Optogenetic analysis of neuromuscular transmission in the colon of ChAT-ChR2-YFP BAC transgenic mice

Propulsion of luminal content along the gut requires coordinated contractions and relaxations of gastrointestinal smooth muscles controlled by the enteric nervous system. Activation of excitatory motor neurons (EMNs) causes muscle contractions, whereas inhibitory motor neuron (IMN) activation causes muscle relaxation. EMNs release acetylcholine (ACh), which acts at muscarinic receptors on smooth muscle cells and adjacent interstitial cells of Cajal, causing excitatory junction potentials (EJPs). IMNs release ATP (or another purine) and nitric oxide to cause inhibitory junction potentials (IJPs) and muscle relaxation. We used commercially available choline acetyltransferase (ChAT)-channelrhodopsin-2 (ChR2)-yellow fluorescent protein (YFP) bacterial artificial chromosome (BAC) transgenic mice, which express ChR2 in cholinergic neurons, to study cholinergic neuromuscular transmission in the colon. Intracellular microelectrodes were used to record IJPs and EJPs from circular muscle cells. We used blue light stimulation (BLS, 470 nm, 20 mW/mm2) and electrical field stimulation (EFS) to activate myenteric neurons. EFS evoked IJPs only, whereas BLS evoked EJPs and IJPs. Mecamylamine (10 µM, nicotinic cholinergic receptor antagonist) reduced BLS-evoked IJPs by 50% but had no effect on electrically evoked IJPs. MRS 2179 (10 µM, a P2Y1 receptor antagonist) blocked BLS-evoked IJPs. MRS 2179 and Nω-nitro-l-arginine (100 µM, nitric oxide synthase inhibitor) isolated the EJP, which was blocked by scopolamine (1 µM, muscarinic ACh receptor antagonist). Immunohistochemistry revealed ChAT expression in ~88% of enhanced YFP (eYFP)-expressing neurons, whereas 12% of eYFP neurons expressed nitric oxide synthase. These data show that cholinergic interneurons synapse with EMNs and IMNs to cause contraction and relaxation of colonic smooth muscle. NEW & NOTEWORTHY Electrical stimulation of interganglionic connectives has been used widely to study synaptic transmission in the enteric nervous system. However, electrical stimulation will activate many types of neurons and nerve fibers, which complicates data interpretation. Optogenetic activation of enteric neurons using genetically modified mice expressing channelrhodopsin-2 in cholinergic neurons offers a new approach that provides more specificity for nerve stimulation when studying myenteric plexus nerve circuitry.

Characterization of the peristaltic reflex in murine distal colon

Ascending and descending neuromuscular reflexes play an important role in gastrointestinal motility. However, the underlying mechanisms in colon are incompletely understood. Nerve stimulation (NS)- and balloon distention (BD)-mediated reflexes in distal colonic circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of mice were investigated using conventional intracellular recordings. In the CSM, NS evoked ascending purinergic inhibitory junction potentials (IJPs), whereas BD induced atropine-sensitive ascending depolarization with superimposed action potentials (APs). The ascending depolarization reached a peak ∼4–7 s after the onset of distention and gradually returned to baseline after termination of the distention. In the LSM, NS produced an ascending biphasic IJP followed by a train of atropine-sensitive APs. Both stimuli produced similar descending IJPs in CSM and LSM, which were blocked by MRS-2500 and MRS-2179, putative purinergic receptor blockers. These data indicate that in the murine distal colon, descending purinergic inhibition in both CSM and LSM occurs. Ascending responses are more complex, with NS producing both inhibition and excitation to CSM and LSM, and BD evoking only cholinergic excitation.

Multiscale prediction of patient-specific platelet function under flow

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A2, recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye–loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y1/P2Y12, TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y1 inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Functional contribution of P2Y1receptors to the control of coronary blood flow

Activation of ADP-sensitive P2Y1receptors has been proposed as an integral step in the putative “nucleotide axis” regulating coronary blood flow. However, the specific mechanism(s) and overall contribution of P2Y1receptors to the control of coronary blood flow have not been clearly defined. Using vertically integrative studies in isolated coronary arterioles and open-chest anesthetized dogs, we examined the hypothesis that P2Y1receptors induce coronary vasodilation via an endothelium-dependent mechanism and contribute to coronary pressure-flow autoregulation and/or ischemic coronary vasodilation. Immunohistochemistry revealed P2Y1receptor expression in coronary arteriolar endothelial and vascular smooth muscle cells. The ADP analog 2-methylthio-ADP induced arteriolar dilation in vitro and in vivo that was abolished by the selective P2Y1antagonist MRS-2179 and the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. MRS-2179 did not alter baseline coronary flow in vivo but significantly attenuated coronary vasodilation to ATP in vitro and in vivo and the nonhydrolyzable ATP analog ATPγS in vitro. Coronary blood flow responses to alterations in coronary perfusion pressure (40–100 mmHg) or to a brief 15-s coronary artery occlusion were unaffected by MRS-2179. Our data reveal that P2Y1receptors are functionally expressed in the coronary circulation and that activation produces coronary vasodilation via an endothelium/nitric oxide-dependent mechanism. Although these receptors represent a critical component of purinergic coronary vasodilation, our findings indicate that P2Y1receptor activation is not required for coronary pressure-flow autoregulation or reactive hyperemia.

Src Family Kinases Contribute to GI and Gq-Mediated Platelet Activation

Abstract 5263 The Src family kinases (SFKs) play an essential role in collagen- and von Willebrand factor (vWF)-mediated platelet activation. However, the role of SFKs in G protein-coupled receptor (GPCR)-mediated platelet activation is not fully understood, and little is known about the molecular mechanisms by which SFKs are activated by GPCR stimulation. Here we demonstrate that SFKs are activated by the Gq and Gi pathways, respectively and SFKs play important roles in Gq- and Gi-dependent secretion and activation. ADP induced SFK phosphorylation in wild type and Gq−/− platelets, and ADP-induced SFK phosphorylation was inhibited by the P2Y12 antagonist AR-C69931MX or P2Y12 knockout but was not affected by the P2Y1 antagonist MRS-2179. Lyn and Fyn were co-immunoprecipitated with Gi2 in platelets, and ADP-induced SFK phosphorylation was diminished in Lyn−/− platelets. These results demonstrate that ADP induces SFK activation mainly depending on the Gi pathway activated via its receptor P2Y12. Furthermore, epinephrine also dose-dependently induced SFK phosphorylation in mouse platelets. A selective inhibitor of Src family kinase PP2 inhibited ADP-induced Akt phosphorylation, fibrinogen binding, and platelet aggregation. Thus, activation of Gi is sufficient to induce SFK activation, which plays an important role in Gi-dependent platelet activation. We further show that the thrombin receptor PAR4 peptide AYPGKF elicited SFK phosphorylation in P2Y12−/−, but not in Gq−/− platelets, and AYPGKF-induced SFK phosphorylation was inhibited by the calcium chelator dimethyl-BAPTA, suggesting that Gq-dependent SFK phosphorylation is downstream from the Ca2+ signaling. The calcium ionophore, A23187-induced TXA2 synthesis, platelet aggregation and secretion were inhibited by pre-incubation of platelets with PP2. PAR4-induced TXA2 synthesis was also abolished by PP2. Moreover, PAR4-mediated granule secretion, integrin aIIbb3 activation, and aggregation of P2Y12 deficient platelets were partially inhibited by PP2 or a PKC inhibitor Ro-31-8220, but were completely abolished by Ro-31-8220 plus PP2 or dimethyl-BAPTA, suggesting that Ca2+/SFKs and PKC represent two parallel signaling pathways mediating Gq-dependent platelet activation. In summary, SFKs can be activated by Gq/Ca2+-dependent mechanisms and by Gi-dependent mechanisms, and SFKS play important roles in Gq- and Gi-dependent platelet activation. Disclosures: No relevant conflicts of interest to declare.

ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5′-nucleotidase inhibitor α,β-methylene adenosine 5′-diphosphate (AOPCP), 2) the A2 receptor blocker ZM 241385, 3) the ADP P2Y1 receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A2 receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y1 activation.

Astrocytic P2Y1 receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia

After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y1 receptor (P2Y 1R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y 1R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y 1R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y 1R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y 1R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y 1R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.

Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation

SummaryToll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam3CSK4 stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam3CSK4-induced platelet aggregation and secretion. Platelet interaction with Pam3CSK4 and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+]i mobilisation and thromboxane B2 (TxB2) production. The results show that Pam3CSK4 but not MALP-2 induces [Ca2+]i increase, TxB2 production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam3CSK4-induced responses. The ATP-secretion and aggregation in Pam3CSK4-stimulated platelets was impeded by the purinergic P2X1 inhibitor MRS 2159, the purinergic P2Y1 and P2Y12 antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB2 production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam3CSK4 did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam3CSK4-induced platelet aggregation and secretion depends on a P2X1-mediated Ca2+ mobilisation, production of TxA2 and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.

Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300–900 ms) or slow (durations 1.3–9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2–4-disulfonic acid (30 μM, N = 3) or the specific P2Y1 antagonist MRS 2179 (10 μM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 ( N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y1, S neurons).