Burnstock and the legacy of the inhibitory junction potential and P2Y1 receptors

The synaptic event called the inhibitory junction potential (IJP) was arguably one of the more important discoveries made by Burnstock and arguably one of his finer legacies. The discovery of the IJP fundamentally changed how electromechanical coupling was visualised in gastrointestinal smooth muscle. Its discovery also set in motion the search for novel inhibitory neurotransmitters in the enteric nervous system, eventually leading to proposal that ATP or a related nucleotide was a major inhibitory transmitter. The subsequent development of purinergic signalling gave impetus to expanding the classification of surface receptors for extracellular ATP, not only in the GI tract but beyond, and then led to successive phases of medicinal chemistry as the P2 receptor field developed. Ultimately, the discovery of the IJP led to the successful cloning of the first P2Y receptor (chick P2Y1) and expansion of mammalian ATP receptors into two classes: metabotropic P2Y receptors (encompassing P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–14 receptors) and ionotropic P2X receptors (encompassing homomeric P2X1–P2X7 receptors). Here, the causal relationship between the IJP and P2Y1 is explored, setting out the milestones reached and achievements made by Burnstock and his colleagues.

Interstitial cells of Cajal mediate nitrergic inhibitory neurotransmission in the murine gastrointestinal tract

Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.

Role of myosin Va in purinergic vesicular neurotransmission in the gut

We examined the hypothesis that myosin Va, by transporting purinergic vesicles to the varicosity membrane for exocytosis, plays a key role in purinergic vesicular neurotransmission. Studies were performed in wild-type (WT) and myosin Va-deficient dilute, brown, nonagouti (DBA) mice. Intracellular microelectrode recordings were made in mouse antral muscle strips. Purinergic inhibitory junction potential (pIJP) was recorded under nonadrenergic noncholinergic conditions after masking the nitrergic junction potentials. DBA mice showed reduced pIJP but normal hyperpolarizing response to P2Y1 receptor agonist MRS-2365. To investigate the mechanism of reduced purinergic transmission in DBA mice, studies were performed in isolated varicosities obtained from homogenates of whole gut tissues by ultracentrifugation and sucrose cushion purification. Purinergic varicosities were identified in tissue sections and in isolated varicosities by immunostaining for the vesicular ATP transporter, the solute carrier protein SLC17A9. The varicosities were similar in WT and DBA mice. Myosin Va was markedly reduced in DBA varicosities compared with the WT varicosities. Proximity ligation assay showed that myosin Va was closely associated with SLC17A9. Vesicular exoendocytosis was examined by FM1–43 staining of varicosities, which showed that exoendocytosis after KCl stimulation was impaired in DBA varicosities compared with WT varicosities. These studies show that SLC17A9 identifies ATP-containing purinergic varicosities. Myosin Va associates with SLC17A9-stained vesicles and possibly transports them to varicosity membrane for exocytosis. In myosin Va-deficient mice, purinergic inhibitory neurotransmission is impaired.

Effect of exogenous ATP on canine jejunal smooth muscle

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 ± 0.6 mV) and EFS evoked a NANC IJP (17 ± 0.4 mV). ω-Conotoxin GVIA (100 nM) and a low-Ca2+, high-Mg2+ solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 μM) and N G-nitro-l-arginine (100 μM). Oxyhemoglobin (5 μM) partially (38.8 ± 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-( N, O-bis-[5-isoquinolinesulfonyl]- N-methyl-l-tyrosyl)-4-phenylpiperazine, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, α,β-methylene ATP, 2-methylthioadenosine 5′-triphosphate tetrasodium salt, and adenosine 5′- O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.

ATP is a mediator of the fast inhibitory junction potential in human jejunal circular smooth muscle

The neurotransmitter(s) that generates the fast component of the inhibitory junction potential (IJP-F) in human jejunal circular smooth muscle is not known. The aim of this study was to determine the role of ATP and purinergic receptors in the generation of the IJP-F in human jejunal circular smooth muscle strips. The P2-receptor antagonist suramin (100 μM) reduced the IJP-F by 28%. Apamin (1 μM) reduced the IJP-F by 25%. Desensitization of muscle strips with the putative P2x-receptor agonist α,β-methylene ATP (α,β-MeATP, 100 μM) decreased the IJP-F by 44%, and desensitization with the putative P2y-receptor agonist adenosine 5′- O-2-thiodiphosphate (ADPβS) completely abolished the IJP-F. Desensitization with the putative P2y-receptor agonist 2-methylthioATP had no effect on the IJP-F. Exogenous ATP evoked a hyperpolarization with a time course that matched the IJP-F. The ATP-evoked hyperpolarization was reduced by apamin and suramin, reduced by desensitization with α,β-MeATP (69% decrease), and abolished by desensitization with ADPβS. These data suggest that the IJP-F in human jejunal circular smooth muscle is mediated in part by ATP through an ADPβS-sensitive P2receptor.

Modulation of cholinergic neuromuscular transmission by nitric oxide in canine colonic circular smooth muscle

This study examines the effect of nitric oxide (NO) on cholinergic transmission in strips of canine colonic circular muscle in which neural plexus-pacemaker regions had been removed. Electrical field stimulation gave rise to atropine- and TTX-sensitive excitatory junction potentials (EJPs), the amplitude of which were frequency dependent. In 47% of control muscles, the EJP was followed by an inhibitory junction potential (IJP), whereas in the presence of atropine all preparations exhibited only IJPs. The NO synthase inhibitor N ω-nitro-l-arginine (l-NNA), the guanylyl cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ), and the protein kinase G (PKG) antagonist Rp-8-bromo-PET-cGMPS all significantly increased EJP amplitude and reduced or abolished IJPs. The potentiation of EJPs by l-NNA was reversed by the NO donors sodium nitroprusside (SNP) and S-nitroso- N-acetylpenicillamine in a manner blocked by ODQ. [14C]ACh overflow was also measured to evaluate the possible prejunctional effects of NO. Both norepinephrine and TTX significantly decreased [14C]ACh overflow; however, l-NNA, ODQ, and SNP were without effect. These data suggest that both cholinergic and nitrergic motoneurons functionally innervate the interior of the circular muscle layer. The inhibitory actions of NO on cholinergic transmission appear to be post- rather than prejunctional and to involve guanylyl cyclase as well as possibly PKG.

Electrical responses of gastric smooth muscles in streptozotocin-induced diabetic rats

Electrical responses of gastric smooth muscles produced by transmural nerve stimulation, acetylcholine, norepinephrine, or K-free solution were investigated in streptozotocin-induced diabetic rats, using intracellular microelectrode techniques. In muscles from diabetic rats, 1) the resting membrane potential remained unchanged, 2) slow waves disappeared or were markedly reduced in amplitude, 3) the excitatory junction potential was absent, and in most cases only an inhibitory junction potential of reduced amplitude was elicited, 4) the amplitude of the hyperpolarization generated after superfusion with K-free solution was reduced, 5) the sensitivity of the acetylcholine-induced membrane depolarization was increased, and 6) the norepinephrine-induced hyperpolarization was reduced because of functional loss of alpha- and beta-adrenoceptors. Thus diabetes mellitus caused functional impairment of neuromuscular transmission, reduced the maximum activity of the electrogenic pump, increased the sensitivity of muscarinic receptors, reduced the sensitivity of adrenoceptors, and reduced the myogenic activity in gastric smooth muscles. These alterations in the properties of smooth muscle may be involved in the diabetes-induced gastroparesis.