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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. Ca(2+) imaging experiments were performed using cells loaded with the Ca(2+) 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 alpha7nAChR and alpha4/beta2 subunits and evoked higher Ca(2+) transients compared with monocultured astrocytes. The Ca(2+) transients referred to are activators of Ca(2+)-induced Ca(2+) release from intracellular stores, both IP(3) and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca(2+) 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-1beta (IL-1beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1beta further attenuated nicotine-induced Ca(2+) response. We also found that LPS and IL-1beta 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 Ca(2+) signaling in inflammatory reactive astrocytes and upregulation of IL-1beta and the rearrangements of actin filaments in the cells.

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.

The non-adrenergic relaxation of carbachol precontracted longitudinal muscle of the rat distal colon was investigated. Intrinsic nerves were activated by the nicotinic, ganglionic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). DMPP at 1 and 4 microM caused a relaxation that was markedly antagonized by the nerve blocker tetrodotoxin (1 microM) or the nicotinic receptor antagonist, hexamethonium (1 mM). The response to DMPP was significantly antagonized by apamin (an inhibitor of ATP-sensitive K+-channels), by reactive blue 2 (a blocker of P2y purinoceptors) and by an inhibitor of nitric oxide (NO)-synthase (N(G)-nitro-L-arginine, L-NNA). The combined treatment with reactive blue 2 and L-NNA reduced the relaxatory response to 1 microM DMPP by 77 +/- 8% and to 4 microM DMPP by 58 +/- 4% of control, but left a residual component. Our results indicate that ATP and NO, together with at least one additional (hitherto unidentified) substance may be inhibitory neurotransmitters in rat distal colon.

Duodenal or jejunal motility (monitored as pressure changes in a saline-perfused intraluminal catheter) was studied in anaesthetized rats, vagotomized and pretreated with adrenergic blocking agents. In the duodenum (but not the jejunum), atropine or the selective muscarinic M1 and M3 receptor antagonists, pirenzepine and 4-diphenyl-acetoxy-N-methylpiperidine (4-DAMP), respectively, augmented the spontaneous contractile activity. This effect could be abolished either by nicotinic ganglionic receptor antagonism with hexamethonium, or with morphine. Moreover, blockade of the synthesis of nitric oxide by N omega-nitro-L-arginine elicited hypermotility both in the duodenum and the jejunum, and also this response was abolished by hexamethonium. It is proposed from the present results that the rat small is controlled by non-adrenergic, non-cholinergic inhibitory as well as excitatory motor neurons. The latter motor neurons seem to be modulated by muscarinic, nitroxergic or opioidergic mechanisms.

Some factors known to affect jejunal motility (recorded as volume changes of an intraluminal balloon) were investigated in anaesthetized cats (ether-chloralose) pretreated with guanethidine and atropine. Indomethacin, morphine (both compounds administered systemically) or vagal nerve stimulation elicited jejunal excitatory motor responses. The effect of indomethacin seemed to be independent of cyclooxygenase inhibition and probably did not involve opioid receptors. It is suggested that the spasmogenic stimuli caused jejunal hypermotility by inhibiting tonically active, inhibitory motor neurons that are intrinsic to the gut. Furthermore, when the jenunal tone had been raised by indomethacin or morphine spontaneous relaxations were observed, and these could be mimicked by vagal stimulation. Hexamethonium antagonized these relaxations but did not attenuate the drug-induced jejunal hypermotility.

The distribution of P1- and P2-purinoceptors in isolated rat femoral artery was studied by comparing responses to ATP, alpha, beta-methylene ATP and adenosine, both when endothelial cells were intact and when they had been removed by mechanical rubbing (as confirmed by histochemical staining and abolition of relaxations to acetylcholine). alpha, beta-Methylene ATP and ATP (but not adenosine or acetylcholine) contracted preparations at resting tone. alpha, beta-Methylene ATP was significantly more potent than ATP. The potency of both alpha, beta-methylene ATP and ATP was significantly increased in the absence of the endothelium. These contractions were unaffected by tetrodotoxin, phentolamine and methysergide in concentrations sufficient to abolish contractions to perivascular nerve stimulation, noradrenaline or 5-hydroxytryptamine. Acetylcholine, ATP and adenosine relaxed arteries whose tone had been raised by 10(-6) M noradrenaline. Removal of the endothelium abolished relaxations to ATP (contractions were seen instead) and acetylcholine, but not to adenosine. alpha, beta-Methylene ATP further contracted the high tone preparation and was again more potent when the endothelium was absent. ATP and alpha, beta-methylene ATP were more potent as contractile agents when noradrenaline was present. These results confirm that endothelial cells can mediate vasodilation. They also show that ATP can act at P2-purinoceptors at two locations in the isolated rat femoral artery; one on the endothelium leading to vasodilation, and the other on the smooth muscle leading to vasoconstriction. P1-Purinoceptors, however, appear to be located on the smooth muscle only and mediate vasodilation. At the smooth muscle P2-purinoceptor, alpha, beta-methylene ATP is more potent than ATP, whereas at the endothelial P2-purinoceptor, the reverse is true.

Stimulation of the sectioned cervical vagal nerve of anaesthetized cats (ether-chloralose), pretreated with guanethidine and atropine, in the peripheral direction produced gastric relaxation as well as jejunal and ileal contraction. The administration of indomethacin markedly enhanced intestinal tone and the amplitude of spontaneous phasic activity while the basal gastric motility was essentially unchanged. This suggests that endogenous prostaglandins exert an inhibitory influence on intestinal motility. The vagally induced gastric relaxation was significantly inhibited by indomethacin, with could suggest that prostaglandins modulate non-adrenergic, non-cholinergic inhibitory neurotransmission in the stomach.