (10 --4 M)
Substance P (10 -5 M)
Compound 48/80 (0. I # g / m l )
Compound 48/80 (I # g / m l )
EDTA
Ca 2+
0 0.5 0 0 0.5 0 0 0.5 0 0 0.5 0 0 0.5 0
0 0 0.9 mM 0 0 0.9 mM 0 0 0.9 mM 0 0 0.9mM 0 0 0.9mM
mM
mM
mM
mM
mM
% Histamine release
% Inhibition
16.7±0.7 15.2±1.1 11.8±2.0 46.7±4.9 46.7± 1.5 36.1 ± 1.9 50.4 -+ 5.4 47.7±3.4 39.6±6.1 37.0± 8.4 38.3--+6.0 29.2-+8.8 60.2 -4-5.2 57.5±4.4 63.7_+7.1
8.9± 6.1 30.2± 9.5 l -3.0_+ 10.1 20.2_+ 8.8 4.1± 4.1 21.7± 6.6 i - 10.0-+ 10.6 25.9± 8.1 I 4.2± 1.4 - - 5 . 1 ± 3.5
i P<0.05 compared with the corresponding values in the Ca2+-free PBS without EDTA.
TABLE 2
TABLE 3
Effect of DSCG on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. DSCG was added to cell suspensions 30 sec before the addition of each releaser. All values are the means ±S.E.M. of 4 experiments on different pools of cells.
Effect of BAC on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were preincubatdd with BAC at 37°C for 10 min before the addition of each releaser. All values are the means ± S.E.M. of 4 experiments on different pools of cells.
Inducers of histamine release (concentration)
DSCG (#M)
%Histamine release
% Inhibition
Inducers of histamine release (concentration)
BAC (#g/ml)
% Histamine release
% Inhibition
NT (10-6 M)
0 10 100 0 10 100 0 10 100 0 10 100 0 10 100
15.7±1.1 0.9±0.8 0.7±0.1 36.3±0.8 7.2±0.7 6.7±0.6 43.5±4.5 10.6-+3.7 8.3±2.2 19.9_+ 5.1 4.2_+ 1.3 4.3±1.1 60.3 _+4.6 51.4-+6.0 49.8±7.4
93.4±5.1 95.8±0.8 80.1±2.0 81.6±1.9 77.4_+6.3 81.7_+3.3 75.2±7.3 77.9±1.6 15.4_+4.9 18.3±7.7
N T ( 1 0 - 6 M)
0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0 0 2.5 5.0
23.2---5.0 19.3---3.7 17.1±4.0 40.6±5.7 31.7±4.3 23.6±4.6 51.4 ± 3.0 26.1±3.6 9 . 8+- 1.8 21.8±5.6 5.2± 1.4 0.9-+0.4 68.8±7.1 61.0-+5.6 38.7±7.3
14.9 + 28.1± 21.4± 41.5± 50.0± 81.1± 75.9± 95.9± 10.9± 45.0±
NT ( 1 0 - 4 M)
Substance P (10-5 M) Compound 48/80 (0.1 # g / m l ) Compound 4 8 / 8 0 (1/~ g / m l )
2 2 NT ( 1 0 - 4 M) 2 2 i 2 i 2
P<0.005, 2 P<0.001 compared with the corresponding control values.
Substance P ( 1 0 - s M) Compound 48/80 (0.1/~g/ml) Compound 48/80 (I p g / m l )
4.3 i 6.5 I 5.91 10.0 J 4.0 4 3.35 2.6 5 2.5 s 2.4 z 6.3 3
I P<0.05, : P<0.02, 3 P<0.01, 4 P<0.005, 5 P<0.001 compared with the corresponding control values.
134
% HISTAMINE RELEASE
t
80T
t t t ~ t
60t f t
t
40-
'"'l
:.::1 :2:1
~t
0
* o
20t
*
0 10-6M
10-4M
I
I
NT
IO-SM I
O.l,~g/ml I
Subsl. P
Ipg/ml l
I
Comp. 48/80
Fig. 4. Effect of antimycin A on the histamine release induced by NT, substance P and compound 48/80 from rat peritoneal mast cells. The cells were washed twice with one of the following media: glucose-free PBS without antimycin A (D); glucose-free PBS with 1 #M antimycin A ( n , the second from the left of each group of columns); ordinary PBS without antimycin A (l~); ordinary PBS with 1 #M antimycin A ([]). They were resuspended in the same fresh medium as used for washing and preincubated at 37°C for 5 rain before the addition of each releaser. Results are the means of 4 experiments on different pools of cells and vertical bars represent the S.E.M. Significantly different from the corresponding values in glucose-free PBS without antimycin A by paired t-test: * P<0.05, • * P<0.01, *** P<0.005, **** P<0.001. Significantly different from the corresponding values in glucose-free PBS with I # M antimycin A: t P<0.05, tt P<0.02, tit P<0.01, t t t t P<0.001. Significantly different from the corresponding values in ordinary PBS without antimycin A: ~ P<0.05, O ~ P<0.01.
lease induced by 10-4M NT or 1/~g/ml compound 48/80 was not significantly affected by the presence of Ca 2+. In CaZ+-free medium, EDTA (0.5 raM) had no significant effect on the release reaction induced by any of the substances tested.
3.6. Effect of disodium cromoglycate (DSCG) The histamine release reactions induced by NT and substance P were markedly inhibited by DSCG, an antiallergic drug (table 2). DSCG also had a marked inhibitory effect on the release caused by 0.1 /~g/ml compound 48/80, but was almost ineffective when a higher concentration (1 #g/ml) of this histamine releaser was used.
3. 7. Effect of benzalkonium chloride (BA C) BAC is an inhibitor of the histamine release caused by releasers such as compound 48/80 (Read and Kiefer, 1979). The histamine-releasing action of NT (10 -6 and 10-4M) was only slightly or moderately inhibited by BAC (table 3). In concentrations higher than 5/~g/ml, BAC itself caused histamine release. On the other hand, BAC produced a marked dose-dependent inhibition of the histamine-releasing effects of substance P and low concentrations (0.1 #g/ml) of compound 48/80, and also of bradykinin (10 -5 and 2.5 × 10 -5 M, data not shown). However, the releasing reaction induced by high concentrations of compound 48/80 was less sensitive to the BAC effect.
135
4. Discussion In the present experiments, it was shown that N T induces the release of histamine from both purified and non-purified peritoneal mast cells of the rat and that this release is associated with the degranulation of the mast cells. The release reaction is dependent on temperature and pH of the medium and requires an energy supply by the metabolic processes of the cells as evidenced by the effect of antimycin A. These facts indicate that N T acts directly on rat mast cells and causes the secretion of histamine by a non-cytotoxic mechanism. N T is effective at a concentration as low as 10 -8 M. The high potency of N T is comparable to that of compound 4 8 / 8 0 which is one of the most potent histamine liberators. Hence, N T may be the most potent of the known substances with histamine-releasing activity which are contained in mammalian tissues. N T can elicit histamine release at far lower concentrations than substance P or bradykinin. Interestingly, N T is more potent than these 2 polypeptides in increasing vascular permeability (Chahl, 1979). The present results are consistent with the view that histamine alone mediates the vascular permeability increase induced by N T (Chahl, 1979). Probably, histamine also mediates to at least some degree the vasodilatation and hypotension induced by NT. The histamine-containing cells in the gut and pancreas are different from typical mast cells such as peritoneal mast cells (Lorenz et al., 1969). It has been shown that these cells are resistant to the histamine-releasing action of compound 48/80 (Mongar and Schild, 1952; Enerb~ick, 1966). Although the hyperglucagonemia-inducing effect of N T has been suggested to be mediated by histamine (Brown et al., 1976; Nagai and Frohman, 1978), the origin of such histamine remains to be determined. The histamine-releasing properties of the substances tested are similar with respect to the following: the release reactions do not depend on extracellular Ca 2÷ , they require an energy supply by the metabolic processes of the cells and are rapidly completed. However, the N T effect is strikingly different from that of the other histamine releasers
both as to shape of the dose-response curve and sensitivity to BAC. NT, substance P and compound 4 8 / 8 0 show somewhat different effects with changes in temperature and pH. These results suggest that there are considerable differences in the mode of action of these histamine releasers. The histamine-releasing action of high concentrations of compound 48/80 is less sensitive to the inhibitory effect of BAC than are low concentrations of this compound. This is compatible with the findings of Read and Kiefer (1979) that BAC is a competitive inhibitor of compound 48/80. In the present experiments, the degree of inhibition by DSCG of the compound 48/80'induced release varied markedly with changes in the concentration of compound 48/80. Similar results have also been reported by Orr et al. (1971) and Ennis et al. (1980). Since extracellular Ca 2+ is not required for the compound 48/80-induced histamine release from rat peritoneal mast cells, Ennis et al. (1980) have questioned the view (Foreman and Garland, 1976; Foreman et al., 1977) that the inhibitory effect of DSCG on the histamine release is based on interference with the calcium-gating mechanism in mast cellsl The opinion of Ennis et al. (1980) may be compatible with the present findings that DSCG markedly inhibited the NTand substance P-induced release reactions which likewise do not require extracellular Ca 2+ .
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1969, Biochemical and histochemical studies on the distribution of histamine in the digestive tract of man, dog and other mammals, Naunyn-Schmiedeb. Arch. Pharmakol. 265, 81. Mongar, J.L. and H.O. Schild, 1952, A comparison of the effects of anaphylactic shock and of chemical histamine releasers, J. Physiol. (London) 118, 461. Nagai, K. and L.A. Frohman, 1976, Hyperglycemia and hyperglucagonemia following neurotensin administration, Life Sci. 19, 273. Nagai, K. and L.A. Frohman, 1978, Neurotensin hyperglycemia: evidence for histamine mediation and the assessment of a possible physiologic role, Diabetes 27, 577. Orci, L., O. Beatens, C. Rufener, M. Brown, W. Vale and R. Guillemin, 1976, Evidence for immunoreactive neurotensin in dog intestinal mucosa, Life Sci. 19, 559. Orr, T.S.C., D.E. Hall, J.M. Gwilliam and J.S.G. Cox, 1971, The effect of disodium cromoglycate on the release of histamine and degranulation of rat mast cells induced by compound 48/80, Life Sci. 10 (Part I), 805. Polak, J.M., S.N. Sullivan, S.R. Bloom, A.M.J. Buchan, P. Facer, M.R. Brown and A.G.E. Pearse, 1977, Specific localization of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry, Nature (London) 270, 183. Quirion, R., D. Regoli, F. Rioux and S. St-Pierre, 1980, The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea-pig atria, Br. J. Pharmacol. 68, 83. Quirion, R., F. Rioux and D. Regoli, 1978, Chronotropic and inotropic effects of neurotensin on spontaneously beating auricles, Can. J. Physiol. Pharmacol. 56, 671. Read, G.W. and E.F. Kiefer, 1979, Benzalkonium chloride: selective inhibitor of histamine release induced by compound 48/80 and other polyamines, J. Pharmacol. Exp. Ther. 211, 711. Read, G.W. and J.F. Lenney, 1972, Molecular weight studies on the active constituents of compound 48/80, J. Med. Chem. 15, 320. Saeki, K., 1964, Effects of compound 48/80, chymotrypsin and antiserum on isolated mast cells under aerobic and anaerobic conditions, Jap. J. Pharmacol. 14, 375. Shore, P.A., A. Burkhalter and V.H. Cohn, Jr., 1959, A method for the fluorometric assay of histamine in tissues, J. Pharmacol. Exp. Ther. 127, 182.