Interactions of morphine with PGE1, isoproterenol,dopamine and aminophylline in rat mast cells; their effect on IgE-mediated14C-serotonin release

The formaldehyde method was used to examine the interactions of morphine with PGE₁, isoproterenol, dopamine and aminophylline in rat mast cells by their effects on IgE-mediated¹⁴C-serotonin release. PGE₁ (2×10^–8–2×10^–5 M), isoproterenol (10^–10–10^–8 M), dopamine (4×10^–8–4×10^–6 M) and aminophylline (6×10^–6–6×10^–4 M) caused dose-related inhibition of the mediator release 1 min after an antigen challenge, and propranolol (10^–7 M) blocked the inhibition by isoproterenol (10^–8 M) but not that by dopamine (4×10^–6 M), while haloperidol (4×10^–6 M) blocked that by dopamine (4×10^–6 M) but not that by isoproterenol (10^–8 M). Morphine (3×10^–7–3×10^–5 M) reversed the inhibitory effects of PGE₁ (2×10^–6 M), isoproterenol (10^–8 M) and dopamine (4×10^–6 M) dose-dependently and stereospecifically; naloxone (2×10^–4 M) antagonized these reversing actions of morphine (3×10^–5 M). Morphine (10^–6–10^–4 M) did not reverse the inhibitory action of aminophylline (6×10^–4 M). These results suggest that the inhibitory responses of mast cells to PGE₁, isoproterenol and dopamine but not to aminophylline in immunological mediator release were reversed by morphine through opioid receptors, and that the inhibition of adenylate cyclase in mast cells is one of the biochemical actions of morphine.     

Characterization of monoclonal antibodies against prostaglandin E2: Fine specificity and neutralization of biological effects

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E₂ (PGE₂) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB₂, a transformation product of PGE₂. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE₂. Cellular hybridizations were performed and five anti-PGE₂ monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the ω-chain of PGE₂ but their specificity toward the α-chain is more limited. The association constants are greater than to 1 × 10⁹M^?1. The monoclonal antibody 8E.57.71 (K_a = 1.3 × 10¹⁰M^?1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE₂ monoclonal antibodies were found to neutralize the specific binding of [³H]PGE₂ to rat brain hypothalamic receptors and to inhibit the PGE₂ induction of rat fundus muscular contraction.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of hyaluronan on prostaglandin E<Subscript>2</Subscript> induction by interleukin-1 in rabbit articular chondrocytes

Effects of hyaluronan (HA) on the prostaglandin E₂ (PGE₂) production induced by recombinant human interleukin-1β (rhIL-1β) in rabbit articular chondrocytes were studiedin vitro. The rhIL-1β-induced PGE₂ production was dose-dependently inhibited by HA. HA with the highest molecular weight (M_r=2.0×10⁶) exhibited an inhibitory effect that was statistically more significant than the same polymer of lower molecular weights (M_r=1.0×10⁶, 0.5×10⁶). This effect was observed in both young and adult rabbit articular chondrocytes. Since PGE₂ has been implicated as a mediator of inflammatory joint diseases, our observations suggest that HA may elicit an anti-inflammatory effect by inhibiting PGE₂ production.     

Comparative studies of the effects of 5-hydroxytryptamine and noradrenaline on the rat anococcygeus muscle

The effects of 5-hydroxytryptamine (5HT) and noradrenaline (NA) have been studied on rat anococcygeus muscle.

1. 5HT and NA produced a dose-dependent contraction of rat anococcygeus muscle. Cyproheptadine (1.0×10^?6 M), a specific 5HT receptor blocker, failed to inhibit the responses to either 5HT or NA.
2. However, phentolamine, a specificalpha receptor antagonist competitively blocked the responses to 5HT and NA.
3. The responses to 5HT were inhibited in the reserpinized (5 mg/kg i.p. 24 h) and 6-hydroxydopamine (6-OHDA) pre-treated preparations. 6-OHDA produced a leftward shift of the dose-response curve of NA. Reserpine pre-treatment potentiated lower doses of NA and the threshold dose of NA was significantly decreased.
4. Nialamide (2.2×10^?6 M), the mono-amine oxidase inhibitor produced a significant leftward shift of the dose-response curve of both 5HT and NA. Pyrogallol (2.3×10^?5 M), the catechol-o-methyl transferase inhibitor also potentiated the responses to both 5HT and NA, but the potentiation was significant at lower doses of 5HT and NA.
5. Our data suggest that 5HT- and NA-induced contractions in rat anococcygeus muscle are mediated through commonalpha adrenoceptors. 5HT actions are probably indirect, mediated through the release of NA.

     

Diamine oxydase in rabbit small intestine: Separation from a soluble monoamine oxidase,properties and pathophysiological significance in intestinal ischemia

From all mammals investigated so far only in rabbits diamine oxidase could not be detected in any tissue except the gut. Thus this species was chosen for studying the physiological and pathophysiological function of this enzyme in the gastrointestinal tract. By gel filtration on Sephadex G 50 and G 200 the enzyme was purified 100-fold, separated from a soluble monoamine oxidase, and the properties of the two enzymes were determined. Diamine oxidase from rabbit small intestine deaminated putrescine (K _m =1.3×10^?4 M, pH-optimum 6.4–6.9) and histamine (K _m =8×10^?5 M, pH-optimum 7.5), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. Soluble monoamine oxidase from rabbit small intestine catabolized serotonin (K _m =1.8×10^?4 M, pH-optimum 8.8), but not putrescine and histamine, and was inhibited by pargyline, but not by aminoguanidine. Based on its properties in vitro intestinal diamine oxidase could inactivate the vasoactive biogenic amine histamine in vivo. To confirm this hypothesis, in rabbits the small intestine was damaged severely by inducing total intestinal ischemia, which occurs as mesenteric infarction also in human subjects and is accompanied by histamine release. Treatment with aminoguanidine and ischemia killed the animals 3-times faster than ischemia alone, which supported our hypothesis on a protective role of intestinal diamine oxidase against histamine.     

Strong Suppression by Mononuclear Leukocytes From Cord Blood of Human Newborns on Maternal Leukocytes Associated With Differences in Sensitivity to Prostaglandin E2*

ABSTRACT: We have tested peripheral mononuclear leukocytes (PML) from the cord blood of newborns, from sera of their mothers, and from sera of nonrelated nonpregnant adult women for sensitivity to suppressive exogenous prostaglandin E₂ (PGE₂). Endogenous PG production was simultaneously inhibited by indomethacin 2.8 μM. The phytohemagglutinin-stimulated (PHA-stimulated) uptake of tritiated thymidine (³H-TdR) by PML from the mothers and the nonpregnant women was suppressed by the exogenous PGE₂ at a concentration of 1.4 × 10^?8 M, 100 times less than the one required to suppress the PML from newborns (1.4 × 10^?6 M). In addition, 1.4 × 10^?7 M or less of PGE₂ reversed the suppression of neonatal PML to stimulation. The maternal PML were reversed into stimulation at 1.4 × 10^?9 of exogenous PGE₂. The amount of endogenous PGE₂ synthesized by 1 × 10⁶ fresh, nonstimulated neonatal PML according to gas chromatography-mass spectrometry assay was 5 ng (1.4 × 10^?8 M). The synthesis increased to 27 ng/10⁶ cells after 18 hours' incubation. These concentrations are similar to the ones of exogenous PGE₂ at which neonatal PML were slightly stimulated but the maternal cells were still suppressed. Preincubation for 18 h at 37°C decreased the PGE₂-induced suppression of the adult PML but did not change the response of the neonatal PML.     

Human Fetal/Neonatal Suppressor Activity: Relation Between OKT Phenotypes and Sensitivity to Prostaglandin E2 in Maternal and Neonatal Lymphocytes

ABSTRACT: T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4⁺ subpopulation exerted only a marginal suppressor activity (12 ± 7 as compared to 73 ± 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8⁺ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E₂ (PGE₂) at doses varying between 1.4 × 10^?5 and 1.4 × 10^?9 M. Both maternal OKT4^? and OKT8^? T-cell subsets were highly sensitive to suppression by PGE₂. In contrast, cord OKT8^? T cells were essentially nonsensitive at all doses of PGE₂ used, whereas cord OKT4^? T cells were significantly suppressed at four out of five concentrations tested (1.4 × 10^?6 through 1.4 × 10^?9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE₂.     

Structural requirements for recognition of vasopressin by antibody; Thermodynamic and kinetic characteristics of the interaction

The thermodynamic and kinetic properties of two conventional antivasopressin antisera were studied. Values for binding affinity were determined by equilibrium measurements to be Kd = 1.4 and 2.7 × 10^?12M. The kinetic parameters were independently determined. The association rate constants (kas) were calculated by a pseudo-first-order analysis of binding kinetics (ka = 3.1 and 1.7 × 10⁷M^?1 sec^?1). The dissociation rate constants (kds) were measured by dissociating antibody-labelled antigen complexes with large excess of unlabelled antigen (kd = 1.6 and 1.7 × 10^?5 sec^?1). A fairly close agreement was achieved between equilibrium and kinetic evaluation of the affinity.The heterogeneity cannot be assessed through equilibrium experiments because of the very low concentrations of reagents to be handled. However, kinetic studies strongly suggested that the molecular heterogeneity with respect to affinity of the antisera is restricted to a narrow range (5 × 10^?13M to 7 × 10^?12M).Despite their very similar physicochemical properties these two antisera exhibited different fine specificities: the study of cross-reactivities with various analogues of the original hapten showed that one antiserum—5—is clearly directed against the C-terminal moiety of the molecule. The antigenic determinant is a sequential one and composed of the last four aminoacids Cys-Pro-Arg-GlyNH₂, while the other antiserum is not so sensitive to modifications of the last residue GlyNH₂.     

Sensitivity of somatic mutations in human umbilical cord blood to maternal environments

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (M_f), and glycophorin A (GPA) variant erythrocytes (V_f) of both allele-loss (ø/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt M_f (1.40 ± 1.11 × 10^?6, N = 66) and GPA V_f (ø/N 4.0 ± 2.2 × 10^?6, N = 114; N/N 2.7 ± 2.0 × 10^?6, N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt M_f was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 ± 0.41 × 10^?6, N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt M_f (0.70 ± 0.49 × 10^?6) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated M_f revealed a significant decrease in the relative contribution of gross structural mutations to the overall M_f (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, x² test), suggesting that the higher M_f resulted from an elevated level of “point” mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt M_f or GPA V_f. © 1995 Wiley-Liss, Inc.     

Antigen valence and Fc-localized secondary forces in antibody precipitation

A tetravalent dinitrophenyl hapten, 1,6,11,16-tetra-(?-N-Dnp)-l-lysine₁₆3 was synthesized. Upon interaction with specific antibody of moderate affinity (K_t = 6 × 10⁶ M^?1), a maximum of 80% precipitation occurred. Low pH or 2.5 M guanidine hydrochloride completely inhibited precipitation without significantly affecting hapten binding. Anti-Dnp (Fab')₂ fragments also formed precipitates when mixed with the tetravalent hapten, but these were not soluble in 2.5 M guanidine hydrochloride, and the precipitation itself occurred over a much narrower range of hapten/antibody ratios than observed with intact molecules. These results suggest that a site located in the Fc region assists aggregation and preciptation in intact antibody molecules.     

Comparison of the effect of PGE2 and somatostatin on histamine stimulated14C-aminopyrine uptake and cyclic AMP formation in isolated rat gastric mucosal cells

In isolated rat gastric cells somatostatin and PGE₂ were compared in respect to their effects on the cAMP system and on the histamine-stimulated H⁺-production, measured by¹⁴C-aminopyrine (¹⁴C-AP) uptake. Like PGE₂ somatostatin activated adenylate cyclase (AC) for all in non-parietal cells. This effect on AC declined in cell fractions with increasing number of parietal cells. Activation of AC or elevation of cellular cAMP and uptake of¹⁴C-AP in response to histamine were inhibited by 10^?9 to 10^?5 mol/l PGE₂ and somatostatin. The results indicate remarkable similarity between somatostatin and PGE₂: both activate a non-parietal cell AC and both inhibit H⁺-production, likely by interfering at the histamine sensitive AC of the parietal cell.     

Histamine modification of spontaneous rate and rhythm in infarcted canine ventricle

1. Histamine (10^?3 M) increased the spontaneous rate similarly in isolated preparations of normal left ventricular tissue from control, i.e. normal and sham-operated, dogs (control preparations) and in preparations consisting of normaland contiguous infarcted left ventricular tissue from dogs with subacute, i.e. 24 hours after left coronary artery ligation, myocardial infarction (infarcted preparations).
2. Histamine (10^?3 M) markedly enhanced the irregular rhythm of infarcted preparations.
3. The H₁-receptor antagonist, chlorpheniramine (10^?4 M), and the H₂-receptor antagonist, cimetidine (10^?3 M), antagonized the effects of histamine (10^?3 M) on the spontaneous rate of both control and infarcted preparations.
4. The H₁-receptor agonist, 2-pyridyl ethylamine (PEA, 10^?4 M), increased the spontaneous rate of control and infarcted preparations; these effects were antagonized by chlorpheniramine (10^?4 M). The H₂-receptor agonist, dimaprit, had no effect.
5. Similar to histamine (10^?3 M), PEA (10^?4 M) enhanced the irregular rhythm of infarcted preparations; dimaprit had no effect.
6. High local concentrations of histamine may occur in poorly perfused ischemic tissue. The enhancement of irregular rhythm produced by histamine, and the specific H₁-receptor agonist, PEA, leads us to suggest its involvement in arrhythmias associated with subacute myocardial infarction.

     

Inhibition of human periodontal prostaglandin E2 synthesis with selected agents

Considerable evidence has demonstrated the importance of PGE₂ synthesis in the pathogenesis of periodontal disease. Although various cyclooxygenase inhibitors have been known to block periodontal PGE₂ synthesis and prevent disease progression in animal models, there are few reports comparing relative efficacies of various inhibitors of arachidonic acid (ARA) metabolism. We have developed a sensitivein vitro assay to measure PGE₂ synthesis in periodontal tissues. The apparent IC₅₀ values (i.e. the concentration of drug which causes 50% inhibition of maximum PGE₂ synthesis) have been determined for a series of arachidonic acid analogues as well as competitive and non-competitive cyclooxygenase inhibitors. Periodontal tissue homogenates were incubated in the presence of³H-arachidonic acid for 45 min at 37°C. Inhibitors were tested at 10^–10–10^–4 M and at zero concentration to measure conversion of³H-arachidonate to³H-PGE₂. Log or half log dilutions of inhibitors were tested in triplicate for each assay. Radiolabeled PGE₂ was extracted from homogenates, purified by reverse phase chromatography and quantitated by double antibody capture. RIA was performed on each homogenate to determine the amount of endogenous unlabeled PGE₂ present in the sample to correct for antibody capture recovery. The apparent IC₅₀ values were determined for each drug by averaging two or more replicate assays. Specific total enzymatic activity of periodontal tissue homogenates was typically 5–11 pg PGE₂/min/mg tissue. The following series of compounds were tested and are listed in order of increasing IC₅₀ values: -tocopherol (3.3×10^–9 M), ketoprofen (5.4×10^–9 M), indomethacin (1.0×10^–8 M), flurbiprofen (1.5×10^–8 M), meclofenamate (1.5×10^–6 M), naproxen (2.5×10^–6 M), docosahexaenoic acid (1.0×10^–5 M), eicosapentaenoic acid (1.5×10^–5 M), and ibuprofen (1.5×10^–5 M). These data will enable the rational design of pharmacological formulations to inhibit periodontal tissue PGE₂ synthesis and resultant inflammation, attachment loss and bone resorption.     

The effects of heavy metal ions (Cd2+, Hg2+, Pb2+, Bi3+) on histamine release from human adenoidal and cutaneous mast cells

The influence of lead (Pb[CH₃COO]₂), mercury (HgCl₂), cadmium (CdSO₄) and bismuth (BiO[ClO₄]) on the spontaneous and stimulated histamine release from human adenoidal and cutaneous mast cells was tested in the concentration range 10⁻⁸–10⁻⁴ M. Lead displayed a bell shaped dose-response relationship in adenoidal mast cells with a maximum at 10⁻⁶ M whereas in cutaneous cells only the spontaneous release was slightly enhanced at 10⁻⁴ M. Mercury induced a presumably toxic histamine release in adenoidal and cutaneous mast cells at 10⁻⁴ M. Cadmium increased the histamine release in adenoidal cells at 10⁻⁴ M but in cutaneous cells only the stimulated release (10⁻⁸–10⁻⁵ M) was affected. Bismuth inhibited the histamine release at 10⁻⁴ M in the adenoidal mast cells only. In conclusion, human adenoidal and cutaneous mast cells are affected differently by metal ions.

     

A Genome‐Wide Search for Type 2 Diabetes Susceptibility Genes in an Extended Arab Family

Twenty percent of people aged 20 to 79 have type 2 diabetes (T2D) in the United Arab Emirates (UAE). Genome‐wide association studies (GWAS) to identify genes for T2D have not been reported for Arab countries. We performed a discovery GWAS in an extended UAE family (N = 178; 66 diabetic; 112 healthy) genotyped on the Illumina Human 660 Quad Beadchip, with independent replication of top hits in 116 cases and 199 controls. Power to achieve genome‐wide significance (commonly P = 5 × 10^?8) was therefore limited. Nevertheless, transmission disequilibrium testing in FBAT identified top hits at Chromosome 4p12‐p13 (KCTD8: rs4407541, P = 9.70 × 10^?6; GABRB1: rs10517178/rs1372491, P = 4.19 × 10^?6) and 14q13 (PRKD1: rs10144903, 3.92 × 10^?6), supported by analysis using a linear mixed model approximation in GenABEL (4p12‐p13 GABRG1/GABRA2: rs7662743, P_adj‐agesex = 2.06 × 10^?5; KCTD8: rs4407541, P_adj‐agesex = 1.42 × 10^?4; GABRB1: rs10517178/rs1372491, P_adj‐agesex = 0.027; 14q13 PRKD1: rs10144903, P_adj‐agesex = 6.95 × 10^?5). SNPs across GABRG1/GABRA2 did not replicate, whereas more proximal SNPs rs7679715 (P_adj‐agesex = 0.030) and rs2055942 (P_adj‐agesex = 0.022) at COX7B2/GABRA4 did, in addition to a trend distally at KCTD8 (rs4695718: P_adj‐agesex = 0.096). Modelling of discovery and replication data support independent signals at GABRA4 (rs2055942: P_{adj‐agesex‐combined} = 3 × 10^?4) and at KCTD8 (rs4695718: P_{adj‐agesex‐combined} = 2 × 10^?4). Replication was observed for PRKD1 rs1953722 (proxy for rs10144903; P_adj‐agesex = 0.031; P_{adj‐agesex‐combined} = 2 × 10^?4). These genes may provide important functional leads in understanding disease pathogenesis in this population.     

Chromosome-mediated gene transfer of HPRT and APRT in an intraspecific human cell system

Chromosome-mediated transfer of genes between human cell lines was accomplished using HeLa cells as chromosome donors and HT1080 fibrosarcoma lines as recipients. This report describes the intraspecific transfer of two genetic markers, hypoxanthine-guanine-phosphoribosyl-transferase (HPRT ⁺)and adenine phosphoribosyltransferase (APRT ⁺).The isolation and characterization of the necessary enzyme-deficient (HPRT ^? and APRT ^?)recipient HT1080 cell lines are also described. The chromosome-mediated gene transfer was carried out using a modification of the procedure of Miller and Ruddle, including treatment of the donor chromosomes with calcium phosphate and subsequent exposure of the recipient cells of dimethyl sulfoxide. In experiments to optimize this procedure for HT1080 cell recipients, we found that a brief (2-min) exposure to high DMSO concentration (20%) was effective for enhancing transfer efficiencies in this system. Transfer frequencies (transferents per recipient cells assayed) averaged approximately 1×10^?6 for HPRT ⁺ and were >2×10^?6 for APRT ⁺.     

Effects of midodrine,ST 1059, methoxamine and glycine on spontaneously beating guinea-pig atria

The chronotropic effects of midodrine, glycine (10^–8 to 3.10^–3 M), ST 1059 and methoxamine (10^–8 to 10^–3 M) were investigated in the spontaneously beating guinea-pig right atrial preparation. Midodrine and glycine produced a slight, but significant rise in atrial rate over a wide concentration range. The midodrine-induced rise in atrial rate was not influenced by the beta-adrenergic receptor blocking drug propranolol (10^–6 M). The histamine (H₂)-receptor blocking drug metiamide (3.10^–5 M) abolished the positive chronotropic actions of both midodrine and glycine. No positive chronotropic effect was seen after the administration of ST 1059 or methoxamine. A decrease in atrial rate was elicited by high concentrations (above 10^–4 to 10^–3 M) of the sympathomimetic agents midodrine, ST 1059, and methoxamine, but not by the amino acid glycine.     

First order dissociation rates between a subpopulation of high affinity rabbit anti-fluorescyl IgG antibody and homologous ligand

The first order dissociation rates of liganded subpopulations of purified hyperimmune rabbit antifluorescyl IgG antibodies were determined using the method of Green (1963). The dissociation of radiolabeled fluorescyl ligands was measured in the presence of excess unlabeled homologous hapten. Results with three different antibody preparations indicated dissociation rates of 1.3 × 10[su?3, 2.4 × 10^?4 and 2.2 × 10^?6 sec^?1 for the liganded subpopulations. Assuming an association rate of 4.6 × 10⁸M^?1 sec^?1 (Levison et al., 1971), average equilibrium constants ranging from 3.1 × 10¹¹M^?1 to 2.1 × 10¹⁴M^?1 were calculated. These values are among the highest reported for antibody-hapten interactions.     

Protein kinase A enhances lipopolysaccharide-induced IL-6, IL-8, and PGE2 production by human gingival fibroblasts

Objective

Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Interleukin (IL)-6, IL-8, and the chemical mediator prostaglandin E₂ (PGE₂) are known to play important roles in inflammatory responses and tissue degradation. Recently, we reported that the protein kinase A (PKA) inhibitor H-89 suppresses lipopolysaccharide (LPS)-induced IL-8 production by human gingival fibroblasts (HGFs). In the present study, the relevance of the PKA activity and two PKA-activating drugs, aminophylline and adrenaline, to LPS-induced inflammatory cytokines (IL-6 and IL-8) and PGE₂ by HGFs were examined.

Methods

HGFs were treated with LPS from Porphyromonas gingivalis and H-89, the cAMP analog dibutyryl cyclic AMP (dbcAMP), aminophylline, or adrenaline. After 24 h, IL-6, IL-8, and PGE₂ levels were evaluated by ELISA.

Results

H-89 did not affect LPS-induced IL-6 production, but suppressed IL-8 and PGE₂ production. In contrast, dbcAMP significantly increased LPS-induced IL-6, IL-8, and PGE₂ production. Up to 10 ??g/ml of aminophylline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at 100 ??g/ml. Similarly, 0.01 ??g/ml of adrenaline did not affect LPS-induced IL-6, IL-8, or PGE₂ production, but they were significantly increased at concentrations of 0.1 and 1 ??g/ml. In the absence of LPS, H-89, dbcAMP, aminophylline, and adrenaline had no relevance to IL-6, IL-8, or PGE₂ production.

Conclusion

These results suggest that the PKA pathway, and also PKA-activating drugs, enhance LPS-induced IL-6, IL-8, and PGE₂ production by HGFs. However, aminophylline may not have an effect on the production of these molecules at concentrations used in clinical settings (8 to 20 ??g/ml in serum). These results suggest that aminophylline does not affect inflammatory responses in periodontal disease.     

H1- and H2-receptor mediated responses to histamine on contractility and cyclic AMP of atrial and papillary muscles from guinea-pig hearts

On guinea-pig heart we investigated whether cyclic AMP serves as a messenger for H₁- and/or H₂-mediated responses to histamine.

1. On papillary muscle histamine elicited positive inotropic responses which were antagonized by burimamide but not by promethazine. The stimulation of H₂-receptors was not only associated with an increase in contractility but also with an increase in cAMP. As shown by the time course of effects for 10^?5 M histamine, the maximal increase in cAMP preceded the maximum in contractility. The mechanical and biochemical responses to histamine were potentiated by the phosphodiesterase inhibitor papaverine, but antagonized by burimamide.
2. On the left guinea-pig atrium containing H₁-receptors the inotropic response to histamine (10^?5 M) was not accompanied by increases in cAMP at stimulation frequencies of 0.5 and 2 Hz, respectively. In addition, in the presence of papaverine (3×10^?5 M) no change in the cyclic AMP level occurred after application of histamine. Papaverine by itself, however, concomitantly increased contractility and cyclic AMP at a stimulation frequency of 0.5 Hz. In contrast, at 2 Hz papaverine increased only cAMP leaving the contractility unchanged. At this frequency the well-known Ca²⁺-antagonistic effect comes into prominence, thus masking the positive inotropic effect atributable to the inhibition of the phosphodiesterase.
3. On the right guinea-pig atrium the mediation of the positive charonotropic response to histamine by H₂-receptors which is partly involved in the inotropic effect via the frequency-force relationship does not lead to a concomitant increase in cAMP. Also, in the presence of papaverine, histamine had no influence on the cAMP. However, papaverine potentiated the cardioacceleration produced by histamine. Although it is very likely that the cAMP in the sinus node rises, we were not able to detect an increase in cAMP in the whole atrial tissue.

From the present results the conclusion can be drawn that the mediation of the inotropic effect due to stimulation of H₂-receptors by histamine is associated with an increase of cyclic AMP, whereas that of H₁-receptors is not. The view that cAMP may be the second messenger in the chronotropic action of histamine needs further elucidation by experiments on sino-atrial cells.