Role of ectodomain lysines in the subunits of the heteromeric P2X2/3 receptor

Lysine residues near each end of the receptor ectodomain (in rat P2X2 Lys69 and Lys308) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits and found that lysine-to-alanine substitutions at equivalent positions in the P2X3 receptor (Lys63 and Lys299) also prevented channel function. Heteromeric P2X2/3 receptors are formed when P2X2 and P2X3 subunits are expressed together; they can be distinguished by their relatively sustained response to alphabeta-methylene-ATP. By coexpression of wild-type P2X3 and mutated P2X2 subunit, we found that the heteromeric P2X2/3 channel functioned normally when either lysine in the P2X2 subunit was mutated to alanine (i.e., [K69A] or [K308A]) but not when both lysines were mutated to alanine (i.e., [K69A, K308A]). However, coexpression of wild-type P2X2 with a mutated P2X3 subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X2 subunit by wild-type P2X3 (but not the converse) suggests that the heteromeric channel contains one P2X2 and two P2X3 subunits and that the receptor functions essentially normally as long as two subunits are not mutated. The failure to rescue function in the P2X2 subunit with both lysines mutated by wild-type P2X3 suggests that these residues from two different subunits interact in agonist binding or channel opening.     

A comparison of the binding characteristics of recombinant P2X1 and P2X2 purinoceptors.

1. We have recently provided evidence that [35S]-adenosine 5'-O-[3-thiotriphosphate] ([35S]-ATP gamma S) can label the human bladder recombinant P2X1 purinoceptor (human P2X1 purinoceptor). In this study we have characterized the binding of [35S]-ATP gamma S to a second P2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2X2 purinoceptor (rat P2X2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2X1 purinoceptors. 2. Infection of CHO-K1 cells with the rat P2X2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pKd = 9.3; Bmax = 18.1 pmol mg-1 protein) binding sites for [35S]-ATP gamma S but not for [3H]-alpha, beta-methylene ATP ([3H]-alpha beta meATP). Since functional P2X purinoceptors could be detected electrophysiologically in these cells, but not in non-infected or CHO-K1 cells infected with SFV containing the LacZ gene, these results suggest that the rat P2X2 purinoceptor can be labelled using [35S]-ATP gamma S. 3. The binding characteristics of the rat P2X2 purinoceptor were compared with those of the human P2X1 purinoceptor, which was also expressed in the CHO-K1 cells using SFV. A major difference between the two recombinant P2X purinoceptor types was in the binding characteristics of alpha, beta-methylene ATP (alpha beta meATP). Thus, in the absence of divalent cations, alpha beta meATP possessed low affinity for both the human P2X1 purinoceptor (pIC50 = 7.2) and rat P2X2 purinoceptor (pIC50 = 7.1) labelled using [35S]-ATP gamma S. However, when the recombinant P2X purinoceptors were labelled with [3H]-alpha beta meATP in the presence of 4 mM CaCl2, the affinity of alpha beta meATP for the human P2X1 purinoceptor increased (pIC50 for alpha beta meATP = 8.2), while the affinity of the rat P2X2 purinoceptor for alpha beta meATP did not change (pIC50 for alpha beta meATP = 6.8). 4. Affinity estimates of 15 other nucleotide analogues for the [35S]-ATP gamma S binding sites on the two recombinant P2X purinoceptor subtypes were surprisingly similar (less than 5 fold difference), the only exception being 2'-deoxy ATP which possessed 8 fold higher affinity for rat P2X2 than for human P2X1 purinoceptors. In contrast dextran sulphate and the P2 purinoceptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid and 4,4'-diisothiocyanatostilbene-2,2' disulphonic acid, possessed 7 to 33 fold higher affinity for the human P2X1 than for the rat P2X2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5. There was some evidence for species differences in the P2X1 purinoceptor. Thus, most nucleotides possessed slightly greater (up to 9-10 fold), while the P2 purinoceptor antagonists possessed slightly lower (up to 7-16 fold), affinity for the endogenous rat vas deferens and rat bladder P2X1 purinoceptors than for the human recombinant P2X1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2X1 purinoceptor and the endogenous P2X1 purinoceptors labelled in either the rat deferens (r = 0.915) or the rat bladder (r = 0.932), than when comparing within species between the endogenous rat vas deferens and rat bladder P2X1 purinoceptors (r = 0.995). 6. In summary, [35S]-ATP gamma S can be used to label the recombinant P2X1 and P2X2 purinoceptors. Despite the marked differences reported between these two forms of P2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.     

Identification of key residues coordinating functional inhibition of P2X7 receptors by zinc and copper

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.     

Evidence that 8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT) is a selective alpha 2-adrenoceptor antagonist on guinea-pig submucous neurones.

1 Intracellular recordings were made from neurones of the submucous plexus and from submucosal arteriolar smooth muscle of guinea-pig ileum for the purpose of examining the the actions of 8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT). 2 8-OH-DPAT (10 nM-20 microM) had no direct presynaptic or postsynaptic actions on submucous plexus neurones. 3 Membrane hyperpolarizations induced in neurones by noradrenaline or UK 14304 were competitively antagonized by 8-OH-DPAT. For dose-ratios up to 40, Schild plots were linear with slopes not significantly different from unity; pA2 values for the 8-OH-DPAT antagonism of postsynaptic alpha 2-adrenoceptors were 6.9-7.2. 4 The inhibitory synaptic potential, which is due to activation of alpha 2-adrenoceptors located on submucous plexus neurones, was selectively inhibited by 8-OH-DPAT; the IC50 value for inhibition of the inhibitory synaptic potential was 250 nM. 5 Neuronal hyperpolarizations mediated through activation of delta-opioid receptors or somatostatin receptors were unaffected by 8-OH-DPAT (0.1-1 microM). 6 The ability of noradrenaline and UK 14304 to inhibit the release of acetylcholine at synapses in the submucous plexus, and to inhibit the release of the transmitter which mediates the excitatory junction potential in the submucosal arteriolar smooth muscle, was also blocked by 8-OH-DPAT. 7 These results suggest that some of the actions of 8-OH-DPAT previously ascribed to agonism at 5-hydroxytryptamine (5-HT) receptors may actually result from blockade of the actions of endogenously released noradrenaline acting on alpha 2-adrenoceptors.     

Corrosion at the interface of cobalt-alloy heads on titanium-alloy stems.

The combination of a cobalt-alloy head on a titanium-alloy femoral hip stem is widely accepted for press-fit and biologic fixation applications. Examination of 30 components retrieved at periods of 0.5 to 66.9 months for histologic examination of tissue ingrowth revealed that 56.6% of the tapered connections between head and stem showed evidence of crevice corrosion leading to concerns of metal ion release and the potential failure of head to stem fixation.     

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation

ATP‐gated P2X₄ receptors (P2X₄R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X₄R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X₄R protein expression by Western blot and biotinylation assays and functional expression by whole‐cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X₄R expression by 2‐ to 7‐fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN‐γ and TNF‐α or IFN‐γ and LPS reduced surface and functional P2X₄R expression by 3‐fold without altering total P2X₄R protein levels. Alternative activation with IL‐4 or IL‐13 did not alter total, surface or functional expression of P2X₄R. This is the first study of the regulation of P2X₄R in macrophages by physiological stimuli and presents a picture whereby P2X₄R become functional in response to initial phagocytic stimuli but return to a non‐functional state during sustained activation by classical macrophage activation.     

Multilevel analysis of couple congruence on pain, interference, and disability

Couple congruence on ratings of pain severity and disability were examined using hierarchical linear modeling. Older community Individuals with Chronic Pain (ICPs) and their spouses completed the Multidimensional Pain Inventory (pain severity, interference, negative spouse responses to pain), Sickness Impact Profile (physical disability, psychosocial disability), and the Mood and Anxiety Symptom Questionnaire (psychological distress). Both spouses reported on ICPs' pain and disability as well as their own psychological distress. Spousal incongruence was observed on interference and physical disability such that ICPs reported greater disability than their spouses reported for them. No significant incongruence was observed in pain severity or psychosocial disability. Predictors of couples' mean ratings of pain and disability were identified. Specifically, couples in which the ICP was female reported higher couples' ratings of pain severity and interference. ICP distress was related to higher couples' ratings of all pain and disability variables whereas spouse distress was related to higher psychosocial disability ratings. ICPs' perceptions of negative spouse responses were also positively associated with couples' ratings of physical and psychosocial disability. In terms of congruence, ICP distress was associated with incongruence on interference, physical disability, and psychosocial disability whereas spouse distress predicted incongruence on pain severity, and interference. This study suggests that understanding couples' pain outcome ratings involves an awareness of factors that might influence their perceptions and behaviors.     

Diagnosis of Helicobacter pylori in bleeding peptic ulcer patients,evaluation of urea-based tests

BACKGROUND/AIMS: The prevalence of Helicobacter pylori (Hp) has been reported to be lower in patients with bleeding peptic ulcers than in patients with nonbleeding peptic ulcers. This might be due to inaccuracy of the urease-based diagnostic tests when used in patients with bleeding peptic ulcers. The aims of this study were to compare the validity of the rapid urease test (RUT) and (13)C-urea breath test in patients with bleeding (group 1) and nonbleeding peptic ulcers (group 2) and to examine whether the presence of blood in the stomach influences the validity of urease-based tests. METHODS: 95 consecutive patients with bleeding peptic ulcers (48 with and 47 without blood in the stomach) and 44 with uncomplicated peptic ulcers. Biopsies for RUT and histology were obtained during endoscopy. After endoscopy a (13)C-urea breath test was performed. Positive histology was used as 'gold standard' defining positive Hp-status. RESULTS: The prevalence of Hp-infection was 44/95 (46%) in group 1 and 29/44 (66%) in group 2 (p = 0.04). The sensitivities and specificities of RUT, (13)C-urea breath test and serology (control) were between 0.72 and 0.96; no difference was found between the groups. In group 1 the sensitivity of the RUT decreased from 0.96 when no blood was present to 0.60 when blood was present (p = 0.006). The sensitivity of (13)C-urea breath test was not affected by blood in the stomach. CONCLUSION: When comparing patients with bleeding and nonbleeding peptic ulcers, we did not find any difference in either sensitivity or specificity of the diagnostic tests for Hp. However, the sensitivity of the RUT was lower when blood was present in the stomach, which was the case in only half of the patients. The sensitivity and specificity of the (13)C-urea breath test was not affected by the presence of blood in the stomach.     

Mu and delta receptors belong to a family of receptors that are coupled to potassium channels.

The effects of agonists at mu and delta opioid receptors were compared by measuring membrane currents under voltage clamp from neurons of the rat nucleus locus coeruleus and guinea pig submucous plexus. In each tissue, the appropriate selective agonist (Tyr-D-Ala-Gly-MePhe-Gly-ol for mu receptors in locus coeruleus or Tyr-D-Pen-Gly-Phe-D-Pen for delta receptors in submucous plexus) increased the conductance of an inwardly rectifying potassium conductance and strongly hyperpolarized the membrane. The properties of the potassium conductance affected by the two opioids could not be distinguished. Experiments with intracellular application of guanosine 5'-[gamma-thio]triphosphate indicated that a guanine nucleotide-binding regulatory protein was involved in the coupling between opioid receptor and potassium channel, but there was no evidence for activation of either cAMP-dependent protein kinase or protein kinase C. It is noted that a number of vertebrate neurotransmitter receptors are coupled to potassium channels. The potassium conductance associated with these channels has properties similar to the conductance activated by mu and delta opioids; this family includes the following receptors: acetylcholine M2, norepinephrine alpha 2, dopamine D2, 5-hydroxytryptamine 5-HT1, adenosine A1, gamma-aminobutyric acid GABAB, and somatostatin. It is suggested that this conductance is a conserved neuronal effector coupled to one of the receptor types that mediates the effects of each of several major transmitters. The mu and delta opioid receptors appear to be unusual in that both utilize this same effector mechanism.