Histamine synthesis by intact mast cells from canine fundic mucosa and liver

GASTROENTEROLOGY 1982;82:254-62

Histamine Synthesis by Intact Mast Cells from Canine Fundic Mucosa and Liver MICHAEL

A. BEAVEN,

ANDREW

H. SOLL,

and

KLAUS J. LEWIN Medical and Research Services, Wadsworth Veterans Administration Hospital and Departments of Medicine and Pathology; UCLA School of Medicine, Los Angeles, California; and National Heart, Blood, and Lung Institute, National Institutes of Health, Bethesda, Maryland

The synthesis and degradation of histamine by dog fundic mucosa was studied by using cells dispersed by enzymatic digestion and separated sequentially by velocity sedimentation in an elutriator rotor, and by density gradient. Histidine decarboxyiase activity was found in appreciable amounts in fractions highly enriched in mast cells when these cells were studied intact, whereas only trace activity was, detected in homogenates of these mucosal mast cells or of whole mucosa. Unlike the rat gastric mucosal histamine cell, dihydroxyphenylalanine decarboxylase activity was not present in the canine fundic mast cell. Serotonin, which is found in the rat peritoneal mast cell, was not detectable in the canine mast cell. The histamine-degrading enzyme, histamine methyltransferase, was also present in gastric mucosal cells, but not diamine oxidase. This methyltransferase activity was primarily associated with parietql cells and was not found in the mast cell-enriched fractions. For comparison, fractions containing 60%--8003, mast cells were enriched by elutriatibn from enzyme-dispersed cells of canine liver. As with the gastric mast cells, histidine decarboxylase activity was found in intact cell, but it was lost upon cell disruption.

Received April 14,1981.Accepted September 25, 1981. Address requests for reprints to: Andrew H. Soll, M.D., Wadsworth Veterans Administration Hospital, Building 115, Room 203, Los Angeles, California 90073. This work was supported by National Institute of Arthritis, Metabolism, and Digestive Disease Grants 17328 and 19984, and by the Medical Research Service of the Veterans Administration. The authors are indebted to the late Morton I. Grossman for his selfless support, encouragement, and many suggestions that underlie the inception and execution of these studies. The authors thank Reynaldo Rodrigo, Brodie Brickie, and June Ferrari for their invaluable technical assistance.

The importance of histamine in the physiologic regu!ation of gastric acid secretion was unequivocally established in 1972 with the finding that Hzreceptor antagonists block the secretory response to all stimulants (1). Little is known, however, about the localization of the various enzymes involved in the synthesis and degradation of histamine in the gastric fundic mucosa. In species other than the rat, the histamine-forming enzyme histidine decarboxylase has not been detected in appreciable amounts in extracts of fundic mucosa. We have recently developed techniques for preparing highly enriched fractions of histamine-containing cells from suspensions of canine fundic mucosa cells by sequential application of velocity and density separation techniques (2). In these fractions, mast cells accounted for most, if not all, of the histamine content (2). Even though these techniques yielded highly enriched fractions of viable mast cells, preliminary studies found only trace activities (~30% above blank values) of histidine decarboxylase in extracts of these cells. In the present work we have addressed the question of whether significant histamine formation occurs in intact mast cells isolated from canine fundic mucosa and liver. Both of these tissues are rich sources of mast cells in dog (2-4). We show that histamine is synthesized from histidine by intact mast cells, but that very little histidine-decarboxylase activity is demonstrable after cell disruption. Dihydroxyphenylalanine (dopa) decarboxylase activity and serotonin have also been studied as markers of enterochromaffinlike cells, and neither are associated with the canine mast cells. We further show that the major histamine degradative enzyme in canine fundic mucosa is histamine methyltransferase and that this enzyme is present primarily in the parietal cell.

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February 1982

Methods and Materials Cell Isolation

and

Separation

Mongrel dogs were fasted overnight and killed by pentobarbital infusion. The fundic mucosa was stripped from the submucosa, then chopped in a McIllwain tissue chopper, and incubated sequentially with collagenase (Sigma collagenase I, 0.25 mg/ml) and ethylenedianinetetraacetate (EDTA) (1 mM) as described elsewhere (5). On a basis of histamine content per microgram of DNA, 69% of the histamine was recovered from the cells dispersed by these techniques (2). Mucosal cells were separated by sedimentation velocity with an elutriator rotor (2,5,6). Dispersed cells (2 x 10')were loaded in a 20-ml volume at a flow rate of 20.1 ml/min and a velocity of 2800 rpm, and the first fraction was collected. Fractions 2 and 3 were also collected at this flow rate at velocities of 2300 and 2100 rpm, respectively. Fractions 4 through 8 were collected at a velocity of 2000 rpm and flow rates of 22.5, 29.0, 34.4, 44.0, and 54.0 ml/min, respectively. A volume of 100 ml was collected for each fraction. This protocol was chosen to allow separation of the small cells without the necessity of the long collection periods required by flow rates under 20 ml/min. For the latter fractions, increasing flow rates were used to further shorten the separation time and because clumping more frequently occurred in the chamber at velocities below 2000 rpm during this initial load of dispersed cells. A second elutriation was used to further enrich parietal cells. Fraction 8 (80-100 x lo6 cells), which contained 50%-65% parietal cells, was loaded at a flow rate of 31 ml/min and a velocity of 2100 rpm, and the first fraction was collected. The subsequent five fractions were then collected at this same flow rate at velocities of 2000, 1900, 1800, 1700, and 1600, respectively. A 250-ml volume was collected for fraction 2. Density separation was accomplished in a Sorvall zonal rotor using an isotonic, linear gradient formed from a heavy solution of 20% bovine serum albumin (BSA) plus 9% Ficoll in distilled water and a light solution of 12% BSA with 0.7x Hanks’ balanced salts solution added to achieve isotonicity (2). A total gradient volume of 1200 ml was used. Fractions of 20 ml were collected, diluted to 50 ml, and then centrifuged. The cells from four consecutive fractions were pooled and resuspended in Hanks’ solution. Identical techniques were used to disperse cells from liver tissue. Liver cells were separated in the elutriator rotor loading 1.5 x 10'cells at a flow rate of 21 ml/min and a velocity of 2800 rpm. Fractions 2 through 4 were collected at a flow rate of 21 ml/min at velocities of 2600, 2400, and 2200 rpm, respectively. Fractions 5 through 7 were collected at a velocity of 2000 rpm and flow rates of 23,29, and 37 ml/min, respectively. Fraction 8 was composed of the cells remaining in the rotor after collecting fraction 7.

Histologic

Studies

and

Identification

of Cell

Types Cells were fixed in 1% glutaraldehyde in o.l-mM cacodylate buffer (pH 7.4), embedded in Epon and pre-

255

pared for light and electronmicroscopy (23. Slides were also prepared from a 0.5-ml suspension with lo5 cells centrifuged at 800 rpm for 2 min in a cytocentrifuge (Shannon-Southern, Runcorn-Cheshire, England). These slides were then fixed in the buffered glutaraldehyde solution for toluidine-blue staining (0.1%; pH 4.0). Mast cells were identified by the characteristic metachromatitally stained granules on light microscopy, and by their granular structure on electronmicroscopy. Periodic acid Schiff- (PAS) stained paraffin sections of Bouin’s fixed-cell pellets or Bouin’s fixed-cytocentrifuge slices were examined to determine the distribution of parietal cells and mucous (PAS-positive) cells (6). Pepsinogen content of the cell fractions was determined with a modification of a hemoglobin assay standardized against hog pepsin (6). Differential counts were done by two observers who were unaware of how the fractions had been prepared.

Measurement of Biogenic Enzyme Activities

Amines

and

Histidine decarboxylase and dopa-decarboxylase activities were assayed in soluble cell extracts by a microprocedure in which 14C02 release from labeled-carboxyl Lhistadine or dopa was measured (7). To assay histidine decarboxylase activity in intact cells, cell suspensions were sedimented at 100 g for 10 min, and then resuspended in Gey’s solution (GIBCO, Grand Island, N.Y.) to give lo6 cells/100 ~1. Ten microliters of a reaction mixture containing [l-14C]L-histidine (New England Nuclear Corp., Boston, Mass.), 50 mM HEPES buffer, and 10 PM pyridoxal phosphate was then added to 10 ~1 of the cell suspension, and the mixture was incubated for 60 min at 37%. 14C02 was trapped in hyamine hydroxide (7). Histamine formation was also assessed by isotope dilution; 20 nCi of [/33H]L-histidine (tritiated on the /3 carbon of the side chain, New England Nuclear Corp.) was included in the incubation medium in addition to the labeled-carboxyl histidine. After the evolution of 14C02, the final reaction mixture was assayed for [3H]histamine by isotope dilution derivative analysis as described elsewhere (8). The concentration of histidine used during these assays was 0.25 mM. Cells were disrupted where indicated by freezing the cell suspension on dry ice 3 times and thawing. Inhibitors were added in a small volume (2 ~1) before addition of the reagent. With either the 14COz-release assay or the isotope dilution assay, Brocresine (NSD 1055, 10 PM), a potent inhibitor of decarboxylase enzymes, completely suppressed histidine decarboxylase activity. The activity measured in the presence of 10 PM Brocresine was thus taken as the blank and was subtracted from the other values obtained. Histamine (9) and serotonin (9a) were assayed by an enzymatic isotopic assay in which the amines were converted to 14C-methylated metabolites by incubation with S-adenosyl-L-methionine ([14C]methyl) and the appropriate methyltransferase enzyme. Histamine methyltranferase was assayed by the method of Beaven and Horakova (lo), and diamine oxidase by measurement of tritium release from [P-3H]histamine (11).

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Results

Histamine Synthesis Preparations

Distribution of Histamine, Serotonin, and Decarboxylase Enzymes in Fundic Mucosal Cell Fractions After separation of the cell fractions by elutriation, histamine and serotonin were recovered in cells of small size (Figure 1A). These small cell fractions (2-4) were then further fractionated by density to produce separate histamine and serotonin peaks in the dense and light portions of the gradient, respectively (Figure 1B). In extracts of fractions with high histamine contents disrupted by sonication (fractions 1, 5, 9, 13 in Figure lB), histidine decarboxylase activity was detectable (about twice the blank value), but it was much lower than that in rat peritoneal mast cell or gastric enterochromaffinlike (ECL) cell (12). In contrast, dopa-decarboxylase activity was present in these disrupted fundic mucosal cells at levels 2000 times that of histidine decarboxylase, and it was distributed mainly in fractions of high serotonin content, although some additional activity was present in fractions which did not contain serotonin (fractions 17, 21, 25 in Figure 1B). Distribution of Histamine Activities in Mucosa

Catabolic

Enzyme

The intact dog fundic mucosa and isolated mucosal cells contained very little diamine oxidase (histaminase) activity (less than twice blank value) but high histamine methyltransferase activity* (Figure 2). A major portion of the methyltransferase activity was associated with large mucosal cells (fractions 6-8, Figure 2A), and a minor portion with cells of smaller size (fractions 2 and 3, Figure 2A). On further separation of large cells by the second elutriator protocol, the distribution of methyltranferase activity paralleled that of parietal cells (Figure 2B). The methyltranferase activity in the smaller cells (fractions 2-4, Figure 2A) was not clearly associated with a single cell type. However, upon density-gradient separation of these small cell fractions, no activity was associated with mast cells, which predominated in fractions l-9 of the gradient (Figure 2C).

* Histamine methyltransferase activity in the unfractionated cell suspension was 239 pmol/h . 10" cells or 1.8 pmol/h . pg protein and in the enriched parietal cell suspension (fraction 8, Figure 2A) was 790 pmol/h .lo6cells or 3.4 pmol/h . pg protein. This value compares to values of 46 and 2.1 pmol/h . pg protein, respectively, for partially purified preparation of histamine methyl transferase from rat kidney and guinea pig brain, which are two sources of enzyme used for the isotopic assay of histamine.

Vol. 82, No. 2

by Intact Cells

In contrast to the cell extracts, intact cells exhibited low but significant histamine synthesis, as measured both by ‘*CO2 release and by [P-3H]histamine formation (Figure 3). The rate of histamine synthesis and histamine content were highly correlated (r = 0.90; p < 0.001) in the different cell fractions (Fig. 3). Decarboxylation was inhibited by cu-methylhistidine, a specific inhibitor of histidine decarboxylase, and also by tryptophan (Table 1). Disruption of cells by freezing and thawing or by sonication decreased histamine synthesis (Table 1). Isolation

of Mast Cells from Liver

Elutriation of cells obtained by enzymatic digestion of canine liver resulted in a discrete distribution of histamine (Figure 4A). Sixty percent to 80% of the cells in the fractions with the maximal content of histamine were mast cells (Figure 5). Their granules demonstrated metachromasia when stained with toluidine blue, and they had a typical variable appearance of mast-cell granules in electronmicrographs+ (13,14). There was a good correlation between the number of mast cells and the amount of histamine in the various fractions (Figure 4A). Correcting for the proportion of mast cells, the histamine content of the hepatic mast cells was estimated at 2.5 pg/cell. Histidine decarboxylation by intact cells was evident with either the ‘*CO2 release assay or with the formation of isotopically-labeled histamine (Figure 4B). This activity was three to four times greater than that of the histamine-containing cells from fundic mucosa. Although the two procedures gave comparable results for cells in the histamine-rich fractions, in the fractions containing large cells the 14COz-release assay gave higher values than were found by isotope dilution (Figure 4B). The rate of histidine decarboxylation was decreased upon cell disruption or by the addition of tryptophan or (Ymethylhistidine (Table 1).

Discussion High concentrations of histamine are found in the acid-secreting portion of the gut of all species with a well-defined stomach (15,16). The ability of + Granular detail in the mast cells (Figure 5B) reveals vacuolization. Although this finding may have resulted from effects of dispersion and cell separation, similar granular morphology was found in mast cells in intact canine fundic mucosa (2) suggesting either fixation artifact or an accurate reflection of granular structure.

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HISTAMINE FORMATION BY INTACT CELLS

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257

A

Figure x EI Ez
1. Distribution of biogenic amines and decarboxylase activities in cell fractions from dog gastric mucosa. The distribution of histamine and serotonin content and histidine decarboxylase and dopa decarboxylase activities are shown for cell fractions separated by elutriation (panel A] and by linear density gradient of elutriator fractions 2 and 3 (panel B). Decarboxylase activities were measured by “%Oz release assay. Data are from one mucosal preparation representative of two oth-

$b =-

ers

u)

5

1

123456

13 17 21 25 29 33 37 41 45 49

9

FRACTION

Figure

rAAAAA4 2

4

3

5

6

7

-0 12

8

3

4

5

8

-4

C

-3

-2

-1

1

5

9

13

17

21

25

29

FRACTION

33

37

41

45

49

2.

Distribution of histamine degrading enzymes in dog mucosal-cell fractions. A. Histamine methyltransferase (HMT), and diamine oxidase (DAO), as well as pepsinogen content and the percentage of parietal cells and mucous (PAS-positive) cells in fractions separated by elutriation are illustrated. B. A greater enrichment of the parietal cells was obtained by a second elutriation of fraction 8 from panel A. C. Data for HMT activity is from the density gradient shown in Figure 1B. Data from one preparation representative of two others.

258

BEAVEN ET AL.

GASTROENTEROLOGY

Table

1.

The Effects of Inhibitors Histidine

Decarboxylase

Vol. 82, No. 2

and Cell Disruption Activity

on

Histidine decarboxylase activity” (pmoI/h . lo6 cells) Gastric mucosa Treatment

2,3b

l-SC

Liver 4d

None cx-Methylhistidine (W3 M) Tryptophan (1Om3M) Freezing and thawinge

7.2k 1.2

12.2

48 k 8

2.9 f 1.8 2.0f 1.4 0.2* 0.1

6.0 1.0 1.4

14 r 5 10 t 4 322

a Histidine decarboxylase activity was determined by the %Ozrelease assay as described in the methods, Values are means * SE for 3 preparations of either gastric or liver cells. b Fractions with peak histamine content from the elutriator separation of gastric mucosa (Figure 1A). ’ Fractions from the density separation (Figure 1B). d Fraction 4 from the elutriator separation of liver cells (Figure 4). e Sonication also caused a similar degree of suppression of activity.

HISTAMINE(pg/cell) Figure 3. Histamine content and histidine decarboxylase activity in canine fundic mucosal mast cells. Decarboxylase activity in cells disrupted by freezing and thawing (0, A) and in intact cells (0, A) is plotted as a function of the histamine content of the fraction. Histidine decarboxylase activity was measured by 14COz release (0,O) or isotope dilution (A, A). For the CO* release assay, data are from elutriator fractions 2 and 3, and for density-gradient fractions l-9 and 37-41 from three separate cell preparations. Data for the isotope dilution are from the same density-gradient fractions from two preparations. Histamine content and histidine decarboxylase activity were highly correlated (r = 9.99, p < O.OOl].

Hz-receptor antagonists to block acid secretion produced in response to feeding or gastrin administration-as well as to histamine-indicates that gastric histamine plays an essential role in the physiologic regulation of acid secretion (l,17). Significant levels of histidine decarboxylase activity, however, have been detected only in the fundic mucosa of rats (18) and possibly rabbits (19) and guinea pigs (20), but not in the fundic mucosa of dogs (4) or humans (2l), although conflicting data on this latter point have been reported (22). In addition, gastrin induction of histidine decarboxylase has been found in rats (20), and possibly in rabbits (19) and guinea pigs (zo), but not in dogs (4) or humans (21). Thus in the latter two species the formation of histamine in the fundic mucosa and the factors regulating this formation remain unclear (23). The differences between these species may reflect the findings that fundic mucosal

histamine is stored in endocrinelike cells in the rat (23-25) and in mastlike cells in dogs (2,4,26) and humans (21,26-28). Intact, isolated canine mast cells were found to synthesize histamine. Because the release of 14C02 from histidine can occur by pathways other than through the action of histidine decarboxylase (20), measurement of enzyme activity was verified by measuring histamine formation with an isotopedilution technique. These two procedures yielded similar results for the fractions examined from dog gastric mucosa. For liver cells, however, histidine decarboxylase activity by the two methods were similar only for the histamine-cell-rich fractions (fractions 2-4, Figure 4B), but not for fractions with large hepatic cells (fractions 5-8, Figure 4B). In these latter fractions, histidine was decarboxylated without the appearance of histamine, indicating either that newly formed histamine was being rapidly degraded or that decarboxylation occurred through routes other than histidine decarboxylase (20). Histamine synthetic activity was lost upon disruption of the canine fundic mucosal and liver mast cells. A similar loss of synthetic activity has been observed in rat peritoneal mast cells (29). This effect has been attributed in part to a loss of a transport system (system N) that concentrates histidine within the cell (29). Tryptophan and glutamine were found to inhibit both histidine uptake and decarboxylation by rat peritoneal mast cells, but not to block histidine decarboxylase activity in cell extracts (29). The present demonstration that tryptophan blocks histamine synthesis in canine mast cells may also indicate the importance of histidine uptake in these cells, although this possibility was not directly test-

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HISTAMINE FORMATION BY INTACT CELLS

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259

12345678

345670 FRACTION

Figure 4. Separation by elutriation of histamine-containing cells from canine liver. A. Histamine content and the percent mast cells are illustrated for the different fractions. Data are from a single separation representative of two others. When histamine content vs. percent mast cells for each fraction were plotted, a high degree of correlation was found (r = 0.98, p < O.OOl], and a value of 2.5 pg histamine/mast cell was obtained by regression analysis. B. Histidine decarboxylase activity obtained by “‘COz-release and isotope dilution assay of [3H]histamine are shown for the same cell fractions as in panel A. The correlation between histamine content and histidine decarboxylase activity (by isotope dilution) was highly significant (r = 0.83, p < O.Ol]. The CO, release assay yielded much higher values for fractions 6-8.

ed. The uptake of histidine and the formation of histamine have been previously demonstrated in basophils (30,31). A component of the loss of decarboxylase activity may also be due to the lability of the decarboxylase enzyme, particularly in light of the high activity of proteolytic enzymes that may be released upon mast cell disruption (32). However, protease inhibitors did not significantly preserve histidine decarboxylase activity upon disruption of rat peritoneal mast cells (29). With canine mast cells the role of proteolysis in the loss of enzyme activity with cell disruption has not been tested, and this factor may be of importance. In contrast to the present data with canine fundic mucosal mast cells, soluble decarboxylase activity can be readily extracted from the rat gastric histamine cell (12). This contrast may reflect the differences between the proteases released upon disruption or a less-labile histidine-decarboxylase enzyme in the rat gastric histamine cell. Correcting for the mast cell,content of the fractions examined, we estimate a histamine content of 2.5 pg/ mast cell for both the canine fundic mucosal and liver mast cells. The histamine synthetic activity for these cells respectively range “from 10 to 20 and from 30 to 90 pmol/106 cells * $ These values are lower than those found in peritoneal mast cells (17 pg/ histamine cell and 500 pm01 histamine/h * 10” cells) (29) and’gastric mucosal histamine cells in the rat (2-8 pg histamine/cell and 150 pmql histamine/h * lo6 cells) (12). Although no direct measurements

have been made, several lines of reasoning suggest that the rate of histamine synthesis and release in dog fundic mucosa in vivo is low. A large portion of the mast cells in dog mucosa lie in close proximity to parietal cells (2): Earlier studies with isolated parieta1 cells have shown that histamine in concentrations as low as 0.1 PM markedly potentiates the actions of both gastrin and cholinergic agents (33), so only small amounts of histamine may be required for adequate functioning of the gastric secretory system in dog. The major degradative pathway for histamine in dog gastric mucosa is methylation of the Nr-imidazole nitrogen, a reaction catalyzed by histamine methyltransferase (34). The activity of this enzyme is especially high in dog gastric mucosa, and the present studies indicate that a major portion of the enzyme is associated with the parietal cell. The presence of an inactivating enzyme in or on the target cell itself may be one reason why only small amounts of histamine leak from the mucosa of this species (35). Further evidence for the rapid degradation of histamine within the gastric mu&a comes from studies in which prior administration of inhibitors of histamine methyltransferase enhanced acid secretion in response to histamine (39) and to pentagastrin (37). In the rat the major source of the gastric mucosal histamine is an endocrinelike cell (24,25,38) that possesses the APUD characteristic (39) of being able to take up and decarboxylate amino-acid precursors

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Figure 5. Mast cells from canine liver. Light [A) and electron micrographs (B) are illustrated for the fraction with maximal histamine content (fraction 4 from Figure 4).

k

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HISTAMINE FORMATION BY INTACT CELLS

1982

to form dopamine and serotonin (25,381. Fractions of dispersed rat gastric mucosal cells enriched in ECL cells by the present techniques contained both high histidine- and dopa-decarboxylase activities, but do not appear to contain significant amounts of serotonin (12). In contrast, the rat peritoneal mast cell contains histamine and serotonin, but little dopadecarboxylase activity in cell extracts (29). The content of serotonin may reflect uptake of preformed serotonin, rather than decarboxylation of precursor (40). The canine fundic mast cell has not been found previously to have APUD characteristics (4), and we detected no serotonin or dopa-decarboxylase activity in the fractions enriched in these fundic mast cells. However, dopa decarboxylase and serotdnin are present in an unidentified (perhaps enterochromaffinlike) cell, which is concentrated in fractions of low density. The interrelationships between the cells that store histamine and serotonin and those that are capable of taking up and decarboxylating amines precursors are complex and remain to be sorted out.

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gastric secretion.

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