Effects of lipoxin A4 on chemotaxis and degranulation of human eosinophils stimulated by platelet-activating factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine

Lipoxins are trihydroxytetraene metabolites derived through a double lipoxygenation of arachidonic acid. Lipoxin A₄ (LXA₄) was prepared by total chemical synthesis, and its capacity to modulate eosinophil migration has been evaluated. LXA₄ is a weak and partial chemotactic agent; at 10⁻⁶ M, it achieved about 20% of the response of 10⁻⁶ M platelet-activating factor (PAF). Preincubation of eosinophils with increasing doses of LXA₄ (10⁻¹⁰−10⁻⁵ M) resulted in a concentration-dependent inhibition of cell migration induced by 10^-6 M formyl-methionyl-leucyl-phenylalanine (FMLP) and 10^-6 M PAF. The concentration of LXA₄ which produced 50% inhibition (IC₅₀) of eosinophil migration was approximately 10^-6 M. LXA₄ (10^-10-10^-6 M) did not elicit ECP release or modulate ECP release induced by 10^-6 M FMLP. LXA₄ may have antiallergic properties in preventing eosinophilic migration.     

What Formyl Peptide Receptors,if Any,Are Triggered by Compound 43 and Lipoxin A4?

In this study, we determined receptor preferences for compound 43, a nitrosylated pyrazolone derivative, and the eicosanoid lipoxin A(4) (LXA(4)), potent anti-inflammatory mediators in many experimental in vivo models. Their effects have been suggested to be mediated through binding to formyl peptide receptor (FPR)2 [earlier known as formyl peptide receptor-like 1 or the lipoxin A(4) receptor (ALXR)], one of the two members of the FPR family expressed in neutrophils. Compound 43 activates all neutrophil functions investigated, whereas LXA(4) induces a unique inhibiting pathway suggested to involve β-arrestin binding as an early signalling step, but not a transient rise in intracellular Ca(2+). We show that compound 43 can activate not only FPR2 but also FPR1, the other neutrophil receptor in the FPR family, and FPR1 is actually the preferred receptor in human neutrophils and possibly also in the murine equivalent. LXA(4) analogues from two commercial sources were used, and neither of these induced any translocation of β-arrestin as measured in an enzyme fragment complementation assay. The conclusions drawn from these experiments are that neither compound 43 nor LXA(4) works as FPR2 agonists in neutrophils, findings of importance for a proper interpretation of results obtained with these compounds as regulators of inflammation.     

Lack of activity of 15‐epi‐lipoxin A4 on FPR2/ALX and CysLT1 receptors in interleukin‐8‐driven human neutrophil function

Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15‐epi‐lipoxin (LX)A₄ interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti‐inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15‐epi‐LXA₄ analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK‐571 binds to FPR2/ALX, so cross‐reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15‐epi‐LXA₄ are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15‐epi‐LXA₄‐mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX‐mediated signalling, enhancing guanosine triphosphate‐gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15‐epi‐LXA₄ was inactive. Furthermore, 15‐epi‐LXA₄ showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15‐epi‐LXA₄ showed a moderate reduction of interleukin (IL)‐8‐mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15‐epi‐LXA₄ is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL‐8‐induced neutrophil survival and produces only moderate inhibition in IL‐8‐mediated neutrophil migration. Our data do not support an anti‐inflammatory role of 15‐epi‐LXA₄‐ FPR2/ALX interaction in IL‐8‐induced neutrophil inflammation.     

A VH-Associated Idiotype in Human Anti-Tetanus Antibodies

An anti-idiotypic (α Id) serum was induced against anti-tetanus toxoid (α TET)-reactivc F(ab')₂ fragments of a single donor. After appropriate adsorption the serum was shown to react only with α TET F(ab')₂ fragments and not with F(ab')₂ fragments deplcted of α TET reactivity or anti-diphtheria toxoid (DIP) or anti-purified protein derivative (α PPD) F(ab')₂ fragments of this donor. Inhibition studies with soluble TET indicated that the anti-idiotypic serum partly reacts with determinants close to or within the antigcnic binding site. When tested on a small panel of α TET F(ab')₂ fragments derived from unrelated donors, one out of four donors tested showed cross-reactivity. When tested on α TET F(ab')₂ fragments derived from related family members, the donor's father and two sisters were found to cross-react, whereas the mother and two other sisters did not. The observed cross-reactivity was independent of the litres of α TET antibodies in the serum. Furthermore, two-dimensional gel electrophoretic studies carried oui on α TET IgG of Id-positive or Id-negative family members indicated that the expression of idiotypic determinants is linked to a particular heavy chain. Immunofluorescent staining with α Id and cytofluoro-graphic analysis of donor T-lymphoblast proliferation in response to TET showed that the T cells did not express Id determinants found on his α TET antibodies.     

Comparative vascular effects of histamine, prostaglandin (PG) D2 and its metabolite 9α, 11β-PGF2 in human skin

In this double-blind study we have investigated the vascular effects of prostaglandin, (PG) D₂, in normal skin and compared these effects with histamine and the initial PGD₂ metabolite 9α, 11β-PGF₂. In eight healthy subjects the vascular response to intradermal injections of histamine, PGD₂, a combination of histamine and PGD₂, and 9α, 11β-PGF₂, was assessed by measurement of the weal and flare area. Histamine caused dose-related increases in weal area ( P <0.01). The weal response due to PGD₂ was greater than saline control only at a dose of 71.0 and 710 nmol ( P <0.05). Because of the small size of the weal produced by PGD₂ when compared with histamine, it was not possible to determine their relative potencies. Histamine and PGD₂ caused dose-related increases in flare area ( P <0.05), and when compared at a response level of 10 cm² and 15 cm², histamine was 45 and 251 ( P <0.01) times more potent than PGD₂ in molar terms. Weal and flare responses due to 9α, 11β-PGF₂ were similar to those observed with the equimolar concentration of PGD₂. The weal and flare responses when PGD₂ and histamine when combined were not significantly different from that predicted by a purely additive effect. We conclude that histamine is likely to be an important mediator contributing towards increased vascular permeability and vasodilatation following immunological activation of skin mast cells in vivo , while PGD₂ and its metabolite 9α, 11β-PGF₂ play only a minor role.     

Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μ m ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺ imaging showed that similarly applied 1 μ m ATP, 30 μ m BzATP (a P2X₇ agonist), or 1 μ m 2MeSATP (a P2Y₁ and P2Y₁₁ agonist) increased intracellular Ca²⁺ concentration, but 100 μ m UTP (a P2Y₂ and P2Y₄ agonist) and α,β-meATP (a P2X agonist except for P2X₂, P2X₄ and P2X₇) did not. RT-PCR suggested the expression of P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₁₃ and P2Y₁₄ among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X₂. The exposure of the basolateral membranes to 3 m m ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μ m PPADS (a non-selective P2 blocker) and 1 μ m KN-62 (a P2X₇ blocker). These results showed for the first time the functional expression of P2X₂ and P2X₇ on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.     

Spatiotemporal patterning of IP3-mediated Ca2+ signals in Xenopus oocytes by Ca2+-binding proteins

Ca²⁺-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP₃ in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP₃-evoked Ca²⁺ signals. Parvalbumin (PV), a CaBP with slow Ca²⁺-binding kinetics, shortened the duration of IP₃-evoked Ca²⁺ signals and 'balkanized' global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca²⁺ responses and promoted 'globalization' of spatially uniform Ca²⁺ signals at high [IP₃]. Oocytes loaded with CR or PV showed Ca²⁺ puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca²⁺ buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP₃ action evident with CR. The ability of Ca²⁺ buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca²⁺ signals is likely to have significant physiological implications.     

Regulation of Newborn and Adult Monocytes by PGE2: Evidence of Comparable Differentiation Status

PGE₂, which is produced in large amounts by monocytes, is considered to be an important autocrine factor in the regulation of the immune response. Altered sensitivity to PGE₂ is known to reflect the differentiation status of monocytes/macrophages. Since newborn monocytes are thought to be less differentiated than adult peripheral blood monocytes, this study was designed to determine whether the diminished newborn monocyte function, relative to adult peripheral blood monocytes, might be the result of an altered autocrine responsiveness to PGE₂. Phosphodiesterase activity as well as cAMP and TNFα levels in response to PGE₂ were measured in newborn and adult monocytes, as an index of sensitivity to PGE₂. Basal intracellular cAMP levels were reduced in cord monocytes compared with adult monocytes. Addition of increasing concentrations of PGE₂ (10  m ⁻¹⁰–10  m ⁻⁶) caused similar fold increases in intracellular levels of cAMP for both populations. Lipopolysaccharide-induced TNFα production was significantly reduced in a similar fashion for both cord and adult monocytes at high (≥ 10⁻⁷  m ) PGE₂ concentration. Phosphodiesterase activity was comparable in lysates from both populations. This study shows that PGE₂ does not differentially influence newborn and adult monocyte responses and therefore that the diminished functional abilities of newborn monocytes is not due to altered autocrine responsiveness.     

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.     

α1-Microglobulin Glycopeptides Inhibit Antigen-Specific Stimulation of Human Peripheral Blood Leucocytes

Glycopeptides were prepared from proteolytically digested human and guinea pig α₁ microglobulin (α₁-m) by gel chromatography on Sephadex G-100 and affinity chromatography on concanavalin A-Sepharose. Amino acid analysis showed that the average glycopeptide was a tripeptide containing the amino acids aspartic acid/asparagine, isoleucine, and serine and/or threonine. It has earlier been shown that human α₁-m carries two or three N-glycosidically linked oligosaccharides of the high-mannose type. These preparations of glycopeptides inhibited the proliferative response of peripheral human blood leucocytes in the antigen purified protein derivative. The dose-response relationship of the inhibitions showed a greater specific activity of the glycopeptides than of whole α₁-m. Mild alkali treatment of human α₁-m did not affect its specific inhibitory activity, suggesting that the peptide backbone is of little importance for the immunosuppressive effect of α₁-m. No difference in inhibitory activity was seen between human and guinea pig native α₁-m or α₁-m glycopeptides. Human asialo α₁-m exerted a suppressive effect on the antigen-specific leucocyte proliferation similar to that of native α₁-m.     

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule-1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins Gα_q/11 also mediate flow-induced responses. Here, we investigated whether PECAM-1 and Gα_q/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of Gα_q/11 and PECAM-1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gα_q/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gα_q/11–PECAM-1 complex within 30 s and a partial relocalization of the Gα_q/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced Gα_q/11–PECAM-1 dissociation. These results allow us to conclude that Gα_q/11–PECAM-1 forms a mechanosensitive complex and its localization suggests the Gα_q/11–PECAM-1 complex is a critical mediator of vascular diseases.     

Rapid Report

Sympathetic vasoconstriction is blunted in the vascular beds of contracting skeletal muscles. We sought to determine whether this blunted vasoconstriction is specific for post-junctional α₁- or α₂-adrenergic receptors. We measured forearm blood flow (Doppler ultrasound) and calculated the vascular conductance (FVC) responses to brachial artery infusions of tyramine (which evokes endogenous noradrenaline release), phenylephrine (an α₁ agonist) and clonidine (an α₂ agonist) in 10 healthy men during rhythmic handgrip exercise (10-15 % of maximum) and during a control non-exercise vasodilator condition (intra-arterial adenosine). Steady-state FVC during exercise and adenosine was similar in all trials (range: 243-272 and 234-263 ml min⁻¹ (100 mmHg)⁻¹, respectively; P > 0.5). During exercise the percentage reductions in FVC in response to tyramine (−24 ± 7 vs. −55 ± 6 %), phenylephrine (−12 ± 8 vs. −37 ± 8 %) and clonidine (−17 ± 6 vs. −49 ± 4 %) were significantly less compared with adenosine (all P < 0.05). The magnitude of the blunted vasoconstrictor responses was similar for both receptor subtypes. These findings are in contrast to those from studies in animals demonstrating that α₂-adrenergic receptor-mediated vasoconstrictor responses are much more sensitive to contraction-induced inhibition than α₁-mediated responses. We conclude that vasoconstrictor responses mediated via both post-junctional α₁- and α₂-adrenergic receptors are blunted in contracting human skeletal muscles.     

Persistent CD3-Crosslinking Down-Regulates Interleukin-2 Responsiveness in Interleukin-2-Competent Cloned T Cells: the Possible Involvement of Protein Kinase C

To investigate the regulation of interleukin-2 (IL-2) responsiveness of T cells, a human CD4⁺ T-cell clone with constitutive expression of IL-2 receptors was stimulated with recombinant IL-2 (rIL-2) in the presence or absence of immobilized anti-CD3 monoclonal antibodies (αCD3_imm MoAb). Incubation of T cells with αCD3_imm MoAb decreased IL-2-induced proliferation which could not be ascribed to the modulation of IL-2 receptor expression nor to cell death. Phorbol-myristate-acetate (PMA), an activator of protein kinase C (PKC), also induced down-regulation of IL-2 responsiveness. The αCD3_sol MoAb, inducing Ca²⁺-mobilization without activating PKC, did not inhibit IL-2 responsiveness whereas cyclosporine A (CsA), a drug that inhibits the Ca²⁺-dependent activation pathway, did not prevent the induction of IL-2 hyporesponsiveness induced by αCD3_imm MoAb. It is concluded that modulation of IL-2 responsiveness of T cells via the T-cell receptor/CD3 complex (TCR/CD3) may be mediated by a PKC-activating signal.     

Subunit composition and role of Na+,K+-ATPases in adrenal chromaffin cells

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na⁺,K⁺-ATPase α and β subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 μ m ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca²⁺ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na⁺,K⁺-ATPase α1 subunit-like and β3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas α2- and β2-like IR materials were present in restricted parts of the cell periphery. The α1 and α2 subunits were mainly immunoprecipitated from AM preparations by anti-β3 and anti-β2 antisera, respectively. Peripheral BODIPY-FL-InsP₃ binding sites were localized below membrane domains with α2- and β2-like IR materials. The results indicate that in AM cells, α1β3 isozymes of Na⁺,K⁺-ATPase were present ubiquitously in the plasma membrane, while α2β2 isozymes were in the membrane domain closely associated with peripheral Ca²⁺ store sites. This close association of the α2β2 isozyme with peripheral Ca²⁺ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 μ m ouabain.     

Immunosuppressive Properties of α1-Microglobulin

α₁-Microglobulin (α₁m), a serum glycoprotein (26,000 d). was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that, α₁m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of α₁m on antigen stimulation did not seem to be due to binding of α₁m to PPD or cells, to altered kinetics of the PPD response, or to non-specific cytotoxicity. In contrast, PPD-induced leucocyte migration inhibition was not reversed by α₁m. α₁m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, α₁m had its own leucocyte migration inhibitory effect.     

Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon

Proteinase-activated receptor 2 (PAR2) is a receptor for mast cell tryptase and trypsins and might participate in brain-gut communication. However, evidence that PAR2 activation can lead to afferent impulse generation is lacking. To address this issue, we examined the sensitivity of jejunal afferent nerves to a hexapeptide agonist of PAR2, SLIGRL-NH₂, and the modulation of the resulting response to treatment with drugs and vagotomy. Multiunit recordings of jejunal afferent activity were made using extracellular recording techniques in anaesthetised male rats. SLIGRL-NH₂ (0.001–1 mg kg⁻¹, I.V.) increased jejunal afferent firing and intrajejunal pressure. The reverse peptide sequence (1 mg kg⁻¹, I.V.), which does not stimulate PAR2, was inactive. Naproxen (10 mg kg⁻¹, I.V.), but not a cocktail of ω-conotoxins GVIA and SVIB (each at 25 μg kg⁻¹, I.V.), curtailed both the afferent response and the intrajejunal pressure rise elicited by the PAR2 agonist. Although neither treatment modulated the peak magnitude of the afferent firing, they each altered the intestinal motor response, unmasking an initial inhibitory component. Nifedipine (1 mg kg⁻¹, I.V.) reduced the peak magnitude of the afferent nerve discharge and abolished the initial rise in intrajejunal pressure produced by SLIGRL-NH₂. Vagotomy did not significantly influence the magnitude of the afferent response to the PAR2 agonist, which involves a contribution from capsaicin-sensitive fibres. In conclusion, intravenous administration of SLIGRL-NH₂ evokes complex activation of predominantly spinally projecting extrinsic intestinal afferent nerves, an effect that involves both direct and indirect mechanisms.     

Inhibition by thromboxane antagonists of airway hyperresponsiveness to histamine induced by 13,14-dihydro-15-keto-PGF2α in guinea-pigs

Summary. We studied the effect of intravenous administration of 13,14-dihydro-15-keto-prostaglandin (PG) F_27alpha; on airway responsiveness to histamine and airway wall thickening in guinea-pigs. Guinea-pigs were killed and the lungs were fixed in formalin. Slides from paraffin-embedded sections of the lungs were stained and the airways that were cut in transverse section were measured by tracing enlarged images using a digitizer. Moreover, airway resistance (Raw) was determined by a pulmonary mechanics analyser and we calculated two indices, an index of airway wall thickening and the one of airway hyperresponsiveness to histamine, from changes of baseline-Raw and peak-Raw following intravenous administration of histamine before and after the intravenous administration of 13,14-dihydro-l5-keto-PGF_2n. Intravenous administration of 10/μg/kg 13,14-dihydro-15-keto-PGF_2α for 1 h did not induce an increase of the relative thickness of the airway wall by the histological examination. In analysis of airway function, intravenous administration of 10μg/kg 13,14-dihydro-15-keto-PGF_2α for 1 h induced airway hyperresponsiveness to histamine without airway wall thickening. Thromboxane A₂ (TXA₂) receptor antagonists ONO-NT-126 and ONO-8809 inhibited the 13,14-dihydro-15-keto-PGF_2α-induced airway hyperresponsiveness to histamine, suggesting that the effect of 13,14-dihydro-15-keto-PGF_2α on bronchial hyperresponsiveness is likely to be mediated through TXA₂.     

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.     

Modulation of K+ currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K⁺ currents by 10 ± 3 %. UTP and the analogues α,β-methylene-ATP and 2-methylthio-ATP also inhibited K⁺ currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K⁺ currents could be inhibited by ADP (9 ± 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that α,β-methylene-ADP, often used to block the ecto-5'-nucleotidase, also inhibited voltage-gated K⁺ currents (7 ± 2.3 %). This inhibition was occluded by ADP, suggesting that α,β-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5'-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, α,β-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of α,β-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K⁺ currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation.