The in vitro effects of H-89, a specific inhibitor of protein kinase A, in the human colonic carcinoma cell line Caco-2.

SUMMARY: H-89 is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A. In the present study, we observed that H-89 induced morphological transformation and caused growth inhibition of the human colon cancer cell line Caco-2 in a dose-dependent manner. However, another protein kinase A inhibitor, H-8, had no effect on Caco-2 cells. To evaluate the possible molecular mechanism of H-89-evoked effects in Caco-2 cells, we analysed the capacity of H-89 to regulate the protein kinase B (Akt/PKB) signalling pathway. H-89 treatment led to an activation of Akt/PKB in Caco-2 cells. This activation was phosphatidylinositol 3 (PI3)-kinase-dependent and promoted survival of Caco-2 cells because the PI3 kinase inhibitor LY294002 inhibited the Akt/PKB activation and induced apoptosis of Caco-2 cells. To test whether Akt/PKB activity promoted resistance to H-89-induced effects, LY294002 was added in combination with H-89. LY294002 greatly potentiated the H-89-induced growth inhibition and apoptosis of Caco-2 cells. These results suggest that the H-89-induced growth inhibition of Caco-2 cells is associated with phosphorylation of Akt/PKB protein and that the cells become more sensitive to H-89 and die by apoptosis upon inhibition of the PI3K/Akt pathway.

Migratory responses of polymorphonuclear leukocytes to kinin peptides.

The present study examines the influence of kinins on the migratory capacity of human polymorphonuclear leukocytes under in vitro conditions using the Boyden chamber technique. By means of checkerboard analysis the migration of neutrophils induced by bradykinin could be characterized as true chemotaxis. The stimulation of human neutrophils with bradykinin, with the nonpeptide B(2) receptor agonist FR190997 as well as with des-Arg(9)-bradykinin and des-Arg(10)-kallidin results in a concentration-dependent migration. Pretreatment of the neutrophils with the B(2) receptor antagonist HOE-140 (icatibant) inhibited the bradykinin-induced migration but not that induced by B(1) receptor agonists, whereas the B(1 )receptor antagonist des-Arg(10)HOE-140 abolished the migration elicited by des-Arg(9)-bradykinin or des-Arg(10)-kallidin but not that evoked by bradykinin. Pretreatment of the neutrophils with the leukotriene B(4) (LTB(4)) antagonist ZK158252 inhibited the LTB(4)-induced chemotaxis as well as the chemotaxis produced by bradykinin and des-Arg(10)-kallidin. An involvement of interleukin-1beta and of the chemokine IL-8 in the bradykinin-induced migration in vitro could be excluded during the migration time of the neutrophils. In conclusion, the present study provides pharmacological evidence showing that B(1) and B(2) kinin receptors are involved in the migration of human neutrophils in vitro, that LTB(4) participates in the downstream pathway and that the B(1) kinin receptor seems to be expressed already under physiological conditions.

Substance P (NK(1)) receptor expression by human colonic epithelial cell line Caco-2.

The peptide substance P (SP) is known to take part in the regulation of the Cl(-)-dependent secretion in the animal and human colonic mucosa. However, no conclusive evidence for the expression of the functional tachykinin NK(1) receptor has been found in the human colonic epithelial cells. Using the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the transcripts of the NK(1) receptor in the human colonic epithelial cell line Caco-2. Furthermore, we characterized the mechanism of substance P-induced intracellular signaling in Caco-2 cells. While substance P had no effect on intracellular calcium concentration as measured by fura-2 AM, it induced the activation of the mitogen-activated protein kinases (MAPKs) in a time- and dose-dependent manner. Surprisingly, the peptide NK(1) receptor antagonist [D-Pro(2), D-Trp(7,9)]SP stimulated the activity of MAPKs in the same manner as substance P. In contrast, the specific nonpeptide NK(1) receptor antagonist CP-96,345 clearly abolished the effect of substance P and [D-Pro(2), D-Trp(7,9)]SP on MAPK activity. CP-96,345 itself did not increase the activity of MAPKs. Thus, we provide the first evidence that a functional NK(1) receptor is expressed in the human colonic epithelial cell line Caco-2. The results show that in Caco-2 cells the peptide antagonist [D-Pro(2), D-Trp(7,9)]SP acts as a NK(1) receptor agonist in contrast to the nonpeptide antagonist CP-96,345.

Delay of neutrophil apoptosis by the neuropeptide substance P: involvement of caspase cascade.

Neutrophil apoptosis is an important event in the resolution of inflammation. The role of substance P (SP) in neutrophil apoptosis has not been previously investigated. We found that substance P delays apoptosis in neutrophils. Human neutrophils were isolated and cultured up to 24 hours. Apoptosis was detected by light and electron microscopy, as well as DNA-fragmentation assays. Substance P delayed the spontaneous apoptosis of neutrophils at 6, 12, 18 and 24 hours in a dose-dependent fashion in the range of 10-100 microM. Whereas the both peptide neurokinin-1 (NK-1) receptor antagonists [D-Pro(2), D-Trp(7,9)]-SP and GR 82334 inhibited the substance P effect on neutrophils, the nonpeptide NK(1) receptor antagonist L-703.606 itself, an analogue of CP-96,345, induced apoptosis of neutrophils. Surprisingly, the effect of L-703.606 could be prevented by substance P. Western blotting results showed that the neuropeptide substance P inhibited the spontaneous apoptosis-associated caspase-3 activation in the same concentration range as described above. Parallel the inhibition of cleavage of focal adhesion kinase (FAK), a substrate of caspases could be observed by substance P. In conclusion, our results extend the range of biological effects of the neuropeptide substance P and provide new insight to the role of this tachykinin in the modulation of the inflammatory response by the nervous system.

Kinins and kinin receptors: importance for the activation of leukocytes.

In this article, we analyzed the role of kinins and kinin receptors with respect to the activation of leukocytes. In these cells, the biological effects of kinin peptides are mediated by kinin receptor subtypes B1, B2, or both, depending on species and cell type. In contrast to the other leukocytes, neutrophils contain the complete system for the synthesis and release of bioactive kinins. Consequently, very high concentrations of these peptides can be reached in the close neighborhood of the kinin receptors, in particular at the site of inflammation. Kinins are responsible for many effects in leukocytes including the release of other inflammatory mediators, such as cytokines, prostaglandins, leukotrienes, and reactive oxygen species. Obviously, the potency of kinins to stimulate leukocytes is dependent on the differentiation and especially on the activation stage of these cells. An upregulation of kinin receptors on neutrophils and macrophages appears to be involved in increasing the sensitivity of these cells to kinins at the site of inflammation.

Influence of interleukin-1 beta on bradykinin-induced responses in guinea pig peritoneal macrophages.

OBJECTIVE AND DESIGN: The endogenous nonapeptide bradykinin is implicated in the pathogenesis of a number of inflammatory diseases. Recently, it could be shown that bradykinin is a potent stimulus for the generation of arachidonic acid, prostaglandin E2 (PGE2) and superoxide radical via the bradykinin B2 receptor from macrophages depending on their stage of maturation or activation. The present study was designed to characterize the effect of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) on responsiveness of macrophages to bradykinin. MATERIAL: Guinea pig peritoneal macrophages were collected 7 days after i.p. injection of paraffinum subliquidum. RESULTS: The bradykinin-induced increase in cytosolic free calcium concentration ([Ca2+]i) and activation of the respiratory burst were significantly enhanced by interleukin-1 beta (100 U/ml) added to cultures of macrophages 24 h before stimulation with bradykinin. However, the EC50 values of bradykinin-induced calcium signal of 23 +/- 8 nM and 29 +/- 17 nM and superoxide radical formation of 64 +/- 22 nM and 34 +/- 29 nM in control and interleukin-1 beta-treated cells, respectively, were not changed. In contrast to the influence of IL-1 beta on these functions, saturation and competition experiments measured by binding of [3H]bradykinin ([3H]BK) to intact macrophages demonstrate that cell exposure to interleukin-1 beta for 24 h caused no change in the density and affinity of the bradykinin B2 receptors. CONCLUSIONS: The most likely explanation for the cytokine effects is an influence on the level of the receptor-effector coupling downstream of the agonist binding.

Differential sensitivity of macrophages to bradykinin.

In this report, we investigated the responsiveness of subpopulations of elicited peritoneal macrophages between each other compared to resident tissue macrophages of alveoli of guinea pig to the action of bradykinin. Bradykinin stimulated the secretion of superoxide radical, arachidonic acid and prostaglandin E2 (PGE2) via the bradykinin B2 receptor subtype in peritoneal macrophages, indicated by complete inhibitory effect of the bradykinin B2 receptor antagonist HOE 140. The extent of the secretion, however, varied substantially between macrophages of different size. The highest level of the secretion was observed in the fraction containing the intermediate-size macrophages, while progressively lower level of the secretion was observed with decreasing size. In contrast, large macrophages obviously lost their secretory ability. Additionally, the bradykinin-stimulated release of cyclooxygenase products exerted an inhibitory action on NADPH-oxidase activity depending on size and stage of maturation/activation of macrophages, as judged by an increase in superoxide radical generation by indomethacin (100 microM) preincubation of cells. Furthermore, the investigation of resident tissue macrophages of alveoli has shown that these cells also express the bradykinin B2 receptor subtype. However, the receptor activity measured by bradykinin-induced increase in intracellular free calcium [Ca2+]i was very low compared to elicited peritoneal macrophages. These findings indicate that the stage of differentiation/maturation and activation of macrophages may be important for the ability of bradykinin to stimulate these cells to inflammatory responses in vivo.

Bradykinin receptors in signal transduction pathways in peritoneal guinea pigs macrophages.

The presence of a bradykinin receptor on guinea pig peritoneal macrophages was evidenced by binding studies and by the effect of bradykinin on activation of the phospholipase C and the increase in intracellular calcium concentration ([Ca2+]i). Binding studies demonstrated a specific, saturable binding for [3H]bradykinin inhibited by the bradykinin B2 (HOe 140) but not bradykinin B1 (des-Arg9[Leu8]bradykinin) receptor antagonist. Scatchard analysis revealed a single class B2 bradykinin binding site with a binding affinity (kd) of 0.8 nM and a receptor concentration (Bmax) of 35 fmol/5 x 10(6) cells, representing approximately 4000 bradykinin receptors per cells. Kinetic studies confirmed the presence of this single binding site by the determination of similar binding affinity. Activation of peritoneal macrophages by bradykinin resulted in a time- and dose-dependent release of inositol phosphates determined by anion exchange chromatography and intracellular calcium analyzed using fura-2/AM. The increase in [Ca2+]i induced by bradykinin was blocked by the specific bradykinin B2 receptor antagonist HOE 140 but not the bradykinin B1 receptor antagonist des-Arg9[leu8]-BK. These studies provide novel information regarding the nature of kinin receptors on guinea pig peritoneal macrophages and their signal transduction pathways.

[Metastatic bile duct cancer in secondary biliary cirrhosis associated with primary sclerosing cholangitis in ulcerative colitis]. / Metastasierendes Gallengangskarzinom bei sekundär biliärer Zirrhose auf dem Boden einer primär sklerosierenden Cholangitis bei Colitis ulcerosa.

By means of a case-report of a 33 years old pat., having colitis ulcerosa for 16 years with serious epitheldysplasia and developing primary sclerosing cholangitis, the typical progress of the disease with manifestation of a secondary biliary cirrhosis is shown. The course was complicated by the development of a bile-duct carcinoma. Present possibilities of diagnosis and therapy are discussed.