CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.     

Dual effect of acid pH on purinergic P2X3 receptors depends on the histidine 206 residue

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X(3) receptors. In HEK293 cells permanently transfected with human (h) P2X(3) receptors (HEK293-hP2X(3) cells), an acidic pH shifted the concentration-response curve for alpha,beta-methylene ATP (alpha,beta-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of alpha,beta-meATP. Further, a low pH value increased the activation time constant (tau(on)) of the alpha,beta-meATP current; the fast and slow time constants of desensitization (tau(des1), tau(des2)) were at the same time also increased. Finally, acidification accelerated the recovery of P2X(3) receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X(3) receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the alpha,beta-meATP currents. The voltage sensitivity and reversal potential of the currents activated by alpha,beta-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X(3) receptor channels. It is suggested that acidification may have a dual effect on P2X(3) channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues.     

Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.     

Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.     

Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits

P2X receptors are ligand-gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo- and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co-injection, P2X4 subunits were co-purified with hexahistidyl-tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two-electrode voltage-clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of alpha,beta-methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X(1+4) receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.     

Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit

The P2X(3) receptor is an ATP-gated ion channel predominantly expressed in nociceptive neurons from the dorsal root ganglion. P2X(3) receptor channels are highly expressed in sensory neurons and probably contribute to the sensation of pain. Kinetics of P2X(3) currents are characterized by rapid desensitization (<100 ms) and slow recovery (>20 s). Thus, any mechanism modulating rate of desensitization and/or recovery may have profound effect on susceptibility of nociceptive neurons expressing P2X(3) to ATP. Here we show that currents mediated by P2X(3) receptor channels and the heteromeric channel P2X(2/3) composed of P2X(2) and P2X(3) subunits are potentiated by the neuropeptides substance P and bradykinin, which are known to modulate pain perception. The effect is mediated by the respective neuropeptide receptors, can be mimicked by phorbol ester and blocked by inhibitors of protein kinases. Together with data from site-directed mutagenesis our results suggest that inflammatory mediators sensitize nociceptors through phosphorylation of P2X(3) and P2X(2/3) ion channels or associated proteins.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

Effects of extracellular nucleotides on renal tubular solute transport

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y₁ receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K⁺ channels in mouse collecting duct, apparently through stimulation of P2Y₂ receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y₂ receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.     

Zinc modulation of glycine receptors in acutely isolated rat CA3 neurons

Glycine and GABA are the primary inhibitory neurotransmitters in the spinal cord and brain stem, with glycine exerting its physiological roles by activating strychnine-sensitive ionotropic receptors. Glycine receptors are also expressed in the brain, including the cortex and hippocampus, but their physiological roles and pharmacological properties are largely unknown. Here, we report the pharmacological properties of functional glycine receptors in acutely isolated rat CA3 neurons using conventional whole-cell patch clamp techniques. Both glycine and taurine, which are endogenous agonists of glycine receptors, elicited Cl(-) currents in a concentration-dependent manner. The glycine-induced current (I(Gly)) was inhibited by strychnine, picrotoxin or cyclothiazide in a concentration-dependent manner. At lower concentrations (0.01-1 microM), ICS-205,930 potentiated I(Gly), but at higher concentrations (>10 microM) it inhibited I(Gly). These pharmacological properties strongly suggest that CA3 neurons express functional strychnine-sensitive glycine receptors containing alpha2 subunits. Furthermore, at lower concentrations (1-30 microM), Zn(2+) potentiated I(Gly), but at higher concentrations (>100 microM) it inhibited I(Gly). Considering that Zn(2+) is synaptically co-released with glutamate from mossy fiber terminals that make excitatory synapses onto CA3 neurons, these results suggest that endogenous Zn(2+) modulation of these glycine receptors may have an important role in the excitability of CA3 neurons.     

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels,the case of ATP-gated P2X receptors

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.     

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.     

2′,3′-<Emphasis Type="Italic">O</Emphasis>-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABA_A and 5-HT₃ receptors, whose membrane currents were unaffected by the tested compounds.     

Purinergic receptors and gastrointestinal secretomotor function

Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP), or uridine diphosphate (UDP) (P2X(1-7), P2Y(2), P2Y(4), P2Y(6), P2Y(12) receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A(1), A(3), P2Y(12)) or excitatory (A(2), P2Y(1)) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y(1), other P2Y, P2X) or inhibitory (A(1), A(3)) receptors are involved in mechanically evoked secretory reflexes or "neurogenic diarrhea." Distinct neural (pre- or postsynaptic) and non-neural distribution profiles of P2X(2), P2X(3), P2X(5), P2Y(1), P2Y(2), P2Y(4), P2Y(6), or P2Y(12) receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A(2b), P2Y(2), P2Y(4), and P2Y(6) receptors are involved in fluid and Cl(-), HCO(3) (-), K(+), or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A(2a) or A(3) receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.     

Zinc effects on NMDA receptor gating kinetics

Zinc accumulates in the synaptic vesicles of certain glutamatergic forebrain neurons and modulates neuronal excitability and synaptic plasticity by multiple poorly understood mechanisms. Zinc directly inhibits NMDA-sensitive glutamate-gated channels by two separate mechanisms: high-affinity binding to N-terminal domains of GluN2A subunits reduces channel open probability, and low-affinity voltage-dependent binding to pore-lining residues blocks the channel. Insight into the high-affinity allosteric effect has been hampered by the receptor's complex gating; multiple, sometimes coupled, modulatory mechanisms; and practical difficulties in avoiding transient block by residual Mg²⁺. To sidestep these challenges, we examined how nanomolar zinc concentrations changed the gating kinetics of individual block-resistant receptors. We found that block-insensitive channels had lower intrinsic open probabilities but retained high sensitivity to zinc inhibition. Binding of zinc to these receptors resulted in longer closures and shorter openings within bursts of activity but had no effect on interburst intervals. Based on kinetic modeling of these data, we conclude that zinc-bound receptors have higher energy barriers to opening and less stable open states. We tested this model for its ability to predict zinc-dependent changes in macroscopic responses and to infer the impact of nanomolar zinc concentrations on synaptic currents mediated by 2A-type NMDA receptors.     

MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC₅₀s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.     

Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.     

Regulation of neuronal ion channels via P2Y receptors

Within the last 15 years, at least 8 different G protein-coupled P2Y receptors have been characterized. These mediate slow metabotropic effects of nucleotides in neurons as well as non-neural cells, as opposed to the fast ionotropic effects which are mediated by P2X receptors. One class of effector systems regulated by various G protein-coupled receptors are voltage-gated and ligand-gated ion channels. This review summarizes the current knowledge about the modulation of such neuronal ion channels via P2Y receptors. The regulated proteins include voltage-gated Ca²⁺ and K⁺ channels, as well as N-methyl-d-aspartate, vanilloid, and P2X receptors, and the regulating entities include most of the known P2Y receptor subtypes. The functional consequences of the modulation of ion channels by nucleotides acting at pre- or postsynaptic P2Y receptors are changes in the strength of synaptic transmission. Accordingly, ATP and related nucleotides may act not only as fast transmitters (via P2X receptors) in the nervous system, but also as neuromodulators (via P2Y receptors). Hence, nucleotides are as universal transmitters as, for instance, acetylcholine, glutamate, or -aminobutyric acid.     

Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP₃ pathway

Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca2? concentration ([Ca2?](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10?? to 10?? M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10?? M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca2?](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10?? M) or CdCl?. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP? receptor antagonist 2-APB (10?? M) or the PLC inhibitor U73122 (10?? M). OT (10?? M) also increased the IP? concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP?-Ca2? signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.