GLUCOCORTICOID-INDUCED GROWTH INHIBITION OF HUMAN NEOPLASTIC SALIVARY GLAND DUCT CELL LINE (HSG)

The purpose of this study was to examine the effect of glucocorticoid on human neoplastic salivary duct epithelial cell line (HSG). Dexamethasone was found to inhibit cell growth and to increase cell size and the ratio of protein content to DNA content in a cell. The inhibition of cell growth was dose-dependent; in comparison to the control (33.8±3.1 h), the population doubling time was 1.57-fold longer in 10⁵ M dexamethasone (P<0.01, N-K test). [³H] thymidine incorporation was inhibited in 45.5% of the control at 10^-5 M. Plating efficiency was 20.5±3.0% in 10⁵ M and 47.0±4.4% in the absence of dexamethasone. Cell diameters increased 1.29 fold in 10^-5 M dexamethasone in comparison to the control size (16.0±2.1 μm). The ratio of total protein content of DNA content increased 1.46 fold in 10^-5 M dexamethasone-treated cells on the seventh day of cultivation. Scatchard plot analysis using [6, 7-³H] -triamcinolone revealed that the HSG cells had apparent cytosolic glucocorticoid receptors with an equilibrium dissociation constant (Kd value) of 6.48 nM, whose number of binding sites (NBS) was 57.8fmol/mg protein.     

The effects of triacetyloleandomycin and oleandomycin phosphate on the glucocorticoid receptor in cultured skin fibroblasts

Triacetyloleandomycin (TAO) has been described as a "steroid-sparing" drug in that poorly controlled asthmatic patients can be stabilized or improved with the addition of TAO despite decreasing dosages of steroids, specifically methylprednisolone (MP). It is not clear whether the beneficial effects of TAO are due to decreased MP clearance or are due to enhanced glucocorticoid effect peripherally. We tested the latter possibility by studying the interaction of TAO and oleandomycin phosphate (OLEO), the active metabolite of TAO in vivo, with glucocorticoid receptors in dispersed, intact cultured human skin fibroblasts. With the use of cells incubated with [3H]dexamethasone at 22 degrees C, we examined the competitive binding properties of TAO and OLEO (at varying concentrations) with and without MP as compared with several other steroids and MP alone. We also studied the effects on cellular glucocorticoid receptor number and affinity when TAO at a concentration of 4 X 10(-5) alone, at 10(-5) M in combination with a receptor saturating concentration of MP (5 X 10(-8) M), with MP alone, or with OLEO (10(-5) M) combined with MP was added to fibroblasts in the growth phase 1 wk before assay. TAO and OLEO did not compete for the binding of [3H]dexamethasone to the fibroblast glucocorticoid receptor, nor did they alter the binding properties of MP. With prolonged cellular exposure, TAO alone did not alter the number of glucocorticoid receptors (per cell) or their affinity for [3H]dexamethasone. Interestingly, prolonged exposure to saturating concentrations of MP alone decreased glucocorticoid receptor density; this effect was not altered by the presence of TAO or OLEO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of thymosin on glucocorticoid receptor activity and glucocorticoid sensitivity of human thymocytes

Incubation with thymosin fraction 5, (TMS F5 at 300 micrograms/ml) a partially purified thymic factor, reduced the steroid binding activity of human infant thymocytes from 9.6 +/- 2.1 fmole/ml to 5.0 +/- 2.0 fmole/ml. The glucocorticoid receptor activity in normal infant thymocytes was found to be 2,146 +/- 726 (s.d.) sites per cell with dissociation constant of 1.4 +/- 0.6 X 10(-8)M. TMS F5 also increased the resistance of human thymocytes to the cytolytic effect of dexamethasone (2.5 X 10(-8)M) to 168.6 +/- 30.2% of control (P less than 0.01). In animals, medullary and peripheral blood T cells are more resistant to glucocorticoids than immature thymic T cells. The results show that thymosin can induce changes consistent with differentiation in human thymocytes. These in vitro results are consistent with a physiological role of thymosin in intrathymic T cell maturation in man. Incubation of a human malignant thymus derived T cell line (MOLT 3) with TMS F5 also resulted in a significant reduction of the number of steroid binding sites to 44.2 +/- 15.3% of control (P less than 0.05), but TMS F5 did not significantly reduce the glucocorticoid sensitivity of MOLT 3 cells.     

Regulation of plasminogen activator secretion, interferon induction and proliferation in murine macrophages

The purpose of this work was to study the interrelationship of proliferation and secretion of plasminogen activator (PA) and interferon (IFN) by murine macrophages. For induction of macrophage proliferation and secretion of PA, concanavalin A (Con A) was used. Secretion of IFN was induced by polyinosinic polycytidylic acid complex. The glucocorticoid dexamethasone acetate (DA) (10(-6)-10(9) M) inhibited Con A-stimulated secretion of PA and synthesis of DNA as evaluated by incorporation of [3H]thymidine. DA did not inhibit IFN induction. Preincubating macrophages with DA for 45 h reduced basal proliferation and secretion of PA but did not reduce responsiveness to Con A. Also retinoic acid, a modulator of carcinogenesis was used in inhibition studies because of its known antagonistic effects on lymphocyte mitogenesis. In macrophages a biphasic effect of retinoic acid (1 X 10(-5) - 5 X 10(-5)M) was found: (a) inhibition of DNA synthesis and secretion of PA during the first 45 h of incubation, and (b) enhancement of DNA synthesis (but not PA secretion) after 72 h. Secretion of IFN was not affected. It is suggested that secretion of PA but not IFN is linked to cell cycle traverse of macrophages.     

Differential effects of adrenocorticotropin(1-24) on [3H]2-deoxy-D-glucose uptake in cultured cells derived from different brain regions

The effect of adrenocorticotropin(1-24) (ACTH(1-24)) on the uptake of [3H]2-deoxy-D-glucose ([3H]2-DG) was compared in cell cultures derived from two regions (hypothalamus, and extrahypothalamic forebrain) of fetal rat brain. Under control conditions, [3H]2-DG uptake was similar in extrahypothalamic (10.9 +/- 1.1 nmol/mg protein/5 min) and hypothalamic (11.9 +/- 1.3) cells. No significant effect of ACTH (1-24) (10(-7) to 10(-5) M) was found on uptake of [3H]2-DG in extrahypothalamic cells. In contrast, in hypothalamic cells, a potent stimulatory effect (P less than 0.0001) up to 174% over the control value of [3H]2-DG uptake was produced by these concentrations of ACTH(1-24). This study suggests that ACTH may be a stimulator of brain glucose uptake, and that this effect varies in different brain regions.     

Interaction of anabolic steroids with glucocorticoid receptor sites in rat muscle cytosol.

While glucocorticoid hormones act catabolically on skeletal muscle through their binding to glucocorticoid-specific receptors in the cytosol, androgens exert anabolic responses but no androgen-specific binding proteins could be detected in this responsive tissue. However, various nonradioactive androgens were effective in displacing labeled dexamethasone or cortisol from their respective cytoplasmic receptors in muscle, both in vitro and in vivo. The inhibition of glucocorticoid binding by androgens is competitive, and could be observed following a single or repeated administration of the androgens to adrenalectomized-castrated animals. The synthetic androgen fluoxymesterone and the hormone testosterone displayed Ki values of 7.5 X 10(-6) M and 1 X 10(-5) M, respectively, for the inhibition of [3H]dexamethasone binding in muscle cytosol. On the basis of competition experiments it is postulated that interaction of androgens with glucocorticoid receptors prevents the binding of glucocorticoids and might be responsible in part for the anabolic effects of pharmacologic doses of androgens in muscle.     

Inhibition of human basophil leukotriene release by antiinflammatory steroids

We have previously shown that 24-hour culture of human basophils with the antiinflammatory steroid dexamethasone produces an inhibition of the IgE-dependent release of histamine. In contrast, similar treatment of purified human lung mast cells does not inhibit the subsequent release of either histamine, prostaglandin D2, or leukotriene (LT) C4. We now show that incubation of mixed leukocytes for 24 h with 10(-7) M dexamethasone produces an inhibition of anti-IgE-induced basophil LTC4 release as detected by radioimmunoassay. In three experiments, control (CON) and dexamethasone (10(-7) M; DEX)-treated cells were challenged with 0.01, 0.03 and 0.1 micrograms/ml of anti-IgE, and histamine and LTC4 were monitored. LTC4 release (ng LTC4/micrograms total cell histamine) from cells stimulated with anti-IgE was: 0.01 micrograms/ml anti-IgE, 6.9 +/- 4, 0.3 +/- 0.1 (CON, DEX); 0.03 micrograms/ml anti-IgE, 13.8 +/- 4.7, 0.9 +/- 0.5; 0.1 micrograms/ml anti-IgE, 19.5 +/- 2.3, 4.9 +/- 1.2. Histamine release was inhibited by 50-75% by treatment with DEX in these experiments. Dose-response studies (n = 4) indicate that the inhibitory actions of DEX on LTC4 release occur in the range of 10(-10) to 10(-7) M. The concentration of DEX at which LTC4 release was inhibited by 50% (IC50) was approximately 2 X 10(-9) M. Another glucocorticoid (betamethasone) inhibited LTC4 release, while the nonglucocorticoids tetrahydrocortisone and beta-estradiol were inactive. High performance liquid chromatography (HPLC) analysis (coupled with RIA) indicated that the relative proportions of LTC4, LTD4, and LTE4 did not differ in supernatants from CON- and DEX-treated, anti-IgE-challenged cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of cell proliferation and DNA synthesis in rat colon: role of glucocorticoids

This study was undertaken to determine whether the rat colon exhibits ontogenic changes in epithelial cell proliferation and DNA synthesis during growth. DNA synthesis was measured at intervals after birth in four colonic segments by the incorporation rates of [3H]thymidine. The labeled crypt cell index was determined by radioautography. New findings from our study are that 1) in each colonic segment of suckling rats, [3H]thymidine incorporation rate overshot the adult levels (49-119%) with a peak occurring at day 14 postpartum, 2) between days 14 and 20, the incorporation rates declined sharply to adult values and remained thereafter unchanged until adulthood; during the same period, the labeled and mitotic index decreased, respectively, from 52 to 19% and from 3.58 to 1.43%, 3) the decrease in DNA synthesis and in cell proliferation rates was concomitant with an upsurge in plasma total corticosterone initiated on day 14, and 4) treatment of 10-day-old sucklings with physiological doses of hydrocortisone for 4 consecutive days significantly depressed (P less than 0.01) colonic DNA content and DNA synthesis rates to levels about 45-67% of the control values. These data indicate that growth of the colon may be under the control of glucocorticoid secretion at the weaning period.     

Quantitative flow cytometry of mouse mammary tumor virus envelope glycoprotein (gp52): alternative measures of hormone-mediated change in a viral cell surface antigen.

An immunofluorescence procedure with C3H mouse mammary tumor cells (Mm5mt/cl) has incorporated flow cytometry to provide a fluorescence-based measurement of changes in the mouse mammary tumor virus (MMTV) cell surface glycoprotein (gp52). A comparison of mean channel fluorescence intensity (delta mean) of cell populations stained with immune sera and NRS permitted a gp52-specific signal to be measured for controls and cells treated with 10(-6) M dexamethasone (Dex). Three different methods have been developed to quantitatively compare gp52-related fluorescence on control and hormone-treated cells. First, delta mean, measured as a gp52-specific difference in channel number was 169-209 for control cells and 299-341 for Dex-treated cells. These fluorescence measurements with 4 different sera demonstrated gp52-specific increases due to Dex treatment of 141, 130, 143, and 115 channels. A second method of gp52 quantitation determined the percentage shift in staining populations over NRS and specified channel intensity markers. Dex treatment resulted in a 6.9 to 32.4% shift over channel 508 (NRS marker) and a more marked shift of 45.5 to 49.2% over channel 676 (control cell marker). A third methodology utilized fluorescein bead standards to calculate molecules of equivalent soluble fluorescein (MESF). These MESF determinations permitted hormonal effects to be measured as fold increases over controls. Dex induction of gp52 for C3H and GR mammary tumor cells ranged from 1.5 to 9.1 fold increases. Alternative steroid treatments and antibody directed against the internal cytoplasmic MMTV P27 provided negative controls for measurements of changing gp52 levels.     

Localization of [3H]ouabain-sensitive Na+ pump sites in cultured pig kidney cells.

Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form monolayers and maintain morphological features characteristic of epithelia. Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by incubation in the presence of 45 mM K+. Freeze-dry radioautographic localization of [3H]ouabain-binding sites revealed grains distributed only along that fraction of the plasmalemma directly facing the culture-dish surface. Binding and localization of [3H]ouabain were correlated with an inhibition of the Na+ pump in these cells because analysis of cellular electrolytes in control cultures versus those exposed to 10(-3) M ouabain revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a reciprocal increase in Na+. There was no change in cell H2O. Similarly, oxygen consumption was reduced by 32% after exposure to ouabain. These results provide direct evidence that in epithelial cells in culture the membrane facing the culture dish corresponds to the basolateral membrane of epithelial cells in vivo.     

Apoptosis in human thymocytes after treatment with glucocorticoids.

Treatment of unfractionated human thymocytes in culture with the synthetic glucocorticoid dexamethasone induced cell death, as measured by trypan blue exclusion, after several hours of incubation. In purified subsets of human cortical and medullary thymocytes dexamethasone caused cell lysis with similar kinetics in both populations; 50% of thymocytes were killed after 20-24 h of incubation with the steroid. The mechanism of dexamethasone-induced cell death seems to correspond to apoptosis since degradation of DNA into oligonucleosome-sized fragments could be observed in the cultures treated with the steroid. A certain degree of DNA fragmentation and cell death could also be observed in control cultures of thymocytes. In contrast, peripheral T lymphocytes were resistant to the cytolytic effect of glucocorticoid hormone. The killing of human thymocytes by dexamethasone was inhibited by cycloheximide, suggesting that this cell death program requires a fully operating protein synthesis machinery and perhaps the induction of new proteins.     

Binding of dexamethasone and its effect on histamine release from rat mast cells

Purified rat peritoneal mast cells were incubated for 20 h with or without dexamethasone (4 x 10(-6) M) and then passively sensitized with serum from Trichinella spiralis-infected rats. The release of histamine using various secretagogues (concanavalin A, crude antigen of T. spiralis and polymyxin B) was determined. Dexamethasone treatment markedly inhibited IgE-dependent release of histamine (from 33.9 +/- 5.0% to 12.4 +/- 5.1% and from 39.8 +/- 7.9% to 14.2 +/- 6.5% of total cellular histamine content, respectively) whereas histamine release stimulated by the nonimmunological stimulus, polymyxin B was unaffected by this steroid. This suggests that the effects of dexamethasone cannot be exclusively explained by inhibition of phospholipases. Specific binding of 3H-dexamethasone to purified mast cells displayed sigmoidal dependence on concentration which may be the result of either negative cooperativity or the presence of a different class of binding sites. Two saturation plateaux at 20-30 x 10(-9) M and 70-90 x 10(-9) M were observed. The equilibrium dissociation constant for the higher affinity binding sites was Kd1 = 1.9 x 10(-8) M and represented 25,290 sites/cell, whereas the apparent Kd2 for lower affinity sites amounted to 5.5 x 10(-8) M and represented about 120,000 sites/cell.     

The effects of nabumetone,a cyclooxygenase-2 inhibitor,on cisplatin-induced 5-hydroxytryptamine release from the isolated rat ileum

In order to elucidate 5-HT release influenced by PGE2 in the background of the anticancer drug-induced emesis, the effect of nabumetone, a COX-2 inhibitor, on the release of 5-HT from the isolated rat ileum was investigated. PGE2 produced a concentration-dependent increase (10(-9) to 10 M) and decrease (10(-8) to 10(-6) M) in 5-HT release. Arachidonic acid also demonstrated a similar bell-shaped 5-HT release. The arachidonic acid-induced 5-HT release at 3 x 10(-6) M (313.04 +/- 25.90%) was significantly inhibited by the concomitant perfusion with BRL10720 (10(-6) M) (161.98 +/- 19.4%, p<0.01), an active metabolite of nabumetone, or indomethacin (3 x 10(-7) M)(190.01 +/- 16.19%, p<0.05). BRL10720 (10(-6) M)(428.57 +/- 51.72%, p<0.05) significantly inhibited the increase in 5-HT release induced by cisplatin (10(-6) M)(748.56 +/- 136.31%), suggesting that PGE2would be involved in cisplatin-induced 5-HT release. The increase in 5-HT release from the isolated ileum 72 hrs after cisplatin administration, in a delayed-emesis animal model, was significantly inhibited by the in vivo 3-day administration of nabumetone or BRL10720, but was not affected by the 3-day administration of dexamethasone. After 72 hours, however, the in vivo 3-days administration of nabumetone, BRL10720 or dexamethasone had no effect on the increase in ileal 5-HT levels induced by cisplatin. The use of COX-2 inhibitors to ameliorate delayed emesis induced by cisplatin-based anticancer chemotherapy has been proposed. On the other hand, there is a possibility that dexamethasone works through a mechanism other than 5-HT release in delayed emesis.     

Factors affecting ammonium uptake by C11 clone of MDCK cells

In several tissues ammonium ions are able to use the transport pathways of other ions, particularly of K+. We investigated this possibility in the C11 clone of MDCK cells, thought to represent intercalated cells, in control and 0 Cl- conditions. Cell pH was measured by ratiometric fluorescence microscopy using the pH indicator BCECF. After preincubating the cells for 10 min in control or 0 Cl- (substituted by gluconate) Ringer, an ammonium pulse was applied to induce cell acidification. The magnitude of the initial alkalinization (DeltapH) was 0.24+/-0.03 ( n=28) pH units in controls, which fell to 0.023+/-0.01 ( n=12) in 0 Cl-, suggesting uptake of NH4+ balancing the alkalinization by NH3. Addition of 10(-3) M bumetanide or furosemide to the 0 Cl- medium, or 10(-4 )M hexamethylene amiloride, did not alter DeltapH. However, with 5 mM Ba+, DeltapH increased to 38% of control. When 2.5x10(-4) M ouabain, an inhibitor of Na+-K+ ATPase, was used, DeltapH increased to 46% of control. Inhibition of H+-K+ ATPase by SCH28080 or by omeprazol caused significant increase in DeltapH. In 0 Cl- solution, these cells underwent a mean volume reduction (-d V) of -10.24+/-1.96% per 10 min as measured by confocal microscopy. To investigate if NH4+ influx was regulated by cell volume or by cell Cl-, volume reduction was avoided by two procedures. When preincubating with NPPB, a Cl- channel blocker, in 0 Cl-, volume reduction was inhibited (d V=-2.12% per 10 min), and DeltapH was 0.24+/-0.04 ( n=5). When the cells were preincubated in hypotonic 0 Cl- (260 mosmol/l), cell volume reduction was abolished (d V=+2.6% per 10 min) and DeltapH was 0.52+/-0.07 ( n=7). Thus, activation of NH4+ influx by several transporters was due to volume reduction rather than to [Cl-] alteration.     

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.     

Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta antibody inhibited (P < 0.001) the morphine-induced apoptosis in J774 cells (control 0.7 +/- 0.4%; 10-6 M morphine 23.5 +/- 0.7%; anti-TGF-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta by morphine-treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine-induced bax expression was inhibited by anti-TGF-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.     

Glucocorticoids suppress the production of tumour necrosis factor by lipopolysaccharide-stimulated human monocytes.

We have investigated the modulating effect of steroids on the in vitro production of tumour necrosis factor (TNF) by lipopolysaccharide (LPS)-stimulated human monocytes. Dexamethasone, at concentrations ranging from 10(-8) to 10(-6) M, and cortisol, at concentrations 10(-7) and 10(-6) M, suppressed the TNF production in a dose-dependent manner. The highest concentrations of dexamethasone or cortisol reduced the TNF production to 21 +/- 2% and 48 +/- 8% of the control value, respectively. The effect of dexamethasone was time dependent, and an incubation time of 48 hr was required to reduce the TNF production to 21% of control. The effect of dexamethasone decreased when the incubation time became shorter, and the mean TNF production ranged from 49% to 72% of control when dexamethasone was added later than 8 hr before LPS addition, at the time of LPS addition, or within 1 hr after LPS addition. The magnitude of the TNF-suppressing effect of dexamethasone varied greatly from donor to donor. Only the glucocorticoids, and not the sex steroids or the mineralocorticoids, significantly reduced the TNF production.     

Identification of adenylate cyclase-coupled histamine H2 receptors in guinea pig tracheal smooth muscle cells in culture and the effect of dexamethasone.

Histamine acts on airway contractile elements through at least two different receptor subtypes: H1, which mediates Ca(2+)-dependent contraction, and H2, which stimulates cyclic adenosine monophosphate (cAMP) synthesis and possibly relaxation. The aim of this study was to determine the relative contribution of the different receptor subtypes to histamine-stimulated cAMP production by guinea pig tracheal smooth muscle (GPTSM) cells in primary culture. Histamine and N-alpha-methylhistamine induced concentration-dependent cAMP synthesis; these effects were entirely blocked by 10(-4) M cimetidine, an H2-receptor antagonist, whereas 10(-6) M thioperamide, a selective H3 blocker, was ineffective. The H3 agonist, R-(alpha)-methylhistamine, did not stimulate cAMP synthesis. Triprolidine, an H1 antagonist, did not modify histamine (10(-5) M)-stimulated cAMP synthesis. Histamine (10(-5) M) doubled [Ca2+]i in GPTSM. A 24-h pretreatment of GPTSM cells with 10(-6) M dexamethasone enhanced cAMP synthesized in response to 10(-5) M histamine and to 5 x 10(-6) M forskolin but did not significantly alter either the affinity or the binding capacity for [3H]-tiotidine, an H2-receptor antagonist. These results indicate that GPTSM cells in culture express H2 but not H3 receptors, which are linked to adenylate cyclase; their functional expression does not seem to be modulated by the concurrent activation of H1 receptors, whose presence in GPTSM is evidenced by a histamine-stimulated increase in [Ca2+]i. The most likely site of action of dexamethasone in enhancing histamine-stimulated cAMP synthesis is at the level of adenylate cyclase since the steroid had no effect on the H2 receptor itself.