Nuclear expression of mu-opioid receptors in a human mesothelial cell line.

1 Possibly acting via mu-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 MOR-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.

Sympathetic nerves do not affect experimental ischemia-reperfusion injury of rat liver.

BACKGROUND: We investigated whether sympathetic, noradrenergic nerves participate in experimental acute ischemia-reperfusion injury of the rat liver. METHODS: Female Wistar rats (200-250 g body weight) were anesthetized with pentobarbital. After tracheotomy, we cannulated a carotid artery and jugular vein. The rats were divided in 2 groups (n = 8 per group). The control group received NaCl IV and the test group received the sympatholytic agent, guanethidine (3 mg/kg, IV). After 30 minutes of drug equilibration, laparotomy was performed to arrange the liver for temporary occlusion (by a ligature) of its vascular supply, corresponding with 70% reduction in hepatic blood flow. The rats were then allowed 60 minutes of equilibration. Thereafter, regional ischemia was induced for 30 minutes. The animals were then monitored for 2 hours of reperfusion. Blood samples for alanine aminotransferase (ALT) estimation (as a measure of injury to the parenchyma) were drawn immediately before ischemia, as well as 60 and 120 minutes after reperfusion. Readings of mean arterial pressure were taken during these times. RESULTS: After 2 hours of reperfusion, there were no significant differences between the groups with regard to ALT or mean arterial pressure. CONCLUSION: Sympathetic, noradrenergic nerves did not affect experimental ischemia-reperfusion injury of rat liver in the current model.

Expression of the endogenous, nicotinic acetylcholine receptor ligand, SLURP-1, in human colon cancer.

1. Secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently discovered endogenous ligand at the alpha7 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.

Changes in muscarinic receptors in the toad urothelial cell line TBM-54 following acrolein treatment.

1. In cyclophosphamide-induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM-54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2. The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3. Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration-dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 +/- 6%. Pre-incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M(1) receptor selective), methoctramine (M(2)/M(4) receptor selective) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M(3)/M(1)/M(5) receptor selective)), gave rise to a concentration-dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4. Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M(1)-M(5)) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M(2) receptors was decreased, whereas the expression of muscarinic M(3) receptors was increased. 5. In conclusion, the urothelial cell line TBM-54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide-induced cystitis.

P2Y2- and P2Y4 purinergic receptors are over-expressed in human colon cancer.

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 P2Y(2)- and P2Y(4) 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 P2Y(2)- and P2Y(4) 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 P2Y(2)- and P2Y(4) purinergic receptors could be identified in tumour-free tissue, and both were significantly over-expressed in each of the 10 colon cancers.

Effect of remote preconditioning on mild or severe ischemia-reperfusion injury to rat liver.

UNLABELLED: In this study we examined the effect of remote ischemic preconditioning (RIPC) on liver ischemia-reperfusion (IR) injury. Anesthetized Wistar rats (200 to 250 g body weight, n = 32) had the right femoral artery (FA) dissected. Protocol I. The hepatic artery (HA) was clamped for 60 minutes; peripheral liver blood flow (PLBF) and alanine aminotransferase (ALT) were measured prior to clamping as well as 60 minutes after reperfusion. The cohorts were group 1 (no RIPC; n = 10) and group 2 (RIPC; n = 10) 35 minutes after surgery, the FA was clamped for 10 minutes. After 15 minutes, the HA was clamped as in group 1. In protocol II, a rubber band was applied around the entire vascular supply to about 70% of the liver, yielding group 3 (no RIPC; n = 6) that 60 minutes after surgery, had vascular occlusion performed for 30 minutes and group 4 (RIPC; n = 6) with the FA clamped as above, in a procedure otherwise identical to that of group 3. RESULTS: In protocol I, there was no significant difference in PLBF between the two groups after reperfusion, but the increased ALT levels in the RIPC group were reduced (.70 +/- .05 vs. 1.0 +/- .15 microkat/L, P = .049). In protocol II, we observed no significant differences in ALT levels or PLBF between the two groups. Thus, a beneficial effect of RIPC was demonstrated in protocol I with relative hypoxemia to the liver. However, the effect could not be demonstrated in protocol II, which induced a more severe IR injury.

Nitric oxide-mediated effects on liver blood flow.

We investigated whether blockade of nitric oxide synthase by the arginine analog l- NAME could affect peripheral liver blood flow (PLBF) or hepatocyte integrity (serum ALT) in either a control series or in a series subjected to mild reduction of liver blood flow by temporary clamping of the hepatic artery (HA). Anesthetized rats were arranged for mean arterial pressure (MAP) recordings via a carotid artery, drug injections, and blood sampling via a jugular vein, and monitoring of PLBF using a laser Doppler flowmeter. In series 1, the rats received either l-NAME (30 mg/kg i.v.) or NaCl. l-NAME caused a significant decrease in PLBF and an increase in MAP compared to NaCl; ALT did not differ. In series 2, l-NAME (30 mg/kg i.v.) or NaCl was administered at the beginning of the experiment. After 60 minutes of equilibration, the HA was clamped for 60 minutes then unclamped for another 60 minutes. As in series 1, the l-NAME group had significantly lower PLBF and higher MAP than the NaCl group. Occlusion of the HA resulted in significantly greater reduction in PLBF in the NaCl versus the l-NAME group. Upon unclamping, there was no difference in ALT levels, PLBF, or MAP. To conclude, NO displayed a positive tonic effect on liver blood flow, reduction of which with l-NAME did not aggravate mild ischemia/reperfusion injury in this model.

Demonstration of P2Y4 purinergic receptors in the HT-29 human colon cancer cell line.

1 The aim of the current study was to investigate the existence of P 2 Y(4) 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 P 2 Y(4) purinergic receptor. 4 Immunocytochemistry showed immunoreactivity for the P 2 Y(4) purinergic receptor localized in the cytoplasm of the HT-29 cells. 5 This is the first demonstration of the protein expression of P 2 Y(4) purinergic receptors in a human colon cancer cell line.

Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats.

In the in vitro study, it was investigated whether the expression of muscarinic receptors and cholinergic responses were altered in the situation of experimental cystitis. Rats were treated with cyclophosphamide intraperitoneally and the bladders were excised 36-100 h later. Immunohistochemistry and immunoblotting showed all subtypes of the muscarinic receptor (M1-M5) to be present in the specimens from inflamed urinary bladders and controls. In the cyclophosphamide-treated rats, the expression of muscarinic M5 receptors was increased by more than 40 times (p<0.01; n=8) both in the smooth muscle and the urothelium. Both the maximal contractile response to carbachol and to a high potassium concentration was approximately halved in cyclophosphamide-treated tissues, whereas the reduction was substantially greater in response to low carbachol concentrations (

Postjunctional modulation by muscarinic M2 receptors of responses to electrical field stimulation of rat detrusor muscle preparations.

1. The aim of the present study was to examine the modulator influence of muscarinic M(2) 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 M(1)/M(3) receptor selective: 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP); muscarinic M(2) receptor selective: methoctramine), a beta-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 beta-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 beta-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 M(2) receptor activation inhibits adenosine receptor- and beta-adrenoceptor-evoked relaxations of the rat detrusor muscle. The inhibition occurs via a transient postjunctional mechanism that mainly affects responses with a short latency.

Demonstration of functional receptors for noradrenaline and adenosine-5'-triphosphate, but not for prostaglandin E2, in HT-29 human colon cancer cell line.

1. 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). 2. 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. 3. Challenge with NA (5.9 microM) resulted in an increase in ECAR by approximately 24% of basal. 4. PGE2 (0.0284, 0.284 and 2.84 microM) hardly affected ECAR. 5. ATP (100 microM) elicited a biphasic effect on ECAR (increase and decrease in ECAR by about 58 and 10% of basal, respectively). 6. HT-29 cells were shown to express COX-2 by immunocytochemistry. 7. These data suggest the presence of functional receptors for NA and ATP, but not for PGE2 in HT-29 human colon cancer cell line.

The anaesthetic agent propofol interacts with GABA(B)-receptors: an electrophysiological study in rat.

The mode of action by which propofol induces anaesthesia is not fully understood, although several studies suggest that the compound acts via potentiation of brain GABA(A)-receptors. The aim of the present study is to investigate a putative GABA(B)-receptor agonistic action of propofol. For this purpose the action of propofol on a GABA-receptor mediated regulation of dopamine neurons was analyzed with extracellular single unit recordings of dopaminergic neurons of the substantia nigra in chloral hydrate anaesthetized rats. Intravenous administration of propofol (1-16 mg/kg) was found to dose-dependently decrease the firing rate and burst firing activity of nigral DA neurons. These effects by propofol were effectively antagonized by pretreatment with the selective GABA(B)-receptor antagonist CGP 35348 (200 mg/kg, i.v.) but not by pretreatment with the GABA(A)-receptor antagonist picrotoxin (4.5 mg/kg, i.v.). It is proposed that an activation of central GABA(B)-receptors may, at least partially, contribute to the anesthetic properties of propofol.

On the functional role of muscarinic M2 receptors in cholinergic and purinergic responses in the rat urinary bladder.

The functional effects of muscarinic receptor and purinoceptor agonists and antagonists were studied on isolated strip preparations of the rat urinary bladder. The muscarinic "M3/M1-selective" receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) most conspicuously inhibited the carbachol-evoked contractile responses (pA2=9.8), while the muscarinic "M1-selective" receptor antagonist pirenzepine and the muscarinic "M2-selective" receptor antagonist methoctramine were less potent (pA2=7.0 and 6.5, respectively). Administration of 4-DAMP in combination with methoctramine in selective dosages gave no significant additional reduction of carbachol-evoked contractile responses. Adenosine 5'-triphosphate (ATP) elicited transient dose-dependent contractile responses and it caused relaxation of the carbachol-contracted detrusor strips. The relaxatory response was enhanced in the presence of methoctramine and furthermore, was attenuated by the adenosine receptor antagonist 8-p-sulfophenyltheophylline. Administration of 2-chloro-adenosine to pre-contracted strips tended to cause dose-dependent relaxations, which were significantly increased in the presence of methoctramine. The purinergic contractile response, on the other hand, was not affected by methoctramine. Thus, the results are consistent with the cholinergic contractile response in the rat urinary bladder being exerted via activation of muscarinic M3 receptors, while the muscarinic M2 receptors exerted a modulator effect on purine-evoked relaxations in the rat urinary bladder.

Functional effects of dextran sulphate sodium (DSS) treatment on the longitudinal muscle of rat distal colon.

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 (P(o)) for the contractile response to carbachol (1 microM), in relation to the severity of the colitis. At 5% DSS, the magnitude of the response to carbachol at P(o) 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.

Relaxation of rat distal colon by activation of muscarinic, neuronal receptors: possible involvement of P(2y)-purinoceptors.

McN-A-343, which is a ligand at muscarinic receptors on myenteric ganglia, was found to concentration-dependently (1-44 microM) elicit non-adrenergic relaxation of the longitudinal muscle of rat distal colon, having been precontracted with carbachol (1 microM). This effect was partly antagonized by the muscarinic receptor antagonist, pirenzepine (0.3 microM), the nerve blocker, tetrodotoxin (1 microM), or by drugs which interfere with purinergic neurotransmission (apamin [0.5 microM], reactive blue 2 [50 microM]). Blockade of nitric oxide synthase (L-NNA [100 microM]), or of the cAMP (H-89 [1 microM]), or cGMP (ODQ [10 microM]) second messenger pathways did not affect the relaxatory response to McN-A-343 (14 microM). An additional, non-neurogenic component of the relaxation to this compound on carbachol induced tone is suggested to reflect a partial antagonism of the muscarinic receptors on the gut muscle by McN-A-343.

Preconditioning protects against ischemia/reperfusion injury of the liver.

Ischemic preconditioning (IPC) of an organ may induce protection against the injury caused by longer duration of ischemia and subsequent reperfusion. In a standardized model of such injury in the rat liver, we used the following protocol to investigate whether adenosine played a role in IPC by preventing its enzymatic degradation by dipyridamole pretreatment according to the following protocol: group 1, nonischemic control rats; group 2, ischemic control rats subjected to 60 minutes of ischemia by clamping of the common hepatic artery followed by 60 minutes of reperfusion; group 3, IPC with 10 minutes of ischemia followed by 15 minutes of reperfusion, prior to the ischemia/reperfusion period as in group 2; group 4, pharmacologic preconditioning with administration of dipyridamole prior to the ischemia/reperfusion period as in group 2. Peripheral liver blood flow was significantly reduced during clamping (groups 2 to 4). After unclamping, blood flow was still reduced in the ischemic rats (group 2) but had returned to preclamp values in the animals that had been subjected to ischemic (group 3) or pharmacologic (group 4) preconditioning. Liver cell injury was significantly increased in the ischemia group (group 2) only. In our experimental model of ischemia/reperfusion injury in the rat liver, we found an equally beneficial effect with ischemic and pharmacologic preconditioning. Adenosine appears to be a crucial factor in IPC.

K(+)-induced neurogenic relaxation of rat distal colon.

Relaxations of segments of rat distal colon were elicited by hypertonic solutions of potassium (K(+); final concentration, 20.8 or 50.8 mM). The initial part of the response to K(+) was antagonized by the nerve blocker tetrodotoxin. This effect could, moreover, be significantly antagonized by apamin (a blocker of K(+) channels), reactive blue 2 (a P(2y)-purinoceptor antagonist), N(G)-nitro-L-arginine (an inhibitor of NO synthase), 1H-[1,2,4]- oxadiazolo[4,3-a]quinoxaline-1-one (ODQ; an inhibitor of soluble guanylyl cyclase), or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; an inhibitor of cAMP-dependent protein kinase). Sodium nitroprusside (a donor of NO) and vasoactive intestinal peptide (VIP) both relaxed the tissues. The response to sodium nitroprusside was abolished by ODQ and unaffected by H-89, and that to VIP was partially inhibited by VIP(10-28) (a VIP receptor antagonist), ODQ, or H-89. When combining reactive blue 2 and N(G)-nitro-L-arginine, the response to 50.8 mM K(+) was reduced by approximately 70% and was abolished by the concomitant administration of these antagonists and VIP(10-28). ATP, NO, and VIP may, thus, be inhibitory neurotransmitters in rat distal colon.

Recombinant or plasma-derived antisecretory factor inhibits cholera toxin-induced increase in Evans blue permeation of rat intestinal capillaries.

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.

Effect of ganglionic blocking compounds on in-vivo fluid secretion in the rat small intestine.

It is well-known that enteric, secreto-motor nerves mediate cholera toxin-induced fluid secretion in the rat small intestine. This notion is, in part, derived from experiments on anaesthetized animals in which the response to cholera toxin was antagonized by the ganglionic nicotinic receptor antagonist, hexamethonium. In the current study, such anti-secretory action of ganglionic blocking compounds was analysed in an experiment designed to minimize any possible negative effect of general anaesthesia on intestinal secretion. Rats were anaesthetized with ether for 5-10 min, during which time a jejunal loop (10-12 cm) was constructed. The loop was challenged with one of the secretagogues, cholera toxin, prostaglandin E1 (PGE1) or okadaic acid. Saline (control) or either of the ganglionic blockers, hexamethonium and chlorisondamine, was administered intravenously. The rats were killed 5 h (cholera toxin) or 1.5 h (PGE1 and okadaic acid) after challenge, and the amount of fluid accumulated in the loops was determined. Cholera toxin-induced secretion was unchanged by hexamethonium but reduced by approximately 80% by chlorisondamine. The difference in effect between the two blockers might relate to the duration of ganglionic blockade. Chlorisondamine blocked secretion induced by either PGE1 or okadaic acid by approximately 60%. It is suggested that the anti-secretory effect of ganglionic blocking compounds might be a result of blockade of secreto-motor nerves but other mechanisms, for example interference with haemodynamic factors, cannot be ruled out.