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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 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]

1 The current study addressed how acute colitis, induced in male Sprague–Dawley rats by the administration of dextran sulphate sodium (DSS) in their drinking water, may affect some functional properties of the longitudinal muscle layer of the distal colon. 2 Dextran sulphate sodium was provided at a concentration of 3% for 3 or 7 days, or 5% for 7 days, and the rats were thereafter killed. Specimens of the distal colon were taken for histology or for organ bath experiments. 3 The colitis score increased significantly with increasing dose of DSS administered. At 5% concentration, there was sometimes even transmural inflammation. Functionally, there was a progressive increase in optimal preload (Po) for the contractile response to carbachol (1 μM), in relation to the severity of the colitis. At 5% DSS, the magnitude of the response to carbachol at Po was significantly increased compared with control rats. Such an effect could not be verified when, instead, K+ (60 mM) was used as a spasmogen. 4 It is concluded, that the colitis score increased in severity progressively with increasing amounts of DSS administered. The longitudinal muscle layer was functionally affected by the inflammation. Thus, there was a progressive increase in optimal preload for muscle contraction. Moreover, severe colitis resulted in an increase of the contractile response to carbachol, while a significant increase in the response to depolarization with K+ could not be found. [ABSTRACT FROM AUTHOR]

Sacral nerve stimulation (SNS) has become an established therapy worldwide for the treatment for fecal incontinence. A large number of papers have been published over the years, and SNS is generally considered very effective with improved continence and quality of life for most patients. However, the results are mostly expressed in the semi-quantitative terms, that is, patients' diaries translated into score points. The clinical value of SNS is questionable, especially as the patient groups are usually small and/or etiologically heterogenic and the follow-up period mostly short. The Health Technology Assessment organization in the west region of Sweden has recently evaluated the SNS with regard to evidence, efficacy and risks. Economic and ethical aspects raise serious questions on this expensive and not entirely risk-free treatment in routine medical care. Similar criticism has also been raised by other reviewers proposing a more thorough scientific assessment with well-designed randomized trials and comparison with other similar methods of treatment. [ABSTRACT FROM AUTHOR]

1 Possibly acting via μ-opioid receptors (MORs), morphine inhibits the formation of experimentally induced postoperative abdominal adhesions in rats. Mesothelial cells may participate in adhesion formation by secreting mediators that interfere negatively with fibrinolysis. Morphine may prevent adhesions by inhibiting the release of pro-adhesion mediators from mesothelial cells. This study aimed to investigate whether human mesothelial cells express ΜΟR-1; if so, such could constitute a site of action for morphine in adhesion prevention. 2 Cells from Met-5A, a human mesothelial cell line were seeded and prepared for immunocytochemistry and Western blotting. 3 Immunocytochemistry showed MOR-1 expression in mesothelial cells, predominantly in the nuclei. Western blotting showed two bands ( c. 35 and 50 kDa) which correspond to those obtained with a control lysate from cells known to express MORs. In addition, we found MOR-1 expression with nuclear and cytoplasmatic localization in biopsies from human abdominal adhesions. 4 The current findings may suggest that morphine could interact directly with mesothelial cells via MOR-1 receptors, and thereby modulate adhesion formation, possibly by interfering with the release of pro-adhesion factors from these cells. [ABSTRACT FROM AUTHOR]

1 Secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently discovered endogenous ligand at the α7 subunit of the nicotinic acetylcholine receptors. Previous reports have shown that SLURP-1 is expressed in normal human keratinocytes seemingly with a pro-apoptotic function. Conversely, such expression was markedly attenuated in transformed cells and it was suggested that the molecule could convey protection against malignant transformation. 2 In this study, we demonstrated the mRNA expression (by RT-PCR) and protein expression (by Western blotting and immunocytochemistry) of SLURP-1 in the human colon cancer cell line, HT-29. 3 Furthermore, we demonstrated the expression of SLURP-1 (by immunohistochemistry) in tumour cells of human colon cancer tissue, and, to a greater extent, in immune and smooth muscle cells of adjacent, macroscopically tumour-free colon tissue. 4 The current findings suggest that SLURP-1 participates in the regulation of gut immune functions and motility, as well as possibly playing a role in colon carcinogenesis/cancer progression. [ABSTRACT FROM AUTHOR]

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]

Abstract: Purpose: We investigated whether pharmacologically induced up-regulation of heme oxygenase 1 by pyrrolidine dithiocarbamate (PDTC) conferred protection against subsequent ischemia-reperfusion injury (IRI) to the rat liver after temporary vascular occlusion of 70% of the organ. Methods: Female Wistar rats (200 to 250 g body weight) anesthetized with pentobarbitone were cannulated in the carotid artery and jugular vein. After laparotomy, a rubber band was applied around the entire vascular supply to the median and left lateral lobes, enabling vascular occlusion of 70% of the liver. A laser Doppler miniprobe was placed on the left lateral lobe to monitor peripheral liver blood flow (PLBF). Immediately upon completion of the surgery, the rats were administered either PDTC (50 mg/kg intravenously; n = 8) or its solvent (isotonic NaCl; n = 8). After 60 minutes, regional ischemia was induced for 30 minutes. The animals were then monitored for 2 hours of reperfusion. Blood samples for alanine transferase (ALT) estimation (as a measure of parenchymal injury) were drawn immediately prior to ischemia and reperfusion, as well as 60 and 120 minutes after reperfusion; PLBF was calculated at these times. Results: ALT increased in the course of the experiments but there was no difference between the groups. The reduction in PBLF due to ischemia-reperfusion was significantly lower in the PDTC group: about 16% versus 40%, after 2 hours of reperfusion. Conclusion: Pretreatment with PDTC attenuated the disturbance of hepatic microcirculation, but not parenchymal injury, in the early phase of IRI. [Copyright &y& Elsevier]