Modulation of murine peritoneal macrophage functions by gastrin

Peptides. Vol. 17, No. 2, pp. 219-224, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196.9781/96 $15.00 + .OO

SD1 Ol%-9781(95)02133-7

ELSEVIER

Modulation of Murine Peritoneal Macrophage Functions by Gastrin MONICA DE LA FUENTE, * ’ JASON DRUMMOND,* MONICA DEL RIO,* MONTSERRAT CARRASCO” AND ANGEL HERNANZT * Departamento de Biologia Animal II (Fisiologia Animal), Facultad de Ciencias Bioldgicas, Universidad Complutense de Madrid, Spain, and fServicio de Bioquimica, Hospital LAIPaz de1 Insalud, Madrid, Spain

Received 17 May 1995 DE LA FUENTE, M., J. DRUMMOND, M. DEL RIO, M. CARRASCO AND A. HERNANZ. Modularion of murk pen’toneal macrophagefunctions by gastrirz. PEPTIDES 17(2) 219-224,1996.-The effect in vitro of gastrin-17 and gastrin-34 was studied at concentrations from lo-‘* to 10m6M on several functions of resting peritoneal macrophages from BALB/c mice: adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis) , and ingestion of inert particles (latex beads) or cells (Can&&~ albicans). Both gastrins, at concentrations from lo-” to lo-* M, inhibited significantly all functions studied with the exception of adherence, which was increased. A dose-response relationship was observed, with a maximum inhibition of macrophage functions found at 1O-9 M. These peptides induced in murine macrophages a significant increase of CAMP levels at 60 and 120 s. Adenosine, an adenylate cyclase inhibitor, significantly increased the ingestion of latex beads, whereas the combined presence of adenosine and either G-17 or G-34 produced similar values to those of control samples without adenosine or gastrin. These results suggest that gastrin is a negative modulator of several macrophage functions, and that the inhibition of these activities is carried out through an increase of intracellular CAMP levels. Peritoneal macrophages

Gastrin

Adherence

Chemotaxis

Phagocytosis

Cyclic AMP

tide concentrations ranging from lo-” to 10e6 M for each step of the phagocytic process: adherence, mobility (spontaneous and directed to the infectious focus or chemotaxis), ingestion of inert particles (latex beads ) , or foreign cells ( Cundidu albicuns) , and oxidative metabolism. Moreover, the possible intracellular signal pathways involved in gastrin action were studied by measuring changes in intracellular CAMP concentration and by using an inhibitor of CAMP synthesis such as adenosine ( 3 1) .

THE idea of a functional connection among the nervous, endocrine, and immune systems is now accepted. This connection has been supported by the observation that peptides from nervous and endocrine systems have receptors on immune cells and modulate the function of such cells ( 1) . Gastrin was firstly characterized in the antrum as a stimulant of gastric acid secretion (9). First found in the G cells of the antrum epithelial cells ( 17), later, various forms in blood and several tissues were identified: big gastrin, a linear polypeptide of 34 amino acids (G-34)) little gastrin (G- 17 ) , and mini gastrin ( G- 14), as well as larger forms such as big-big gastrin and component I ( 12). Gastrin immunoreactivity has also been found in the porcine pituitary gland (19) and in the vagus nerve of dogs, cats, and humans (29). Besides gastric acid secretion, gastrin stimulates pancreatic enzyme secretion (26) and antral motility (24). Moreover, gastrin has a wide range of actions on epithelial and smooth muscle targets in the gastrointestinal tract, but many of these effects require pharmacological doses ( 30). Until now, only one work has studied the effects of gastrin on leukocytes, in which human and porcine G-17 did not affect lymphoid proliferation ( 18). We have studied the effect of gastrin, G-17 and G-34, on macrophage functions because the phagocytic function of macrophages represents the beginning of other biological activities in the immune response (28). This study was carried out in murine peritoneal macrophages in vitro with pep-

METHOD

Animals

Male and female BALB /c mice (Mus musculus) (Iffa Credo, St. Germain Sur L’Arbrese, France) aged 20 ? 2 weeks were maintained at a constant temperature (22 t 2°C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France) on a 12-h light/dark cycle and fed Sander Mus and water ad lib. Only those mice without signs of malignancy and other pathologic manifestations were used. Collection of Resident Cells From Peritoneum The resident cells from peritoneum were obtained, after cervical dislocation of the animal, following a method previously described (4). The peritoneal resident cells removed contained 60% macrophages and 40% lymphocytes. Resting macrophages,

’ Requests for reprints should be addressed to Prof. Monica De la Fuente. 219

220

DE LA FUENTE

TABLE

ET AL.

1

ADHERENCE INDEX OF MACROPHAGES INCUBATED WITH G-II OR G-34 Peptide Peptide

G-17

G-34

Time

0

,0-l?

31 23 43 ? 6 53 5 5 63 5 8 3123 41 -t4 49 2 4 58 -+ 6

32 2 3 4414 51 z-6 61 +6 34 2 4 42 ” 5 48 It 6 58 5 7

(min)

5 10 20 60 5 10 20 60

IO-”

34 46 52 62 36 43 49 59

? 2 2 ? ? + 5 ?

Concentration

,04

4 5 6 5 4 4 5 6

37 51 54 63 40 49 49 58

2 5* *5* 2 5 2 7 2 5* 2 5* 2 6 2 7

(M)

IO 4

39 52 58 65 41 52 53 60

+ i 2 + t ? 2 t

IOmX

4* 6* 57 8 5* 6* 5 7

40 46 52 63 39 49 51 58

10-7

? 5* lr 5 2 6 ? 4 t 5* + 5* ?7 5 6

33 2 42 c 52 ir 6426 33 t 43 k 50 2 57 2

IO-”

4 5 7

32 2 3 41 r5 53 2 5 64k4 33 k 5 42 + 4 51 ~6 58 -t 7

4 6 6 7

The results represent the mean 2 SD of eight experiments performed in duplicate. * p < 0.01 and ?p < 0.05 with respect to the control values.

determined by morphology and nonspecific esterase staining, were counted and adjusted in the same medium at 5 x 10’ macrophages/ml. Cellular viability was routinely measured before and after each experiment by trypau-blue exclusion test, and experiments were discarded in which cell viability was less than 95%. All the incubations were performed at 37°C in a humidified atmosphere of 5% CO*. Assay of Adherence

Capacity

For the quantification of substrate adherence capacity, we have used the adherence to a smooth plastic surface because it resembles adherence to animal tissue ( 19). The method was carried out as previously described by De la Fuente et al. (4). Briefly, aliquots of peritoneal suspension (5 X lo5 macrophages/ ml Hank’s medium) were placed in Eppendorf tubes and incubated with G-17 or G-34 (Sigma, St. Louis, MO) at a final concentration ranging from lo-” to 10m6 M or Hank’s medium (Flow, McLean, VA) (controls). At 5, 10, 20, or 60 min of incubation, the number of nonadhered macrophages was determined. The adherence index, AI, was calculated according to the following equation: AI = 100 - (macrophages/ml supematant)/ (macrophages/ml original sample) x 100. Assay of Chemotaxis

Assay of Chemoattractant

Activity

The chemoattractant activity of both gastrins was evaluated according to the technique above described for chemotaxis but in this case the peptides were deposited in the lower compartment of chamber instead of f-Met-Leu-Phe. Assay of Phagocytosis Phagocytosis assay of inert particles (latex beads; Sigma; 1.09 pm diluted to 1% in PBS) was carried out according to the technique described by De la Fuente (4). Aliquots of cell suspension

MacrophagesIfilter 750

1

A

:I::::,:i I

CC

**

*

*

it

2501)

Chemotaxis was evaluated according to a modification (4) of the original technique described by Boyden (2), which uses a chamber of two compartments separated by a Millipore filter with a pore diameter of 3 ,um. Aliquots of peritoneal cells were deposited in the upper compartment with G-17 or G-34 at final concentrations ranging from 10 _ I2 to 10 -6 M or Hank’s medium (controls). Then, f-Met-Leu-Phe ( lo-’ M; Sigma), as a positive chemotactic agent (23), was put into the lower compartment. The chambers were incubated for 3 h, then the filters fixed in methanol for 5 min and stained with eosin-hematoxilin (Merck). The chemotaxis index was determined by counting in an optical microscope (immersion objective) the number of macrophages or lymphocytes in one-third of the lower face of the filter.

0

12

11

10

9

7

6

B

ig-+G& ** *** *

25OJ

/ 0

/ 12

, 11

, IO

**

**

/ 9

, 6

Concentration

Assay of Spontaneous

6

750

, 7

, 6

(1O’M)

Mobility

Spontaneous mobility was evaluated with the technique used in the assay of chemotaxis. Instead of f-Met-Leu-Phe, Hank’s medium was placed in the lower compartment of the Boyden chamber.

FW. 1. Effect of different concentrations of gastrin-17 (A) and gastrin34 (B) on macrophage mobility: chemotaxis (m) and spontaneous mobility (0). The results represent the mean -t SD of eight experiments performed in duplicate. *p < 0.05, **p < 0.01 with respect to control values.

221

GASTRIN EFFECT ON MACROPHAGES Assay of Nitroblue Tetrazolium Reduction Test

No. of latex beads phagocylized 350

The quantitative nitroblue tetrazolium (NBT) reduction test was carried out according to the technique described by De la Fuente et al. (4). Macrophage suspensions were mixed with NBT (Sigma; 1 mg/ml Hank’s medium) and different concentrations of G-17 or G-34 ( lo-l2 to lo-’ M) or Hank’s medium (controls) . Latex beads were added to one sample set (stimulated), whereas only PBS were added to the other sample set (nonstimulated) . After 60 mitt of incubation, the absorbance of reduced NBT was determined in a spectrophotometer at 525 nm. Data obtained were expressed as pmol of NBT reduced by lo8 macrophages by extrapolating in a standard curve different concentrations of NBT reduced with 1,Cdithioerythritol (BoehringerMannheim, Mannheim, Germany).

I

150 b

1;

1’1

lb

4

b

7

b

350

Isolation of Peritoneal Macrophages for CAMP Measurements 300

Peritoneal cells were obtained as indicated above, but with RMPI 1640 (Gibco) instead of Hank’s medium. Aliquots of pooled peritoneal suspensions containing 5 X 105 macrophages/ ml medium were dispensed into Eppendorf tubes, and incubated for 60 min with the addition of trypsin (0.25%; Sigma) to remove nonmacrophage cells and debris. Then adherent macrophages were gently washed three times, and finally incubated with RPM1 1640 for up to 24 h.

250

200

150

I

0

12

11

I

I

I

I

10

9

8

7

Concentration

6

Determination of CAMP

(1O”M)

2. Effect of gastrin-17 (A) and gas&in-34 (B) on the number of latex beads phagocytized per 100 peritoneal macrophages(phagocytosis

Macrophages prepared as indicated above were incubated with G-17 at final concentrations between lo-l2 and 10e6 M or RMPI 1640 (controls). In all cases, samples were accompanied by a nonincubated sample (basal) to determine the intracellular CAMP levels from which we started in each experiment. After the incubation times of 30, 60, or 120 s, the samples were processed following the methods previously described (5) and subjected to immunoassay according to the Amersham ELISA kit ( Amersham, Bucks, UK) for CAMP.

FIG.

index). The results represent the mean + SD of eight experiments formed in duplicate. **p < 0.01 with respect to control values.

per-

were incubated in MIF (migratory inhibitory factor) plates (Sterilin, Teddington, UK) for 30 min. To the adherent monolayer were added latex beads and G-17 or G-34 ( lo-l2 to 10e6 M) or medium (controls). After 30 mm of incubation the plates were washed, fixed, and stained, and the number of particles phagocytized per 100 macrophages was counted. Phagocytosis of Candida albicans was carried out by the method previously described (4). The technique was similar to that used in the phagocytosis assay of latex beads. In the present assay C. albicans ( 10 X lo6 cells/ml medium) were added to the adherent monolayer. The yeasts were previously incubated for 30 mitt with a pool of human serum. Immediately, G-17 or G-34 at final concentrations ranging from lo-‘* to 10e6 M or medium (controls) was added. After 60 min of incubation, the plates were washed, fixed, and stained, and the number of C. albicans ingested per 100 macrophages counted.

Effect of Adenosine on Phugocytosis Phagocytosis of latex beads was evaluated as indicated above. G-17 and G-34 were used at a final concentration of lo-” M and adenosine ( Boehringer-Mannheim) was added simultaneously to G-17 or G-34 at a final concentration of 1O-7 M. Statistical Analysis All values are expressed as the mean + SD of the number of experiments, performed in duplicate, as indicated in the corresponding tables and figures. The data were evaluated statistically by the one-factor ANOVA of repeated measures for paired ob-

TABLE 2 NUMBER

OF Candida

olbicans

INGESTED

PER 100 MACROPHAGES Peptide

Peptide

0

IO-‘2

G-17 G-34

256 2 30 246 -c 33

250 t- 36 240 2 31

lo-”

245 k 39 234 2 34

The results represent the mean + SD of eight experiments * p < 0.05 with respect to the control values.

IO

Concentration

I”

233 -t 38 228 -c 29* performed

in duplicate.

INCUBATED

WITH G-l?

OR G-34

(M)

10-q

10-X

10-7

10-f

194 ? 40** 185 2 27**

215 2 30* 203 2 30*

232 + 35 228 2 37

243 f 32 239 + 34

222

DE LA FUENTE ET AL.

servations of parametric data and the Scheffe F-test between each two groups, with p < 0.05 being the minimum significant level. RESULTS

Table 1 shows the adherence index of macrophages incubated with G-17 or G-34 at 5, 10, 20, or 60 min. G-17 stimulated significantly the adherence of macrophages to substrate at 5 min of incubation at the concentrations lo-” M (p < O.Ol), lO-9 M (p < O.Ol), and lo-* M (p < O.Ol), as well as at 10 min at lo-” M (p < 0.01) and 10m9M (p < 0.01). G-34 also stimulated significantly the adherence at 5 and 10 min of incubation, in both cases at lo-” M (p < O.Ol), 10m9M (p < O.Ol), and lo-’ M (p < 0.01). To discard the possibility that gastrin attaches to the tube surface and attracts the macrophages through its positive charges, a group of control tubes was pretreated with G-34 at lo-” M. There were no significant differences between the pretreated tubes and the control tubes: 32 ? 5 (5 min), 44 ? 6(lOmin),51 ?4(20min),59?3(60min). Once the stimulation of G-17 and G-34 on the adherence capacity was determined, we studied their effect on the mobility of macrophages. Figure 1 shows that G-17 and G-34 concentrations of lo-” M, 10m9 M, and lo-* M inhibited significantly both spontaneous mobility and chemotaxis. However, neither gastrin showed any chemoattractant capacity on peritoneal macrophages. When G-17 or G-34 ( 10e9 M) was placed in the lower chamber as chemoattractant instead of f-Met-Leu-Phe, they did not induced chemokinesis, obtaining chemotaxis indexes of 421 k 47 and 419 k 46 for G-17 and G-34, respectively, in comparison with 429 2 51 2 21 in control samples with only Hank’s medium. The results obtained in the phagocytosis of latex beads are indicated in Fig. 2. Both G-17 and G-34 inhibited the phagocytosis of latex beads by macrophages, being statistically significant at concentrations of lo-” M (p < O.Ol), 10m9 M (p < O.Ol), and 10e8 M (p < 0.01). G-17 inhibited the phagocytosis of latex to a significantly higher degree than G-34 at the same concentrations (p C 0.01). The results obtained in the phagocytosis of C. albicans are indicated in Table 2. G-17 and G-34 inhibited the phagocytosis of C. albicuns. This inhibition was statistically significant with G-17 at the concentrations of lo-'M (p< 0.05) and 10m9M (p < O.OS), and with G-34 at lo-” M (p < 0.05), lo-“ M (p < 0.05), and lo-” M (p< 0.05). Thereduction of NBT in both stimulated and nonstimulated macrophages incubated with G- 17 or G-34 was similar to that in the controls (18 + 3 ,umol/lO* cells for stimulated cells and 10 +- 2 pmol/ 10’ cells for nonstimulated).

Table 3 shows the CAMP levels in macrophages incubated 30, 60, or 120 s with G-17 or G-34. Generally, higher levels of CAMP were found in cells incubated with G-17 or G-34 than in the controls. With G- 17, the significantly statistical differences were obtained at the concentration of lo-” M and lOmyMat all times, and at lo-* M at 60 and 120 s (p < 0.01). With G-34, CAMP levels were significantly higher than those of controls at the concentration of lo-‘* Mat 60 s (p < 0.05), and lo-“‘, 10-,9, and 10-XMat60and120s(p<0.01). Table 4 shows the indirect determination of the implication of CAMP in the action of G-17 or G-34 using an inhibitor of CAMP synthesis such as adenosine in the test of phagocytosis of latex beads in macrophages. Adenosine significantly increased (p < 0.001) the ingestion of latex, whereas G-17 at lo-” M and G-34 at lo-“’ M significantly inhibited (p < 0.01) the ingestion of latex, as seen in previous experiments. The combined presence of adenosine and either G-17 or G-34 produced similar values to those of controls without adenosine or gastrin. DISCUSSION

To our knowledge, this is the first study of gastrin effects on macrophage function. We have used peritoneal macrophages because they are easily available, and are representative of other macrophage populations (27). We have shown that the incubation of murine peritoneal macrophages with gastrin stimulated adherence, whereas it inhibited mobility, both spontaneous and directed to an infectious focus or chemotaxis, ingestion of both inert particles (latex beads) and cells (Candida albicans), and did not influence the production of superoxide anion. In general, the most effective concentration of both gastrins was 10e9 M. Gastrin concentrations between lo-” and 10-I’ M arefound in areas with access to immune cells as it occurs in peripheral blood and cerebrospinal fluid ( 12,16). The lower response to higher gastrin concentrations ( 10m6 M) might be ascribed to a process of cell desensitization, which is characteristic of sequestration and/or “downregulation” of different receptors (32). A similar fact has been found with other regulatory peptides with activity in the digestive tract such as gastrin-releasing peptide (GRP) (4), neurotensin (7), and VIP (6). Adherence, the first step in the immune response of phagocytes (33), was stimulated by gastrin in vitro, which could represent an activation stage of macrophages in vivo. Other peptides also stimulate adherence such as NPY and PYY (5)) bombesin, neuromedin C and GRP (4)) neurotensin and neuromedin N (7). andCCK(8). On the contrary, both gastrins inhibited in vitro the mobility step of the phagocytic process. The peptides decreased the mo-

TABLE 3 LEVELS

OF CAMP (pmol/lOx

CELLS)

IN MACROPHAGES

INCUBATED

WITH G-17 OR G-34

Peptide Concentrations CM) Peptide

G-17

G-34

Time (S)

30 60 120 30 60 120

,0-I?

0

26 26 28 26 27 27

t t 2 ? 2 -c

4 5 4 4 3 5

27 28 29 24 32 30

-c * + k -c 2

10-q

IO-“’

4 4 5 4 4* 3

31 33 33 27 39 42

t4* -c 5* k 5* 5 5 -c 4t -t 6t

32 40 42 29 42 44

The results represent the mean z SD of eight experiments performed in duplicate. * p < 0.05 and tp < 0.001 with respect to the control values.

-c 2 rt t -t -c

10-O

10-X

5* 61 .5t 4 5t 7t

27 38 40 25 41 39

t i_ -t ? 2 f

3 5t 6t 2 6-I 7t

26 27 29 27 33 31

2 4 2 4 t- 5 2 3 2 5 %6

223

GASTRIN EFFECT ON MACROPHAGES

TABLE 4 EFFECT OF ADENOSINE OR PHAGOCYTOSIS OF LATEX BEADS MACROPHAGES INCUBATED WITH G-17 OR G-34 (IO-“’ M)

None P

Adenosine

BY

C0ntrd

G-17

G-34

279 t 46
234 2 35* NS 265 2 50**

236 2 20* NS 250 +- 36**

The results represent the mean 2 SD of eight experiments performed in duplicate. * p < 0.01 and fp < 0.001 with respect to the corresponding control values.

such as f-Met-Leu-Phe or chemotaxis, whereas they were not chemoattractant for peritoneal macrophages. For this reason, the effect found is really a chemotaxis inhibition. Thus, both gastrins would prevent in vivo the accumulation of phagocytes in the inflamed area, thereby decreasing the phagocytic process. In addition, gastrin inhibited the ingestion of both latex beads and C. albicuns. Opposite effects on these macrophage functions were found for other peptides such as GRP (4)) neurotensin (7)) NPY (5), and VIP (6), which stimulated the peritoneal macrophage mobility and ingestion. However, CCK, a peptide that shares five amino acids with gastrin, was found to inhibit in vitro these macrophage functions (8). Although gastrin-17 inhibits adenylate cyclase in some cells, such as murine tumor pancreatic AR4-2J cells (22), in other cellular lines from human colon cancer this peptide increases CAMP levels, whereas in others of similar origin it increases inositol phosphate hydrolysis (14). Our data show that gastrin-17 and gastrin-34 increase CAMP levels in murine peritoneal macrophages. bility directed by chemical gradient of a chemoattractant

It is known that agents that decrease CAMP levels produce an increase in chemotaxis ( 11) , whereas increased CAMP levels inhibit several functions of the phagocytic process (21). Thus, we have found that some peptides such as GRP (4), NPY (5), and VIP (6) decreased CAMP levels in peritoneal macrophages and increased the phagocytic process. On the contrary, CCK-8s increases CAMP levels in murine peritoneal macrophages and decreases their phagocytic functions ( 8). Gastrin seems to act in a similar manner as CCK-8s in these cells. Thus, gastrin-17 and gastrin-34 produce a decrease in the phagocytic process of murine peritoneal macrophages through an elevation of CAMP. Moreover, we have shown in this work that adenosine, which decreases CAMP levels (31), produced an augmentation in the ingestion capacity of peritoneal macrophages; this effect was reversed by gastrin addition. The reason why adherence is the only activity increased by gastrin could be the fact that adherence is stimulated by an increase of both IP1 and CAMP levels (13,25,33). However, an increased adherence capacity of macrophages could be an immunological disadvantage because it would stop the cells and prevent their arrival to the infectious focus. Our results show for the first time that gastrin-17 and gastrin-34 are negative modulators of several macrophage functions. Thus, gastrin could inhibit in vivo the stimulating action on immune cells of other regulatory peptides. In the particular case of GRP, it stimulates macrophage functions and also the release of gastrin in vivo that in turn could inhibit the effects produced by GRP. ACKNOWLEDGEMENTS

We thank N. Guayerbas, J. F. G. Arriaza, and M. Catalti for their technical assistance. This work has been supported by grant No. C239/ 91 from Plan Regional de Investigacibn de la Consejeria de Educaci6n de la Comunidad de Madrid and grant No. 0697/92 from Fond0 de Investigaciones Sanitarias de la Seguridad Social (FISss).

REFERENCES 1. Blalock, 1. E. Molecular basis for bidirectional communications between the immune and neuroendocrine systems. Physiol. Rev. 69:132; 1989. 2. Boyden, S. V. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 11:453457; 1962. 3. De la Fuente, M. Changes in the macrophage function with aging. Comp. Biochem. Physiol. [A] 81:935-938; 1985. 4. De La Fuente, M.; Del Rio, M.; Ferrandez, D.; Hemanz, A. Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C. Immunology 73:205-211; 1991. 5. De la Fuente, M.; Bemaez, I.; Del Rio, M.; Hemanz, A. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C. Immunology 80:259265; 1993. 6. De La Fuente, M.; Delgado, M.; de1 Rio, M.; Martinez, C, Hemanz, A.; Gomariz, R. P. Stimulation by vasoactive intestinal peptide (VIP) of phagocytic function in rat macropbages. Protein kinase C involvement. Regul. Pept. 48:345-353; 1993. De La Fuente, M.; Garrido, J. J.; Arahuetes, R. M.; Hemanz, A. Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N. J. Neuroimmunol. 42:97-104, 1993. De La Fuente, M.; Campos, M.; de.1Rio, M.; Hemanz, A. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul. Pept. 55:47-56; 1995. Edkins, J. S. On the chemical mechanism of gastric secretion. Proc. R. Sot. Land. Biol. Sci. 76:376; 1905

10. Eysselein, V.; Maxwell, V.; Reedy, T.; Wunsch, E.; Walsh, J. Similar acid stimulatory potencies of synthetic human big and little gastrins in man. J. Clin. Invest. 73:1284-1290; 1984. 11. Hatch, G. E.; Nichols, W. K.; Hill, H. R. Cyclic nucleotic changes in human neutrophils induced by chemoattractants and chemotactic modulators. J. Immunol. 119:450-456: 1977. 12. Henderson, A. R.; Tietz, N. W.; Rinker, A. D. Gastric, pancreatic, and intestinal function. In: Tietz, N. W., ed. Texbook of cl&al chemistry. Philadelphia: W. B. Saunders Company; lYY4:1576-1620. 13. Isaad, C.; Ventura, M. A.; Thomopoulos, P. Biphasic regulation of macrophage attachment by activators of cyclic adenosine monophosphate-dependent kinase and protein kinase C. J. Cell Physiol. 140:317-322; 1989. 14. Ishizuka, J.; Townsend, C. M.; Bold, R J.; M&z, J.; Rodriguez, M.; Thompson, J. C. Effects of gastrin on 3’,5’-cyclic adenosine monophosphate, intracellular calcium and phosphatidylinositol hydrolysis in human colon cancer cells. Cancer Res. 542129-2135; 1994. 15. Jensen, D. M.; McCallum, R.; Walsh, J. H. Failure of atropine to inhibit gastrin-17 stimulation of the lower esophageal sphincter in man. Gastmenterology 70:825-827; 1978. 16. Martinez, M.; Hemanz, A.; Grande, C.; Pallardo, L. F. Plasma molecular forms of gastrin, neurotensin in pregnancy and gestational diabetes after oral glucose load or a mixed meal. Regul. Pept. 47: 73-80; 1993. 17. McGuigan, J. Gastrin mucosal intracellular localization of gastrin by immunoflourescence. Gastroenterology 55:315-327; 1968. 18. Mihas, A. A.; Gibson, R. G.; Mihas, T. A. Effects of gastrointestinal hormonal peptides on the transformation of human peripheral lym-

224

19.

20.

21.

22.

23.

24.

25.

DE LA FUENTE

phocytes. Res. Commun. Chem. Pathol. Pharmacol. 73: 123- 126; 1991. Noga, S. J.; Norman, S. J.; Weiner, R. S. Methods in laboratory investigation: Isolation of guinea pigs monocytes and Kurloff cells. Characterization of monocyte subsets by morphologic, cytochemistry, and adherence. Lab. Invest. 5 I:244249; 1984. Powell, C. T.; Ney, C.; Aran, P.; Agarwal, K. A gastrin gene is expressed in both porcine pituitary and antral mucosal tissues. Nucleic Acids Res. 13:7299-7305; 1985. Rivkin, I.; Rosemblatt, J.; Becker, E. L. The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. J. Immunol. 115: 1126- 1134; 1975. Scemama, J. L.; Robberectb, P.; Waelbroek, M.; et al. CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxinsensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4-21. FEBS Lett. 242:61-&l; 1988. Showell, H. J.; Freer, R. J.; Zigmond, S. H.; et al. The structureactivity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J. Exp. Med. 143:1154-1159; 1976. Soumarmon, A.; Chere,t A.; Lewin, M. Localization of gastrin receptors in intact isolated and separated rat fundic cells. Gastroenterology 73900903; 1977. Sung, C. P.; Arleth, A. J.; Storer, B.; Feuerstein, G. Z. Modulation

26.

27.

28.

29.

30.

31.

32. 33.

ET AL.

of U937 cell adhesion to vascular endothelial cells by cyclic AMP. Life Sci. 49:375-382; 1991. Tepperman, B.; Walsh, J.; Preshaw, R. Effect of antral denervation on gas&in release by sham feeding and insulin hypoglycemia in dogs. Gastroenterology 63:973-980; 1972. Unanue, E. R. Macrophages, antigen-presenting cells, and the phenomena of antigen handling and presentation. In: Paul, W. E., ed. Fundamental immunology. New York: Raven Press; 1989:95-l 15. Unanue, E. R.; Allen, P. The basis for the immunoregulatory role of macrophages and other other accessory cells. Science 236:551-557; 1987. Uvnus-Wallensten, K.; Refeld, J.; Larsson, L.; Uvnas, B. Heptadecapeptide gastrin in the vagal nerve. Proc. Nat]. Acad. Sci. USA 74:5707-5710; 1977. Valenzuela, J.; Bugat, R.; Grossman, M. Effect of big and little gastrins on pancreatic and gastric secretion. Proc. Sot. Exp. Biol. Med. 159:237-238; 1978. Williams, R. S.; Bishop. T. Enhanced receptor-cyclase coupling and augmented catecholamine-stimulated lipolysis in exercising rats. Am. J. Physiol. 234:E345-E351; 1982. Yu, S. S.; Lef kowitz, R. J.; Hausdorff, W. P. P-Adrenergic receptor sequestration. J. Biol. Chem. 268:337-341; 1993. Zabrenetzky, V.; Gallen, E. K. Inositol 1,4,5&phosphate concentrations increase after adherence in the macrophage-like cell line J774.1. Biochem. J. 255:1037-1043; 1988.