- Email: [email protected]
Gizzerosine Raises the Intracellular Cyclic Adenosine-3′,5′-Monophosphate Level in Isolated Chicken Proventriculus
Gizzerosine Raises the Intracellular Cyclic Adenosine-3',5'-Monophosphate Level in Isolated Chicken Proventriculus YOSHINORIITO, HIDETO TERAO, TADASHI NOGUCHI,1 and HIROSHI NAITO Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT Gizzerosine, which was originally found in fish meal, is a compound that causes gizzard erosion and ulceration in chicks. The action of gizzerosine on the isolated cells of chicken proventriculus was studied in the present investigations. Gizzerosine increased intracellular cyclic adenosine-3',5'-monophosphate (cAMP) levels, reaching a plateau within 30 min. Similar maxima of cAMP level were observed in the presence of histamine or gizzerosine. However, the potency of gizzerosine was approximately 1,000-fold higher than that of histamine. The action of gizzerosine was depressed by cimetidine, a histamine H2-receptor antagonist, but not by pyrilamine, a histamine H,-receptor antagonist, indicating that gizzerosine is a very strong histamine H2-receptor agonist. The mucosal cells isolated from the proventriculus by the present procedure had both histamine H, and H2-receptors. Gizzerosine showed a higher affinity to the cell surface histamine receptor than histamine. These results partly explain the potent activity of gizzerosine in inducing gastric acid secretion and causing gizzard erosion and ulceration in chickens. {Key words: histamine, gizzerosine, cyclic adenosine-3',5'-monophosphate, gastric mucosal cells, gizzard erosion) 1988 Poultry Science 67:1290-1294 INTRODUCTION
Gizzerosine [2-amino-9-(4-imidazolyl)-7azanonanoic acid] is a compound which causes gizzard erosion and ulceration in chicks (Okazaki et al., 1983). This compound, originally found in overheated fish meal, has been shown to stimulate gastric acid secretion in young chicks. This effect of gizzerosine is depressed by cimetidine, an antagonist of the histamine H2-receptor. Cimetidine also prevented gizzard erosion caused by fish meal in the diet (Masumura et al., 1985). Furthermore, gizzerosine enhanced oxygen consumption in the isolated mucosal cells of the chick proventriculus (Hino et al., 1987). These observations show that gizzerosine stimulates gastric acid secretion in a mode similar to that of histamine via histamine H2-receptor, and induces gizzard erosion. Gizzerosine is about 10 times as active as histamine in stimulating gastric acid secretion in vivo (Masumura et al., 1985). However, gizzerosine is almost as potent as histamine in en-
'To whom correspondence should be addressed.
hancing oxygen consumption in vitro (Hino et al., 1987). To clarify this discrepancy, we studied the effect of gizzerosine on intracellular concentration of cyclic adenosine-3',5'monophosphate (cAMP), which is the main second messenger of histamine action through H2receptor (Soil and Wollin, 1979; Wollin et al, 1979), employing isolated mucosal cells of the chicken proventriculus. At the same time, the interaction of gizzerosine with histamine H2-receptor in the same cells was studied. MATERIALS AND METHODS
Earle's balanced salt solution (EBSS) and Eagle's minimum essential medium (this medium will be referred to as basal medium) were purchased from Nissui Pharmaceutical Co., Tokyo, Japan. Collagenase I, histamine, 3-isobutyl-l-methyl xanthine (IB MX), cimetidine, and pyrilamine were obtained from Sigma Chemical Co., St. Louis, MO. The cAMP assay kit was purchased from New England Nuclear (NEN, Boston, MA). All other chemicals were of the purest grade available commercially. The (L)-gizzerosine, which was synthesized as previously described (Mori et al., 1985), was a kind gift of Professor K. Mori of The University of Tokyo, Tokyo, Japan.
1290
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
(Received for publication July 14, 1987)
INTRACELLULAR CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE
EBSS) were incubated in 3.5 mL EBSS for 15 min. Then 100 JJLL of the reagents were added at the indicated concentrations, the cells were further incubated for 30 min, except for the timecourse experiments. The incubation was terminated by addition of 400 uX trichloroacetic acid (final concentration at 5%). The samples were stored at -20 C until they were used for cAMP determination. [3H] Cyclic AMP (NEN) marker was added to each tube for determination of the recovery, and the suspension was centrifuged for 10 min at 3,000 x g. The supernatant was extracted four times with 10 mL of water-saturated ether. The water phase was then lyophilized and the residue was resuspended in an adequate volume of acetate buffer (pH 6.2). The content of c AMP was measured by radioimmunoassay employing a NEN assay kit. Binding Assay of Gizzerosine to the Isolated Cells. The binding of gizzerosine to the isolated cells was studied as described by Batzri and coworkers (Batzri, 1981; Batzri etal., 1982a,b). The mucosal cells (1-2 x 106 cells/mL) were incubated in .5 mL EBSS containing .2 |xCi [3H]histamine (NEN, 5.7 pmol, specific activity was 35 Ci/mmol) for the defined time at 37 C. At the end of the incubation, 10 mL ice cold EBSS was added and the incubation mixture was centrifuged at 1,000 x g for 2 min. After removing the supernatant, the same volume of the buffer was added to the mucosal cells and the mixture was again centrifuged at the same speed. The pellet was solubilized by mixing with 500 (JLL of 1% Triton X-100, and then, the radioactivity of the solubilized pellet was measured employing a liquid scintillation counter (Aloka Co., Tokyo, Japan). The nonspecific binding of histamine was measured separately by adding histamine (final concentration at 10 mM) together with the labeled histamine. The amount of nonspecifically bound histamine was subtracted from the total bound histamine. RESULTS
Time Course of the Formation of Cyclic Adenosine-3' ,5'-Monophosphate. Figure 1 shows the time course of the formation of c AMP incubated with 10 yiM (L)-gizzerosine (natural form) containing 10 uJtflBMX. Cellular cAMP level increased during 20 min, and after 30 min the level reached a plateau. From this result, 30 min was selected as the incubation time for subsequent assays. Effect of Histamine and Gizzerosine on the Formation of Cyclic Adenosine-3',5'-
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
Isolation of Mucosal Cells of Chicken Proventriculus. The cell isolation method used was similar to Soil's (Soil, 1978) except that the chicken proventriculus was used in place of dog stomach. Male White Leghorn chickens with a body weight of 1.5 to 2.0 kg (2 to 3 mo old) were fasted for 24 h before the experiment and killed by cervical dislocation. The proventriculus was quickly excised, wrapped in a plastic sheet, chilled on ice, and freed from serosal membrane and muscle layer. The mucosal tissue was dissected into small pieces (2 mm) with scissors. Tissue fragments were rinsed 2 to 3 times in the basal medium and incubated for 15 min at 37 C in 15 mL of the basal medium supplemented with collagenase at .4 mg/mL. The incubation flask was kept under the stream of 95% 0 2 -5% C0 2 and shaken continuously. The tissue suspension was centrifuged at 100 X g for 2 min, and the supernatant was removed. Mucosal fragments were subsequently incubated in the basal medium containing 1 mMEDTA under the same condition as that of the first incubation. The suspension was centrifuged again to remove the supernatant. The precipitate was resuspended in the same volume of the basal medium containing collagenase. The suspension was again incubated at 37 C for 15 min. Afterward, the supernatant was removed by centrifugation, and the cells were resuspended in 25 mL of the same collagenase solution and further incubated for 45 min. After this incubation, the suspension was stirred gently for about 15 min at room temperature, then added to the same volume of ice-cold EBSS containing 25 vaM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and cooled on ice. The cells were pipetted 50 times keeping the flask on ice. The cell suspension was passed through a nylon cloth (200 mesh). The filtrate containing mucosal cells was centrifuged at 100 x g for 5 min. The sedimented cells were washed three times with cold EBSS with repeated centrifugation at 100 x g for 5 min. The final cell preparation was suspended in ice cold EBSS and used for the experiments. Approximately 1 x 108 cells were obtained from one proventriculus by this method. The viability of the isolated cells was usually around 90%, as determined by a Trypan blue exclusion test. Assay for Cyclic Adenosine-3', 5' -Monophosphate Formation. Assay of cAMP formation was done following the method of Soil and Wollin (1979). In brief, the cells (106 cells/mL
1291
ITO ET AL.
1292
1BMXW
Si J2 t, o_ ~Z Z u < o
r
20 40 Incubation Time (min)
60
o o 2 a, «—
O
FIGURE 1. Time course of the formation of cyclic adenosine-3',5'-monophosphate (cAMP) by the isolated chicken mucosal cells incubated with gizzerosine and in the presence or absence of 3-isobutyl-l-methyl xanthine (IBMX). Each point is the average for three experiments, with a vertical line representing the SE. The open square shows the cellular cAMP level observed at 0 time in the absence of gizzerosine and IBMX. The SE not shown by vertical lines are within the marks.
Monophosphate. Figure 2 shows the relationship between the concentration of histamine or gizzerosine in the medium on the concentration of cAMP in the mucosal cells. Both histamine and gizzerosine stimulated cellular cAMP
co°10r o o £
1 1
1
-o-
Q.
0/
//
o
E
,'
/
k
-o-
u
<>
Gizzerosine
/
I
•1
3 8 "a*
\ \
Q_
Z <
T
-4
imetidineT""~^^
t
1
3
_
=
5
<-> 0
J
fi
• ff- -
o a>
lr-i
5
^ ^ J Pyri lamine
> —
0_
<
>
*
A
i
//
* 7
\
-
1 L..-I
1—
0 8
Histamine
7
6
5
4
Concentration of Pyrilamine or Cimetidine -log(M) I
i
i
i
0 8 7 6 5 4 3 Concentration of Gizzerosine or Histamine -log(M) FIGURE 2. Effect of gizzerosine or histamine on the formation of cyclic adenosine-3',5'-monophosphate (cAMP) by the isolated mucosal cells of the chicken proventriculus. Each point is the average for three experiments with a vertical line representing the SE.
FIGURE 3. Inhibitory effect as a function of the concentration of pyrilamine and cimetidine on the formation of cyclic adenosine-3',5'-monophosphate (cAMP) induced by gizzerosine. The open square shows the cellular cAMP level observed in the absence of gizzerosine and antagonists (in the presence of 3-isobutyl-l-methyl xanthine). Each point is the average for three experiments with a vertical line representing the SE. Standard errors not shown by vertical lines are within the marks.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
, ' IBMX(-)T
/ i
formation, and the maximal content of cAMP was almost the same. However, maximum cAMP level was attained at .1 and 100 \hM for gizzerosine and histamine, respectively. This shows that the potency of gizzerosine to raise the cellular cAMP level was about 1,000 times higher than that of histamine. Effect of Histamine Receptor Antagonists on the Formation of Cyclic Adenosine-3',5'Monophosphate. Mucosal cells were incubated with gizzerosine (20 yM) and IBMX (10 \LM) together with various concentrations of pyrilamine or cimetidine. As shown in Figure 3, the histamine H2-receptor antagonist, cimetidine, strongly inhibited the formation of cAMP elicited by gizzerosine. In contrast, the histamine H,-receptor antagonist, pyrilamine, exhibited a lower potency in inhibiting the formation of cAMP. Binding and Dissociation of [3H]Histamine to the Mucosal Cells. Histamine was maximally
INTRACELLULAR CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE
DISCUSSION
Gizzerosine has been shown to induce gastric acid secretion in chicks in vivo (Masumura et al., 1985) and to enhance oxygen consumption in isolated mucosal cells (Hino et al., 1987). These activities were inhibited by the histamine
H2-receptor antagonist, cimetidine. These results suggested that gizzerosine is a histamine H2-receptor agonist. The results, following three kinds of observations in the present studies employing isolated mucosal cells of chicken proventriculus, confirmed this assumption. First, gizzerosine displaced the cell bound histamine. Second, both histamine and gizzerosine stimulated cAMP formation in the presence of IBMX (Figure 2). Third, the effects of gizzerosine on the cells were depressed by the histamine H2-receptor antagonist, cimetidine, but not so strongly by the H!-receptor antagonist, pyrilamine (Figure 3). At the same time, the results of binding assays showed that mucosal cells prepared in this report had both H! and H2-receptors (Figure 6). However, as described above, the Hj-receptor is presumably not involved in the action of gizzerosine on these cells. There are some discrepancies between the potency of the activity of gizzerosine and that of histamine on isolated cells. The KD value of gizzerosine for displacing the bound histamine was approximately 10 times lower than that of histamine. However, the ability to raise cellular cAMP level was 1,000 times more potent in gizzerosine. Furthermore, there were no clear differences between the activities of these two compounds in enhancing cellular oxygen consumption (Hino et al., 1987). Batzri et al. showed that, in some cases, binding of some reagents to the histamine receptor does not necessarily induce the formation of 500 « L 4 C
600
400 r
--o" ^.400
u
O / I
/
/
/
" "
•|200 o o
•o
o a.
Total
••= 2 0 0 o o
Nonspecific
-Q_. 37 C
S. 100
I / 20
40 60 Time (min)
80
10 20 Time (min)
30
FIGURE 4. [3H]Histamine binding to and dissociation from the gastric mucosal cells of the chicken proventriculus: a) binding of the [3H]histamine to the cells in the presence or absence of histamine (10 mAf); b) dissociation of the labeled histamine from the cells. Each point is the average of four experiments. The SE are within the circles.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
bound after 50 min of incubation. The amount of histamine which was nonspecifically bound was about one-third of the specifically bound histamine (Figure 4a). Membrane bound histamine did not dissociate when incubated at 4 C, but dissociated at 37 C (Figure 4b). From these results, the incubation time for the histamine binding assay was determined to be 60 min. The processing of cells after incubation was performed at 4 C in order to prevent the dissociation of the bound histamine. Effect of Histamine and Gizzerosine on the [3H] histamine Binding. As seen in Figure 5, both gizzerosine and histamine inhibited the binding of [3H]histamine. The KD was defined as the potency of an agonist on inhibiting tracer binding (Batzri et al., 1982b). "Potency" is measured in terms of the concentration of an agonist required to produce a half maximal response. The KD value for gizzerosine was 6.1 x lO^M, and that of histamine was 5.1 x 10~5M. These results indicate that gizzerosine was bound to histamine receptors with a higher affinity than histamine. Figure 6 shows that binding of histamine to the mucosal cells is prevented by both pyrilamine and cimetidine. The results show that the present preparation of mucosal cells has both H, and H2-receptors.
1293
1294 - 1 0 0 CiM'i 7/£oa o
E X
ITO ET AL.
ioof^-^---------vgN -O- Histamine - • - Gizzerosine
o
E CD x
50 -
w 50
-O-Pyrilamine -#-Cimetidine
c C
in —
X
Q 0
Q.
-•_ Jt^z _1_ 7 6 5 4 3 -log(M) Concentration of Histamine or Gizzerosine
FIGURE 5. Effect of histamine or gizzerosine on the [3H]histamine binding to the mucosal cells of the chicken proventriculus. Each point is the average of four experiments. The SE are within the circles.
cAMP. They also suggested that there is some receptor other than the H2-receptor in guinea pig parietal cells (Batzri et al., 1982a,b). Their observations may partly explain the apparent discrepancy between the activities of displacing bound histamine and raising cellular cAMP level. In any case, available evidence cannot explain clearly the discrepancy between the potency of activity of gizzerosine and that of histamine in vitro. Gizzerosine exhibited activities that enhance gastric acid secretion in vivo. Gastric acid content was shown to be higher after gizzerosine administration to chickens. The ability to raise gastric acid secretion was higher for gizzerosine than for histamine. Although higher potency of gizzerosine compared with histamine was shown in some activities in vitro, it will be very difficult to relate directly the in vitro observations to the differences in activities in vivo. Differences in vivo can be caused by differences of activities in vitro and can also be brought about by differences in metabolism of these two compounds by chickens. Gizzerosine has been shown to stay in chicken blood far longer than histamine after administration (Ito et al., unpublished results). This suggests that metabolism or excretion of gizzerosine by chicken is far slower than histamine. Therefore, the activities of gizzerosine will last longer than those of histamine after administration or ingestion of this compound to chickens. This probably explains the far stronger activity of gizzerosine in causing gizzard erosion and ulceration in chickens. The above results
8 7 6 5 A Concentration of Pyrilamine or Cimetidine -log(M)
FIGURE 6. Effect of pyrilamine or cimetidine on the [3H]histamine binding to the mucosal cells of the chicken proventriculus. Each point is the average of four experiments. The SE are within the circles.
concerning the properties of gizzerosine as a histamine H 2 agonist will contribute to the development of a way to prevent gizzard erosion and ulceration in the poultry industry. REFERENCES Batzri, S., 1981. Interaction of histamine with specific membrane receptors on cells. Biochem. Pharmacol. 30:3013-3016. Batzri, S., J. W. Harmon, J. Dyer, and W. F. Thompson, 1982a. Interaction of histamine with gastric mucosal cells. Effect of histamine H2 antagonists on binding and biological response. Mol. Pharmacol. 22:A\-A1'. Batzri, S., J. W. Harmon, and W. F. Thompson, 1982b. Interaction of histamine with gastric mucosal cells. Effect of histamine agonists on binding and biological response. Mol. Pharmacol. 22:33-40. Hino, T., T. Noguchi, and H. Naito, 1987. The effect of gizzerosine on acid secretion by isolated mucosal cells of the chicken proventriculus. Poultry Sci. 66:548551. Masumura, T., M. Sugahara, T. Noguchi, K. Mori, and H. Naito, 1985. The effect of gizzerosine, a recently discovered compound in overheated fish meal, on gastric acid secretion in chicken. Poultry Sci. 64:356-361. Mori, K., T. Sugai, Y. Maeda, T. Okazaki, T. Noguchi, and H. Naito, 1985. Synthesis of the racemic and optically active forms of gizzerosine, the inducer of gizzard erosion in chicks. Tetrahedron 41:5307-5311. Okazaki, T., T. Noguchi, K. Igarashi, Y. Sakagami, H. Seto, K. Mori, H. Naito, T. Masumura, and M. Sugahara, 1983. Gizzerosine, a new toxic substance in fish meal, causes severe gizzard erosion in chicks. Agric. Biol. Chem. 47:2949-2952. Soil, A. H., 1978. The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. J. Clin. Invest. 61:381-389. Soil, A. H., and A. Wollin, 1979. Histamine and cyclic AMP in isolated canine parietal cells. Am. J. Physiol. 237:E444-E450. Wollin, A., A. H. Soil, and I. M. Samloff, 1979. The action of histamine secretion and PGE2 on cyclic AMP production by isolated canine fundic mucosal cells. Am. J. Physiol. 237:E437-E443.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
in
o •NO
ABSTRACT Gizzerosine, which was originally found in fish meal, is a compound that causes gizzard erosion and ulceration in chicks. The action of gizzerosine on the isolated cells of chicken proventriculus was studied in the present investigations. Gizzerosine increased intracellular cyclic adenosine-3',5'-monophosphate (cAMP) levels, reaching a plateau within 30 min. Similar maxima of cAMP level were observed in the presence of histamine or gizzerosine. However, the potency of gizzerosine was approximately 1,000-fold higher than that of histamine. The action of gizzerosine was depressed by cimetidine, a histamine H2-receptor antagonist, but not by pyrilamine, a histamine H,-receptor antagonist, indicating that gizzerosine is a very strong histamine H2-receptor agonist. The mucosal cells isolated from the proventriculus by the present procedure had both histamine H, and H2-receptors. Gizzerosine showed a higher affinity to the cell surface histamine receptor than histamine. These results partly explain the potent activity of gizzerosine in inducing gastric acid secretion and causing gizzard erosion and ulceration in chickens. {Key words: histamine, gizzerosine, cyclic adenosine-3',5'-monophosphate, gastric mucosal cells, gizzard erosion) 1988 Poultry Science 67:1290-1294 INTRODUCTION
Gizzerosine [2-amino-9-(4-imidazolyl)-7azanonanoic acid] is a compound which causes gizzard erosion and ulceration in chicks (Okazaki et al., 1983). This compound, originally found in overheated fish meal, has been shown to stimulate gastric acid secretion in young chicks. This effect of gizzerosine is depressed by cimetidine, an antagonist of the histamine H2-receptor. Cimetidine also prevented gizzard erosion caused by fish meal in the diet (Masumura et al., 1985). Furthermore, gizzerosine enhanced oxygen consumption in the isolated mucosal cells of the chick proventriculus (Hino et al., 1987). These observations show that gizzerosine stimulates gastric acid secretion in a mode similar to that of histamine via histamine H2-receptor, and induces gizzard erosion. Gizzerosine is about 10 times as active as histamine in stimulating gastric acid secretion in vivo (Masumura et al., 1985). However, gizzerosine is almost as potent as histamine in en-
'To whom correspondence should be addressed.
hancing oxygen consumption in vitro (Hino et al., 1987). To clarify this discrepancy, we studied the effect of gizzerosine on intracellular concentration of cyclic adenosine-3',5'monophosphate (cAMP), which is the main second messenger of histamine action through H2receptor (Soil and Wollin, 1979; Wollin et al, 1979), employing isolated mucosal cells of the chicken proventriculus. At the same time, the interaction of gizzerosine with histamine H2-receptor in the same cells was studied. MATERIALS AND METHODS
Earle's balanced salt solution (EBSS) and Eagle's minimum essential medium (this medium will be referred to as basal medium) were purchased from Nissui Pharmaceutical Co., Tokyo, Japan. Collagenase I, histamine, 3-isobutyl-l-methyl xanthine (IB MX), cimetidine, and pyrilamine were obtained from Sigma Chemical Co., St. Louis, MO. The cAMP assay kit was purchased from New England Nuclear (NEN, Boston, MA). All other chemicals were of the purest grade available commercially. The (L)-gizzerosine, which was synthesized as previously described (Mori et al., 1985), was a kind gift of Professor K. Mori of The University of Tokyo, Tokyo, Japan.
1290
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
(Received for publication July 14, 1987)
INTRACELLULAR CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE
EBSS) were incubated in 3.5 mL EBSS for 15 min. Then 100 JJLL of the reagents were added at the indicated concentrations, the cells were further incubated for 30 min, except for the timecourse experiments. The incubation was terminated by addition of 400 uX trichloroacetic acid (final concentration at 5%). The samples were stored at -20 C until they were used for cAMP determination. [3H] Cyclic AMP (NEN) marker was added to each tube for determination of the recovery, and the suspension was centrifuged for 10 min at 3,000 x g. The supernatant was extracted four times with 10 mL of water-saturated ether. The water phase was then lyophilized and the residue was resuspended in an adequate volume of acetate buffer (pH 6.2). The content of c AMP was measured by radioimmunoassay employing a NEN assay kit. Binding Assay of Gizzerosine to the Isolated Cells. The binding of gizzerosine to the isolated cells was studied as described by Batzri and coworkers (Batzri, 1981; Batzri etal., 1982a,b). The mucosal cells (1-2 x 106 cells/mL) were incubated in .5 mL EBSS containing .2 |xCi [3H]histamine (NEN, 5.7 pmol, specific activity was 35 Ci/mmol) for the defined time at 37 C. At the end of the incubation, 10 mL ice cold EBSS was added and the incubation mixture was centrifuged at 1,000 x g for 2 min. After removing the supernatant, the same volume of the buffer was added to the mucosal cells and the mixture was again centrifuged at the same speed. The pellet was solubilized by mixing with 500 (JLL of 1% Triton X-100, and then, the radioactivity of the solubilized pellet was measured employing a liquid scintillation counter (Aloka Co., Tokyo, Japan). The nonspecific binding of histamine was measured separately by adding histamine (final concentration at 10 mM) together with the labeled histamine. The amount of nonspecifically bound histamine was subtracted from the total bound histamine. RESULTS
Time Course of the Formation of Cyclic Adenosine-3' ,5'-Monophosphate. Figure 1 shows the time course of the formation of c AMP incubated with 10 yiM (L)-gizzerosine (natural form) containing 10 uJtflBMX. Cellular cAMP level increased during 20 min, and after 30 min the level reached a plateau. From this result, 30 min was selected as the incubation time for subsequent assays. Effect of Histamine and Gizzerosine on the Formation of Cyclic Adenosine-3',5'-
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
Isolation of Mucosal Cells of Chicken Proventriculus. The cell isolation method used was similar to Soil's (Soil, 1978) except that the chicken proventriculus was used in place of dog stomach. Male White Leghorn chickens with a body weight of 1.5 to 2.0 kg (2 to 3 mo old) were fasted for 24 h before the experiment and killed by cervical dislocation. The proventriculus was quickly excised, wrapped in a plastic sheet, chilled on ice, and freed from serosal membrane and muscle layer. The mucosal tissue was dissected into small pieces (2 mm) with scissors. Tissue fragments were rinsed 2 to 3 times in the basal medium and incubated for 15 min at 37 C in 15 mL of the basal medium supplemented with collagenase at .4 mg/mL. The incubation flask was kept under the stream of 95% 0 2 -5% C0 2 and shaken continuously. The tissue suspension was centrifuged at 100 X g for 2 min, and the supernatant was removed. Mucosal fragments were subsequently incubated in the basal medium containing 1 mMEDTA under the same condition as that of the first incubation. The suspension was centrifuged again to remove the supernatant. The precipitate was resuspended in the same volume of the basal medium containing collagenase. The suspension was again incubated at 37 C for 15 min. Afterward, the supernatant was removed by centrifugation, and the cells were resuspended in 25 mL of the same collagenase solution and further incubated for 45 min. After this incubation, the suspension was stirred gently for about 15 min at room temperature, then added to the same volume of ice-cold EBSS containing 25 vaM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and cooled on ice. The cells were pipetted 50 times keeping the flask on ice. The cell suspension was passed through a nylon cloth (200 mesh). The filtrate containing mucosal cells was centrifuged at 100 x g for 5 min. The sedimented cells were washed three times with cold EBSS with repeated centrifugation at 100 x g for 5 min. The final cell preparation was suspended in ice cold EBSS and used for the experiments. Approximately 1 x 108 cells were obtained from one proventriculus by this method. The viability of the isolated cells was usually around 90%, as determined by a Trypan blue exclusion test. Assay for Cyclic Adenosine-3', 5' -Monophosphate Formation. Assay of cAMP formation was done following the method of Soil and Wollin (1979). In brief, the cells (106 cells/mL
1291
ITO ET AL.
1292
1BMXW
Si J2 t, o_ ~Z Z u < o
r
20 40 Incubation Time (min)
60
o o 2 a, «—
O
FIGURE 1. Time course of the formation of cyclic adenosine-3',5'-monophosphate (cAMP) by the isolated chicken mucosal cells incubated with gizzerosine and in the presence or absence of 3-isobutyl-l-methyl xanthine (IBMX). Each point is the average for three experiments, with a vertical line representing the SE. The open square shows the cellular cAMP level observed at 0 time in the absence of gizzerosine and IBMX. The SE not shown by vertical lines are within the marks.
Monophosphate. Figure 2 shows the relationship between the concentration of histamine or gizzerosine in the medium on the concentration of cAMP in the mucosal cells. Both histamine and gizzerosine stimulated cellular cAMP
co°10r o o £
1 1
1
-o-
Q.
0/
//
o
E
,'
/
k
-o-
u
<>
Gizzerosine
/
I
•1
3 8 "a*
\ \
Q_
Z <
T
-4
imetidineT""~^^
t
1
3
_
=
5
<-> 0
J
fi
• ff- -
o a>
lr-i
5
^ ^ J Pyri lamine
> —
0_
<
>
*
A
i
//
* 7
\
-
1 L..-I
1—
0 8
Histamine
7
6
5
4
Concentration of Pyrilamine or Cimetidine -log(M) I
i
i
i
0 8 7 6 5 4 3 Concentration of Gizzerosine or Histamine -log(M) FIGURE 2. Effect of gizzerosine or histamine on the formation of cyclic adenosine-3',5'-monophosphate (cAMP) by the isolated mucosal cells of the chicken proventriculus. Each point is the average for three experiments with a vertical line representing the SE.
FIGURE 3. Inhibitory effect as a function of the concentration of pyrilamine and cimetidine on the formation of cyclic adenosine-3',5'-monophosphate (cAMP) induced by gizzerosine. The open square shows the cellular cAMP level observed in the absence of gizzerosine and antagonists (in the presence of 3-isobutyl-l-methyl xanthine). Each point is the average for three experiments with a vertical line representing the SE. Standard errors not shown by vertical lines are within the marks.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
, ' IBMX(-)T
/ i
formation, and the maximal content of cAMP was almost the same. However, maximum cAMP level was attained at .1 and 100 \hM for gizzerosine and histamine, respectively. This shows that the potency of gizzerosine to raise the cellular cAMP level was about 1,000 times higher than that of histamine. Effect of Histamine Receptor Antagonists on the Formation of Cyclic Adenosine-3',5'Monophosphate. Mucosal cells were incubated with gizzerosine (20 yM) and IBMX (10 \LM) together with various concentrations of pyrilamine or cimetidine. As shown in Figure 3, the histamine H2-receptor antagonist, cimetidine, strongly inhibited the formation of cAMP elicited by gizzerosine. In contrast, the histamine H,-receptor antagonist, pyrilamine, exhibited a lower potency in inhibiting the formation of cAMP. Binding and Dissociation of [3H]Histamine to the Mucosal Cells. Histamine was maximally
INTRACELLULAR CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE
DISCUSSION
Gizzerosine has been shown to induce gastric acid secretion in chicks in vivo (Masumura et al., 1985) and to enhance oxygen consumption in isolated mucosal cells (Hino et al., 1987). These activities were inhibited by the histamine
H2-receptor antagonist, cimetidine. These results suggested that gizzerosine is a histamine H2-receptor agonist. The results, following three kinds of observations in the present studies employing isolated mucosal cells of chicken proventriculus, confirmed this assumption. First, gizzerosine displaced the cell bound histamine. Second, both histamine and gizzerosine stimulated cAMP formation in the presence of IBMX (Figure 2). Third, the effects of gizzerosine on the cells were depressed by the histamine H2-receptor antagonist, cimetidine, but not so strongly by the H!-receptor antagonist, pyrilamine (Figure 3). At the same time, the results of binding assays showed that mucosal cells prepared in this report had both H! and H2-receptors (Figure 6). However, as described above, the Hj-receptor is presumably not involved in the action of gizzerosine on these cells. There are some discrepancies between the potency of the activity of gizzerosine and that of histamine on isolated cells. The KD value of gizzerosine for displacing the bound histamine was approximately 10 times lower than that of histamine. However, the ability to raise cellular cAMP level was 1,000 times more potent in gizzerosine. Furthermore, there were no clear differences between the activities of these two compounds in enhancing cellular oxygen consumption (Hino et al., 1987). Batzri et al. showed that, in some cases, binding of some reagents to the histamine receptor does not necessarily induce the formation of 500 « L 4 C
600
400 r
--o" ^.400
u
O / I
/
/
/
" "
•|200 o o
•o
o a.
Total
••= 2 0 0 o o
Nonspecific
-Q_. 37 C
S. 100
I / 20
40 60 Time (min)
80
10 20 Time (min)
30
FIGURE 4. [3H]Histamine binding to and dissociation from the gastric mucosal cells of the chicken proventriculus: a) binding of the [3H]histamine to the cells in the presence or absence of histamine (10 mAf); b) dissociation of the labeled histamine from the cells. Each point is the average of four experiments. The SE are within the circles.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
bound after 50 min of incubation. The amount of histamine which was nonspecifically bound was about one-third of the specifically bound histamine (Figure 4a). Membrane bound histamine did not dissociate when incubated at 4 C, but dissociated at 37 C (Figure 4b). From these results, the incubation time for the histamine binding assay was determined to be 60 min. The processing of cells after incubation was performed at 4 C in order to prevent the dissociation of the bound histamine. Effect of Histamine and Gizzerosine on the [3H] histamine Binding. As seen in Figure 5, both gizzerosine and histamine inhibited the binding of [3H]histamine. The KD was defined as the potency of an agonist on inhibiting tracer binding (Batzri et al., 1982b). "Potency" is measured in terms of the concentration of an agonist required to produce a half maximal response. The KD value for gizzerosine was 6.1 x lO^M, and that of histamine was 5.1 x 10~5M. These results indicate that gizzerosine was bound to histamine receptors with a higher affinity than histamine. Figure 6 shows that binding of histamine to the mucosal cells is prevented by both pyrilamine and cimetidine. The results show that the present preparation of mucosal cells has both H, and H2-receptors.
1293
1294 - 1 0 0 CiM'i 7/£oa o
E X
ITO ET AL.
ioof^-^---------vgN -O- Histamine - • - Gizzerosine
o
E CD x
50 -
w 50
-O-Pyrilamine -#-Cimetidine
c C
in —
X
Q 0
Q.
-•_ Jt^z _1_ 7 6 5 4 3 -log(M) Concentration of Histamine or Gizzerosine
FIGURE 5. Effect of histamine or gizzerosine on the [3H]histamine binding to the mucosal cells of the chicken proventriculus. Each point is the average of four experiments. The SE are within the circles.
cAMP. They also suggested that there is some receptor other than the H2-receptor in guinea pig parietal cells (Batzri et al., 1982a,b). Their observations may partly explain the apparent discrepancy between the activities of displacing bound histamine and raising cellular cAMP level. In any case, available evidence cannot explain clearly the discrepancy between the potency of activity of gizzerosine and that of histamine in vitro. Gizzerosine exhibited activities that enhance gastric acid secretion in vivo. Gastric acid content was shown to be higher after gizzerosine administration to chickens. The ability to raise gastric acid secretion was higher for gizzerosine than for histamine. Although higher potency of gizzerosine compared with histamine was shown in some activities in vitro, it will be very difficult to relate directly the in vitro observations to the differences in activities in vivo. Differences in vivo can be caused by differences of activities in vitro and can also be brought about by differences in metabolism of these two compounds by chickens. Gizzerosine has been shown to stay in chicken blood far longer than histamine after administration (Ito et al., unpublished results). This suggests that metabolism or excretion of gizzerosine by chicken is far slower than histamine. Therefore, the activities of gizzerosine will last longer than those of histamine after administration or ingestion of this compound to chickens. This probably explains the far stronger activity of gizzerosine in causing gizzard erosion and ulceration in chickens. The above results
8 7 6 5 A Concentration of Pyrilamine or Cimetidine -log(M)
FIGURE 6. Effect of pyrilamine or cimetidine on the [3H]histamine binding to the mucosal cells of the chicken proventriculus. Each point is the average of four experiments. The SE are within the circles.
concerning the properties of gizzerosine as a histamine H 2 agonist will contribute to the development of a way to prevent gizzard erosion and ulceration in the poultry industry. REFERENCES Batzri, S., 1981. Interaction of histamine with specific membrane receptors on cells. Biochem. Pharmacol. 30:3013-3016. Batzri, S., J. W. Harmon, J. Dyer, and W. F. Thompson, 1982a. Interaction of histamine with gastric mucosal cells. Effect of histamine H2 antagonists on binding and biological response. Mol. Pharmacol. 22:A\-A1'. Batzri, S., J. W. Harmon, and W. F. Thompson, 1982b. Interaction of histamine with gastric mucosal cells. Effect of histamine agonists on binding and biological response. Mol. Pharmacol. 22:33-40. Hino, T., T. Noguchi, and H. Naito, 1987. The effect of gizzerosine on acid secretion by isolated mucosal cells of the chicken proventriculus. Poultry Sci. 66:548551. Masumura, T., M. Sugahara, T. Noguchi, K. Mori, and H. Naito, 1985. The effect of gizzerosine, a recently discovered compound in overheated fish meal, on gastric acid secretion in chicken. Poultry Sci. 64:356-361. Mori, K., T. Sugai, Y. Maeda, T. Okazaki, T. Noguchi, and H. Naito, 1985. Synthesis of the racemic and optically active forms of gizzerosine, the inducer of gizzard erosion in chicks. Tetrahedron 41:5307-5311. Okazaki, T., T. Noguchi, K. Igarashi, Y. Sakagami, H. Seto, K. Mori, H. Naito, T. Masumura, and M. Sugahara, 1983. Gizzerosine, a new toxic substance in fish meal, causes severe gizzard erosion in chicks. Agric. Biol. Chem. 47:2949-2952. Soil, A. H., 1978. The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. J. Clin. Invest. 61:381-389. Soil, A. H., and A. Wollin, 1979. Histamine and cyclic AMP in isolated canine parietal cells. Am. J. Physiol. 237:E444-E450. Wollin, A., A. H. Soil, and I. M. Samloff, 1979. The action of histamine secretion and PGE2 on cyclic AMP production by isolated canine fundic mucosal cells. Am. J. Physiol. 237:E437-E443.
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on April 26, 2015
in
o •NO