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Abstract: The aim of this study was to investigate whether nicotine acetylcholine receptors (nAChRs) are expressed in a more pronounced way in astrocytes co-cultured with microvascular endothelial cells from adult rat brain, compared with monocultured astrocytes, as a sign of a more developed signal transduction system. Also investigated was whether nicotine plays a role in the control of neuroinflammatory reactivity in astrocytes. Ca2+ imaging experiments were performed using cells loaded with the Ca2+ indicator Fura-2/AM. Co-cultured astrocytes responded to lower concentrations of nicotine than did monocultured astrocytes, indicating that they are more sensitive to nicotine. Co-cultured astrocytes also expressed a higher selectivity for α7nAChR and α4/β2 subunits and evoked higher Ca2+ transients compared with monocultured astrocytes. The Ca2+ transients referred to are activators of Ca2+-induced Ca2+ release from intracellular stores, both IP3 and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca2+ transients were attenuated after incubation with the inflammatory mediator lipopolysaccharide (LPS), but were not attenuated after incubation with the pain-transmitting peptides substance P and calcitonin-gene-related peptide, nor with the infection and inflammation stress mediator, leptin. Furthermore, LPS-induced release of interleukin-1β (IL-1β) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1β further attenuated nicotine-induced Ca2+ response. We also found that LPS and IL-1β induced rearrangement of the F-actin filaments, as measured with an Alexa488-conjugated phalloidin probe. The rearrangements consisted of increases in ring formations and a more dispersed appearance of the filaments. These results indicate that there is a connection between a dysfunction of nicotine Ca2+ signaling in inflammatory reactive astrocytes and upregulation of IL-1β and the rearrangements of actin filaments in the cells. [Copyright &y& Elsevier]

1 A characteristic of cancer is altered signal transduction leading to uninhibited growth. Adenosine-5′-triphosphate (ATP), a natural ligand at P2X- and P2Y purinergic receptors may regulate cell growth in non-neoplastic, as well as neoplastic tissues. In the human colon cancer cell line, HT-29, we previously demonstrated the expression of purinergic receptors of the P2Y2- and P2Y4 subclasses. 2 The aim of the current study was to investigate whether these two purinergic receptors are expressed also in human colon cancer, and, if so, how such expression is related to that in tumour-free colonic tissue. 3 The immunohistochemical findings of both P2Y2- and P2Y4 receptors in the tumours from three patients, prompted us to conduct an investigation of a consecutive series of patients utilizing Western blotting for protein detection and densitometry for quantitation. 4 Both P2Y2- and P2Y4 purinergic receptors could be identified in tumour-free tissue, and both were significantly over-expressed in each of the 10 colon cancers. [ABSTRACT FROM AUTHOR]

1 The aim of the current study was to investigate the existence of P2Y4 purinergic receptors in the HT-29 human colon cancer cell line. 2 We utilized Western blots and immunocytochemistry for the analysis. 3 Western blotting demonstrated two bands that could not be found after the antibody had been preabsorbed with the control peptide, suggesting that both bands are related to the P2Y4 purinergic receptor. 4 Immunocytochemistry showed immunoreactivity for the P2Y4 purinergic receptor localized in the cytoplasm of the HT-29 cells. 5 This is the first demonstration of the protein expression of P2Y4 purinergic receptors in a human colon cancer cell line. [ABSTRACT FROM AUTHOR]

1 The aim of the present study was to examine the modulator influence of muscarinic M2 receptors on responses of rat urinary bladder detrusor muscle evoked by endogenous stimuli, i.e. by stimulation of the bladder innervation. 2 Responses were evoked by electrical field stimulation (EFS; 2–20 Hz, 0.8 ms, 60 V) of isolated strip preparations mounted in organ baths. The tension of the muscle strips was recorded digitally. EFS was performed by applying stimulation with either a short duration (5 s) or a longer duration (to reach peak response; approximately 20 s). 3 Effects of muscarinic receptor antagonists (muscarinic M1/M3 receptor selective: 4-diphenylacetoxy- N-methylpiperidine methobromide (4-DAMP); muscarinic M2 receptor selective: methoctramine), a β-adrenergic antagonist (propranolol) and an adenosine receptor antagonist (8- p-sulfophenyltheophylline) were assessed on contractile activity and on poststimulatory relaxations. 4 Low concentrations of methoctramine (10−8 m) reduced or tended to reduce the EFS-induced contraction, e.g. at 2 Hz by 12% while methoctramine at 10−7 m had no significant effect. In addition, in the presence of 4-DAMP (10−9 m), which tended to inhibit contractions at all frequencies (2–20 Hz;−17 to−25%), methoctramine at 10−8 and 10−7 m induced a further reduction of the contractile responses (−5 to−10%; 2–20 Hz). 5 The β-adrenergic receptor antagonist propranolol (10−6 m) and the adenosine receptor antagonist 8- p-sulfophenyltheophylline (10−6 m) both increased contractile responses by 9–21% (2–10 Hz, long duration; P < 0.05–0.001) as a consequence of antagonizing relaxatory stimuli. Neither antagonist affected the contractile responses to EFS with the short duration stimulation. Poststimulatory relaxations were reduced by 30–60% ( P < 0.05) by propranolol and by 40–60% ( P < 0.001) by 8- p-sulfophenyltheophylline, but for 8- p-sulfophenyltheophylline only after stimulation with the short duration. 6 In the presence of methoctramine (10−7 m), the 8- p-sulfophenyltheophylline-induced increases of the contractile response to long duration EFS were significantly enhanced at 10 Hz (+12 ± 4%; P < 0.05), whereas no such enhancement of the propranolol inhibitory effect occurred in the presence of methoctramine. However, poststimulatory β-adrenoceptor-evoked relaxations after short duration EFS were increased by about 35% in the presence of methoctramine, but not those after long duration. 7 Thus, muscarinic M2 receptor activation inhibits adenosine receptor- and β-adrenoceptor-evoked relaxations of the rat detrusor muscle. The inhibition occurs via a transient postjunctional mechanism that mainly affects responses with a short latency. [ABSTRACT FROM AUTHOR]

The aim of the current study was to investigate in HT-29 human colon cancer cell line, the existence of functional receptors for the signalling molecules, noradrenaline (NA), prostaglandin E2 (PGE2), and adenosine-5′-triphosphate (ATP). We utilized microphysiometry, which monitors on-line extracellular acidification rate (ECAR) as a measure of cellular metabolic activity, and how this variable is altered by signalling molecules. Challenge with NA (5.9 μ m) resulted in an increase in ECAR by approximately 24% of basal. PGE2 (0.0284, 0.284 and 2.84 μ m) hardly affected ECAR. ATP (100 μ m) elicited a biphasic effect on ECAR (increase and decrease in ECAR by about 58 and 10% of basal, respectively). HT-29 cells were shown to express COX-2 by immunocytochemistry. These data suggest the presence of functional receptors for NA and ATP, but not for PGE2 in HT-29 human colon cancer cell line. [ABSTRACT FROM AUTHOR]

The effect of cholera toxin on small intestinal capillary function, utilizing the Evans blue dye method, was analyzed. The modulatory influence of plasma-derived or recombinant human antisecretory factor on this variable was also investigated. Male Sprague-Dawley rats were briefly anesthetized with ether, and a jejunal loop was constructed that was challenged for 90 min with phosphate-buffered saline or cholera toxin. Five minutes prior to death, the rats received an intravenous injection of Evans blue. The tissue content of dye in the loop was quantitated spectrophotometrically or demonstrated histochemically. Cholera toxin increased the recovery of Evans blue; extravasation of the dye was prominent in the top of the villi, while the crypts were spared. It is suggested that the toxin caused increased transcapillary permeation of albumin in a heterogenous fashion in the gut wall. This effect of the toxin was prevented by pretreatment with the antisecretory factor. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) has emerged as one of several important intraovarian regulatory factors. In particular, NO has been implicated in the processes of ovulation and atresia-related apoptosis. The aim of the present study was to investigate the presence and distribution of the NO-generating nitric oxide synthase (NOS) enzymes in the ovary during follicular development, ovulation and luteal formation of the equine chorionic gonadotrophin (ECG)/human chorionic gonadotrophin (HCG)-primed rat. NADPH diaphorase activity was used as a histochemical marker for NOS within the ovary. Diaphorase reactivity was most abundant in the stroma (S) of the ovary and in the theca (T) layer of the follicle. In luteinized ovaries, weaker diaphorase reactivity was present within the corpora lutea (CL). Two different isoforms of NOS, the constitutively expressed endothelial NOS (eNOS) and the inducible isoform of NOS (iNOS), were immunolocalized in ovaries of immature rats and in ECG/HCG-primed rats during the periovulatory period from HCG injection until 2 days after ovulation. In addition, ovarian concentrations of eNOS and iNOS were quantified by immunoblotting. Immunoblotting with a monoclonal anti-eNOS antibody demonstrated the presence of eNOS mainly in the residual ovary (ROV) during the periovulatory period. In luteinized ovaries, higher concentrations of eNOS were seen in CL, while those in the ROV at this stage were lower than in the periovulatory ovary. Immature ovaries contained diminutive amounts of eNOS, detectable mostly in the ROV compartment. In contrast, iNOS was barely detectable during follicular development to the preovulatory stage. A slight elevation of iNOS was observed in the granulosa cells at 6 h after the HCG injection. The levels of iNOS during the luteal phase were also low. Immunohistochemical analysis using polyclonal eNOS and iNOS antibodies revealed the localization of these two isoforms primarily in the S and the T of the periovulatory ovary. In luteinized ovaries, positive immunoreactivity was also seen within the CL. With a monoclonal antibody against eNOS, intense immunoreactivity was observed in the S, T and within CL. There was a particularly strong staining in blood vessels. These data demonstrate the presence of an intraovarian NO-generating system. The localization of this system to the S, T and CL suggests a role for NO in the ovulatory process and in the regulation of CL function. [ABSTRACT FROM AUTHOR]