Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.     

In vitro and in vivo analysis of the Effects of corticotropin releasing factor on rat dorsal vagal complex

In vivo and in vitro electrophysiological experiments were performed on the rat dorsal vagal complex (DVC, i.e. nucleus of the tractus solitarius, NTS, and dorsal motor nucleus of the vagus, DMV) to examine the effects of corticotropin releasing hormone (CRF) on the central components of the vago-vagal reflex control of gastric function. When applied to gastrointestinal projecting DMV neurones, CRF (10-300 n m ) induced a concentration-dependent membrane depolarization, an increase in action potential firing rate and decrease in amplitude of the action potential afterhyperpolarization ( P < 0.05). Pretreatment with the non-selective CRF antagonist, astressin (0.5-1 μM) or the selective CRF₂ receptor antagonist, astressin 2B (500 n m ) attenuated the CRF-induced increase in firing rate but did not alter basal discharge rate. CRF (30-300 n m ) increased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by stimulation of the NTS ( P < 0.05). An alteration in the paired pulse ratio indicated the EPSC's increase occurred due to actions at presynaptic sites. In the in vivo anaesthetized rat preparation, bilateral microinjections (20 fmol in 20 nl for each site) of CRF in the DVC decreased gastric motility in rats pretreated with the muscarinic agonist, bethanecol ( P < 0.05). The effects of CRF were abolished by systemic administration of the NOS inhibitor, L -NAME, or by bilateral vagotomy. We concluded that CRF had both a direct and an indirect excitatory effect on DMV neurones via activation of CRF₂ receptors and the decrease in gastric motility observed following microinjection of CRF in the DVC is due to the activation of an inhibitory non-adrenergic non-cholinergic input to the gastrointestinal tract.     

GABAB receptor activation desensitizes postsynaptic GABAB and A1 adenosine responses in rat hippocampal neurones

Whole-cell recordings of EPSCs and G-protein-activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long-term agonist treatment on the presynaptic and postsynaptic responses mediated by GABA_B receptors (GABA_BRs). GABA_BR-mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one-half after 96 h. In contrast, GABA_BR-mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A₁ adenosine receptors (A₁Rs) was unaffected by prolonged GABA_BR activation, whereas A₁R-mediated GIRK currents were desensitized. Desensitization of postsynaptic GABA_BR and A₁R responses was blocked by the GABA_BR antagonist (1-(S)-3,4-dichlorophenylethyl)amino-2-(S) hydroxypropyl-p-benzyl-phosphonic acid (CGP 55845A), but not by the A₁R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non-hydrolysable GTP analog 5'-guanylylimidodiphosphate (Gpp(NH)p). Short-term (4-24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage-gated calcium channels by activation of GABA_BRs or A₁Rs. These results show that responses mediated by GABA_BRs and A₁Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.     

Central 5-HT7 receptors are critical for reflex activation of cardiac vagal drive in anaesthetized rats

5-Hydroxytryptamine (5-HT; serotonin)-containing neurones contribute to reflex activation of parasympathetic outflow in a number of species, but the 5-HT receptors mediating these effects have yet to be fully determined. The present experiments demonstrate that central 5-HT₇ receptors are involved in the vagal bradycardia evoked during the cardiopulmonary reflex, baroreflexes and the chemoreflex, as well as other autonomic changes caused by these reflexes. The experiments examined the effects of the selective 5-HT₇ receptor antagonists SB-269970 and SB-656104 on these reflexes. For the cardiopulmonary reflex, when compared to time-matched vehicle control experiments, intracisternal application of SB-269970 (30–300 μg kg⁻¹, i.c. ) dose-dependently attenuated the evoked bradycardia. At the highest dose, SB-269970 also attenuated the reflex hypotension and sympathoinhibition. The structurally different 5-HT₇ receptor antagonist SB-656104 (100 μg kg⁻¹, i.c. ) similarly attenuated the reflex bradycardia and hypotension. SB-269970 (100 μg kg⁻¹, i.c. ) also attenuated the bradycardias evoked by electrical stimulation of aortic nerve afferents and the baroreflex evoked by the pressor response to phenylephrine (3–25 μg kg⁻¹, i.v. ). The gain of the baroreflex was also significantly attenuated (0.15 ± 0.06 versus 0.34 ± 0.06 ms mmHg⁻¹). Finally, SB-269970 (100 μg kg⁻¹, i.c. ) significantly attenuated both the bradycardia and sympathoexcitation evoked by the chemoreflex. These data indicate that central 5-HT₇ receptors play an important facilitatory role in the reflex activation of vagal outflow to the heart.     

D1 and D2 receptor-mediated dopaminergic modulation of visual responses in cat dorsal lateral geniculate nucleus

The modulatory effects of dopamine (DA) on the visual responses of relay cells of the dorsal aspect of cat lateral geniculate nucleus (dLGN) were tested using local micro-iontophoretic application of DA and application of the receptor-specific agonists SKF38393 (SKF, D₁/D₅) and quinpirole (QUIN, D₂/D₃/D₄) in the anaesthetized alcuronium-treated cat. The effects of DA and QUIN were clearly dose-dependent: small amounts caused a weak and transient facilitation of visual activity (10–30 % increase) preferentially in Y-type relay cells, which changed to a moderate reduction of visual responses when the dose was increased (50 %, maximal 70 %). The effect of SKF was mainly suppressive and increased with the amount of drug applied (up to 90 % reduction). The selective antagonists SCH23390 (SCH, D₁) and sulpiride (SULP, D₂) reduced the effects of co-applied DA agonists. We found little evidence for a specific dopaminergic modulation of the surround inhibition (stimulus-driven lateral inhibition) although DA slightly facilitated the transmission of weak signals (small stimuli). Nevertheless, some dopaminergic effects seem to be mediated via inhibitory interneurons regulating the strength of sustained or recurrent inhibition. Application of DA agonists during blockade of GABA_A receptors indicates a direct suppression of relay cells via D₁ receptors, an excitation of relay cells via D₂ receptors and - with increasing amounts of D₂ agonist - probably also an excitation of inhibitory interneurons, which results in an indirect inhibition of dLGN relay cells (predominantly of the X-type). The results are discussed in relation to the impairment of visual functions in Parkinson's disease.     

Role of tachykinin NK1 and NK2 receptors in allergen-induced early and late asthmatic reactions, airway hyperresponsiveness, and airway inflammation in conscious, unrestrained guinea pigs

Using a guinea pig model of allergic asthma, we investigated the effects of the inhaled, highly selective nonpeptide tachykinin NK₁ and NK₂ receptor antagonists SR 140333 and SR 48968, respectively, on allergen-induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions, and infiltration of inflammatory cells in the airways. Both SR 140333 (100 nM, 3 min) and SR 48968 (100 nM, 3 min) had no effect on the severity of the EAR, while the NK₂ receptor antagonist SR 48968, but not the NK₁ receptor antagonist SR 140333, caused significant inhibition of the LAR. SR 140333 significantly reduced the allergen-induced AHR to histamine, both after the EAR and the LAR. By contrast, SR 48968 did not affect the AHR after the EAR, but significantly attenuated the AHR after the LAR. Bronchoalveolar lavage studies performed after the LAR indicated that SR 140333 caused significant inhibition of allergen-induced infiltration of eosinophils, neutrophils and lymphocytes, while SR 48968 attenuated the infiltration of neutrophils and lymphocytes, but not of eosinophils. Both NK receptor antagonists tended to reduce the accumulation of ciliated epithelial cells in the airways. These results indicate that NK₁ and NK₂ receptors are importantly, but differentially, involved in the development of allergen-induced airways obstruction, AHR and infiltration of inflammatory cells in the airways. Therefore, both NK₁ and NK₂ receptor antagonists, or dual NK₁ and NK₂ antagonists, could be useful in the treatment of allergic asthma.     

Pathways involved in gut mucosal barrier dysfunction induced in adult rats by maternal deprivation: corticotrophin-releasing factor and nerve growth factor interplay

Neonatal maternal deprivation (NMD) increases gut paracellular permeability (GPP) through mast cells and nerve growth factor (NGF), and modifies corticotrophin-releasing factor (CRF) and corticosterone levels. CRF, corticosterone and mast cells are involved in stress-induced mucosal barrier impairment. Consequently, this study aimed to specify whether corticosteronaemia and colonic expression of both preproCRF and CRF are modified by NMD, and to determine if altered expression may participate in the elevated GPP in connection with NGF and mast cells. Male Wistar rat pups were either separated from postnatal days 2–14, or left undisturbed with their dam. At 12 weeks of age, adult rats were treated with mifepristone (an antagonist of corticoid receptors), α-helical CRF_(9-41) (a non-specific CRF receptor antagonist), or SSR-125543 (CRF-R₁ receptor antagonist). We also determined corticosteronaemia and both colonic preproCRF and CRF expression. Then, control rats were treated by CRF, doxantrazole (mast cell stabilizer), BRX-537A (a mast cell activator) and anti-NGF antibody. NMD did not modify colonic CRF level but increased colonic preproCRF expression and corticosteronaemia. Peripheral CRF, via CRF-R₁ receptor, but not corticosterone, was involved in the elevated GPP observed in these rats, through a mast-cell-mediated mechanism, since the increase of GPP induced by exogenous CRF was abolished by doxantrazole. Anti-NGF antibody treatment also reduced the elevated GPP induced by CRF or BRX-537A. CRF acts through CRF-R₁ receptors to stimulate NGF release from mast cells, which participates in the elevated GPP observed in NMD adult rats. This suggests that early traumatic experience induced neuro-endocrine dysfunction, involved in alterations of gut mucosal barrier.     

Dopamine selectively reduces GABAB transmission onto dopaminergic neurones by an unconventional presynaptic action

The functioning of midbrain dopaminergic neurones is closely involved in mental processes and movement. In particular the modulation of the inhibitory inputs on these cells might be crucial in controlling firing activity and dopamine (DA) release in the brain. Here, we report a concentration-dependent depressant action of dopamine on the GABA_B IPSPs intracellularly recorded from dopaminergic neurones. Such effect was observed in spite of the presence of D₁/D₂ dopamine receptor antagonists. A reduction of the GABA_B IPSPs was also caused by noradrenaline (norepinephrine) and by l -β-3,4-dihydroxyphenylalanine ( l -DOPA), which is metabolically transformed into DA. The DA-induced depression of the IPSPs was partially antagonised by the α2 antagonists yohimbine and phentolamine. DA did not change the postsynaptic effects of the GABA_B agonist baclofen, suggesting a presynaptic site of action. Furthermore, DA did not modulate the GABA_A-mediated IPSP. The DA-induced depression of the GABA_B IPSP occluded the depression produced by serotonin and was not antagonized by serotonin antagonists. The DA- and 5-HT-induced depression of the GABA_B IPSP persisted when calcium and potassium currents were reduced in to the presynaptic terminals. These results describe an unconventional presynaptic, D₁ and D₂ independent action of DA on the GABA_B IPSP. This might have a principal role in determining therapeutic/side effects of l -DOPA and antipsychotics and could be also involved in drug abuse.     

Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Aquaporin-1 and HCO3−-Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO₃⁻-Cl⁻ transporter may be involved in CO₂ transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO₂ transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO₂-containing gas above a stirred cell suspension caused an outside-to-inside directed CO₂ gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO₂ passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO₂-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl₂ at 65 μM) or the HCO₃⁻-Cl⁻ transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO₂, nearly the entire CO₂ transport across the red cell membrane is mediated by AQP1 and the HCO₃⁻-Cl⁻ transporter. Therefore, these proteins may function as high affinity sites for CO₂ transport across the erythrocyte membrane.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Activation of Cloned Human CD4+ Th1 and Th2 Cells by Blood Dendritic Cells

Dendritic cells (DC) have been reported to be the most potent antigen-presenting cells (APC) for the activation of naive T cells and to be 10–100-fold more potent APC than monocytes (Mφ) in the mixed lymphocyte reaction. In this study the authors compared human blood DC with Mφ and B cells for their ability to activate cloned rye grass allergen Lol p I specific CD4⁺ Th₁ and Th₂ cells. In the presence of Lol  p I, all three types of APC activated Th₁ and Th₂ cells to a similar extent, as shown by T-cell proliferation and interferon-γ, interleukin-2 (IL-2) or IL-4 secretion. However, at low APC : T cell ratios, Mφ were the most potent APC for both Th₁ and Th₂ cells followed in decreasing order by DC and B cells. This hierarchy was observed with APC preparations isolated by negative selection or highly purified by positive selection using fluorescent cell sorting for HLA-DR^high-DC, CD14^pos-Mφ and CD19^pos-B cells. The data demonstrate that, in contrast to what has been reported for naive T cells, human blood DC activate cloned memory Th₁ and Th₂ cells to a similar extent as Mφ and B cells presumably because the requirements for activation of memory type T cells are less stringent than those for naive T cells.     

Localization and function of histamine H3 receptor in the nasal mucosa

Background Histamine is an important chemical mediator of allergic rhinitis (AR). Histamine H₃ receptors (H₃R) are located on cholinergic and NANC neurons of the myenteric plexus, and activation of H₃R regulates gastric acid secretion. However, little is known about the localization and function of H₃R in the upper airway.
Objective The objective of this study was to examine the localization and possible function of H₃R in the nasal mucosa.
Methods We extracted total RNA from the inferior turbinate mucosa of patients with AR. H₃R mRNA and β-actin mRNA were amplified by RT-PCR. We used immunohistochemistry to examine localization of H₃R protein in the inferior turbinate mucosa excised during clinically indicated surgery. We used alcian blue/periodic acid-shiff staining to examine the effects of the H₃R agonist (R)-α-methylhistamine and the H₃R antagonist thioperamide on secretion from rat submucosal glands.
Results H₃R protein was expressed around submucosal gland cells. Thioperamide induced degranulation in the submucosal gland in the nasal septum.
Conclusion The present results suggest that H₃R is localized mainly around submucosal glands, and that H₃R plays an important role in the secretion of submucosal glands in the nose.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Both Murine SAA1 and SAA2 Yield AA Amyloid in Alveolar Hydatid Cyst-Infected Mice

Amyloid susceptible C57BL/6 and partially amyloid resistant A/J mice, infected intraperitoneally with 250 alveolar hydatid cyst (AHC), the larval stage of a cestode parasite Echinococcus multilocularis , develop multiple organ amyloid deposits at approximately 1 and 4 weeks post infection (p.i.), respectively. Pooled spleens and livers from each mouse strain, at 8 and 10 weeks p.i., were used for the purification of protein AA utilizing a HiLoad Superdex 200 column equilibrated with 5 M guanidine-HCl. Protein AA from each mouse strain was separated on 16% Tris-tricine SDS-PAGE gels and immunoblotted with monospecific rabbit anti-mouse AA IgG; five and six immunoreactive AA subspecies were detected in the C57BL/6 and A/J materials, respectively. N-Terminal amino acid sequence analysis was performed on the bulk column-purified protein AA as well as on the electroblotted AA subspecies from each mouse strain. The results show a mixture of serum amyloid A₁ (SAA₁) and (SAA₂)-derived AA protein from each mouse strain; SAA₁-derived AA, although alluded to, has never been demonstrated as tissue deposits in mice. These findings suggest that the intense and persistent inflammatory processes in AHC-infected mice may have induced conversion of weakly amyloidogenic SAA₁ to AA. This conversion could be detected by amino acid sequencing of electrophoretically separated AA subspecies.     

Lipoxin A4 Metabolites/Analogues from Two Commercial Sources have No Effects on TNF-α-mediated Priming or Activation through the Neutrophil Formyl Peptide Receptors

The eicosanoid lipoxin A₄ (LXA₄) is a potent anti-inflammatory mediator in many in vivo experimental models, and it has been proposed that the effects of this molecule are mediated through binding to FPR2 (also termed FPRL1 or ALXR), a member of the formyl peptide receptor family. Research has shown that LXA₄ inhibits neutrophil function, which has been suggested to be an important mechanism in the anti-inflammatory activity of this lipoxin. However, experiments demonstrating such an impact of LXA₄ have not always been convincing. In this study, we examined the influence of metabolically stable LXA₄ analogues on the biological activities induced by a previously characterized FPR2 agonist (WKYMWM) and a commonly used FPR1 agonist (fMLF). We also investigated the analogues regarding their direct effect on TNFα-mediated neutrophil mobilization of the complement receptor 3 (CR3) and their indirect effect on cytokine-dependent priming of the cells. The LXA₄ analogues we used came from two commercial sources. In our experiments, they did not induce any direct neutrophil response, nor did they affect the increase in the number of CR3 molecules on the neutrophil surface or the primed response. Therefore, we conclude that these LXA₄ analogues do not have an impact on TNF-α induced signalling in neutrophils. We also applied a recently described technique that has proven to be a valuable tool for identifying selective FPR1 and FPR2 agonists and antagonists. We found that the lipoxin analogues did not induce any changes in the neutrophil response, which implies that LXA₄ does not act through FPR2 in these cells.     

P2X3 receptors and peripheral pain mechanisms

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X₃ subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X₂ subunits into hetero-oligomers. Studies with antagonists selective for P2X₃-containing receptors, experiments with antisense oligonucleotides to reduce P2X₃ subunit levels, and behavioural testing of P2X₃ knock-out mice, all suggest a role for the P2X_2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.     

Anti-Parental-Strain Lymphocyte Responsiveness of F1 Hybrid-Strain Lymphocytes

The interactions between immunocompetent parental-strain tells and the F₁ hybrid rat into which these cells have been injected were studied. This involved examination of the behavior of both parental- and host-strain cells. Host lymphocytes reactive against parental-strain lymphocytes appeared in the thoracic duct lymph within 12 h of the intravenous injection of parental thoracic duct lymphocytes or thymus cells. The activity of these F₁ hybrid-strain lymphocytes was directed preferentially against parental-strain lymphocytes with anti-F₁ hybrid potential. This subpopulation of parental cells was absent from the thoracic duct but was well represented in the spleen, this enrichment being masked by a reversible F₁ hybrid anti-parental-strain lymphocyte response. If the anti-parental-strain lymphocyte activity of F₁ hybrid cells was interrupted by the destruction of host-strain cells with antiserum, parental-strain lymphocytes in the tissues of F₁ hybrid rats could be reactivated. By this procedure it was possible to passage graft-versus-host reactions initiated by parental-strain lymphocytes through several F₁ hybrid rats, thereby implying that anti-parental lymphocyte activity is of importance in limiting the serial passage of these reactions. Some of the anti-parental lymphocyte activity observed on the part of F₁ hybrid-strain cells was probably mediated by anti-idiotypic antibodies, but other phenomena, especially selective migration of both donor and host lymphocyte subpopulations, are of major importance in the interaction between parental-strain lymphocytes and an F₁ hybrid host.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.