Purinoceptor Antagonist Research Articles

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-β,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

STAT1- and NFAT-independent amplification of purinoceptor function integrates cellular senescence with interleukin-6 production in preadipocytes.

Senescent preadipocytes promote adipose tissue dysfunction by secreting pro-inflammatory factors, although little is known about the mechanisms regulating their production. We investigated if up-regulated purinoceptor function sensitizes senescent preadipocytes to cognate agonists and how such sensitization regulates inflammation. Etoposide was used to trigger senescence in 3T3-L1 preadipocytes. CRISPR/Cas9 technology or pharmacology allowed studies of transcription factor function. Fura-2 imaging was used for calcium measurements. Interleukin-6 levels were quantified using quantitative PCR and ELISA. Specific agonists and antagonists supported studies of purinoceptor coupling to interleukin-6 production. Experiments in MS1 VEGF angiosarcoma cells and adipose tissue samples from obese mice complemented preadipocyte experiments. DNA damage-induced senescence up-regulated purinoceptor expression levels in preadipocytes and MS1 VEGF angiosarcoma cells. ATP-evoked Ca2+ release was potentiated in senescent preadipocytes. ATP enhanced interleukin-6 production, an effect mimicked by ADP but not UTP, in a calcium-independent manner. Senescence-associated up-regulation and activation of the adenosine A3 receptor also enhanced interleukin-6 production. However, nucleotide hydrolysis was not essential because exposure to ATPγS also enhanced interleukin-6 secretion. Pharmacological experiments suggested coupling of P2X ion channels and P2Y12 -P2Y13 receptors to downstream interleukin-6 production. Interleukin-6 signalling exacerbated inflammation during senescence and compromised adipogenesis. We report a previously uncharacterized link between cellular senescence and purinergic signalling in preadipocytes and endothelial cancer cells, raising the possibility that up-regulated purinoceptors play key modulatory roles in senescence-associated conditions like obesity and cancer. There is potential for exploitation of specific purinoceptor antagonists as therapeutics in inflammatory disorders.

Roles for α1-adrenoceptors during contractions by electrical field stimulation in mouse vas deferens.

We have investigated the relative roles of α1-adrenoceptors and purinoceptors in contractions to low and high frequency stimulation of the mouse vas deferens, in terms of the time course of responses. In separate experiments, isometric contractile responses were obtained to 10 pulses at 1 Hz and 40 pulses at 10 Hz. Responses to 1 Hz stimulation consisted of a series of discrete peaks. The α1A-adrenoceptor antagonist RS100329 (10–9M–10–7M) significantly reduced the response to the first pulse, the α1D-adrenoceptor antagonist BMY7378 (10–7M–10–6M) significantly reduced the response to the first two pulses, and the non-selective α1-adrenoceptor antagonist prazosin (10–8M) reduced the response to the first 4 pulses at 1 Hz. Responses to 10 Hz stimulation consisted of an early peak response and a maintained plateau response. RS100329 significantly reduced the peak response but did not significantly affect the plateau response. Prazosin, significantly reduced both the peak and plateau responses. The α1A-adrenoceptor antagonist RS17053 in high concentrations reduced mainly the plateau response leaving a clear early peak response. The plateau response of contraction was almost abolished by the purinoceptor antagonist suramin. These results suggest that there is a relatively minor early α1D-adrenoceptor and a larger early α1A-adrenoceptor component to stimulation-evoked contractions of mouse vas deferens, but the major α1-adrenoceptor component is revealed by prazosin to be α1B-adrenoceptor mediated. α1B-Adrenoceptor activation probably facilitates contractions mediated by other α1-adrenoceptors and by purinoceptors. These results suggest that combined non-selective α1-adrenoceptor blockade, particularly α1B-adrenoceptor blockade, in addition to P2X1-purinoceptor blockade is useful in reducing male fertility.

"Tripartite Synapses" in Taste Buds: A Role for Type I Glial-like Taste Cells.

In mammalian taste buds, Type I cells comprise half of all cells. These are termed "glial-like" based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. Using Gad2;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ≈28% Type III cells expressing weakly). In dissociated taste buds, GCaMP+ Type I cells responded to bath-applied ATP (10-100 μm) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 μm cycloheximide, 1 mm denatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 μm). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses.SIGNIFICANCE STATEMENT Most studies of taste buds view the chemosensitive excitable cells that express taste receptors as the sole mediators of taste detection and transmission to the CNS. Type I "glial-like" cells, with their ensheathing morphology, are mostly viewed as responsible for clearing neurotransmitters and as the "glue" holding the taste bud together. In the present study, we demonstrate that, when intact taste buds respond to their natural stimuli, Type I cells sense the activation of the chemosensory cells by detecting the afferent transmitter. Because Type I cells synthesize GABA, a known gliotransmitter, and cognate receptors are present on both presynaptic and postsynaptic elements, Type I cells may participate in GABAergic synaptic transmission in the manner of astrocytes at tripartite synapses.

Pyridoxine stimulates filaggrin production in human epidermal keratinocytes.

Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

In vivo paracrine effects of ATP-induced urothelial acetylcholine in the rat urinary bladder.

Mechanical stretch of the urothelium induces the release of ATP that activates bladder afferent nerves. In the rat urinary bladder, ATP is also a contractile co-transmitter in the parasympathetic innervation. In isolated preparations, ATP evokes a urothelial release of acetylcholine that substantially contributes to ATP-evoked contractile responses. Currently we aimed to further examine the interactions of ATP and acetylcholine in the rat urinary bladder in two in vivo models. In the whole bladder preparation, atropine reduced ATP-evoked responses by about 50% in intact but denervated bladders, while atropine had no effect after denudation of the urothelium. In a split bladder preparation, reflex-evoked responses of the contralateral half were studied by applying stimuli (agonists or stretch) to the ipsilateral half. Topical administration of ATP and methacholine as well as of stretch induced contralateral reflex-evoked contractions. While topical administration of atropine ipsilaterally reduced the ATP- and stretch-induced contralateral contractions by 27 and 39%, respectively, the P2X purinoceptor antagonist PPADS reduced them by 74 and 84%. In contrary, the muscarinic M2-(M4)-selective receptor antagonist methoctramine increased the responses by 38% (ATP) and 75% (stretch). Pirenzepine (M1-selective antagonist) had no effect on the reflex. In vitro, in the absence of the reflex, methoctramine did not affect the ATP-induced responses. It is concluded that urothelial ATP potently induces the micturition reflex and stimulates urothelial release of acetylcholine. Acetylcholine subsequently acts on afferents and on the detrusor muscle. While muscarinic M2 and/or M4 receptors in the sensory innervation exert inhibitory modulation, muscarinic M3 receptors cause excitation.

α1D–Adrenoceptor Mediated Components to Nerve‐Evoked Contractions in Mouse Vas Deferens

Contractions of the mouse vas deferens are mediated by α1‐adrenoceptors and P2X1‐purinoceptors (Brown et al., 1983). α1D‐Adrenoceptors are involved in the nerve‐evoked contraction to a single stimulus (Bexis et al., 2008). In this study, the role of these receptors in contractions to low and high frequency nerve stimulation have been investigated. Whole vas deferens from C57 mice were set up between electrodes in organ baths under 0.5 g tension in Krebs‐Henseleit solution, and stimulated electrically with 10 pulses at 1Hz or 40 pulses at 10 Hz using a Grass stimulator. The response to 1Hz stimulation consisted of discrete peaks of response corresponding to each stimulus. Threshold concentrations of the α1A‐adrenoceptor antagonist RS100329 (1nM) and the α1D‐adrenoceptor antagonist BMY7378 (100nM) significantly reduced contractions to the first pulse, and the non‐selective antagonist prazosin (10 nM) significantly reduced contractions to the first 4 pulses, at 1 Hz, but the combination of prazosin and the purinoceptor antagonist suramin produced marked inhibition of the response to all 10 pulses. The response to 10 Hz stimulation consisted of an early peak and later plateau of contraction. BMY7378 (100 nM) did not significantly affect the 10 Hz response but prazosin (10 nM) and RS100329 (1 nM) significantly reduced contractions, particularly the peak response, and higher concentrations markedly reduced responses. In the presence of the L‐type calcium channel blocker nifedipine, contractions to 10Hz stimulation were markedly reduced, particularly the later plateau component. In the presence of nifedipine, the peak response to 10 Hz stimulation was significantly reduced by prazosin (1nM) and by BMY7378 (100nM), demonstrating a small α1D‐adrenoceptor mediated component. In summary, contractions to 1 Hz stimulation have early α1A‐ and α1D‐adrenoceptor mediated components, but later responses are predominantly purinergic. Contractions even to 10 Hz stimulation have an early small α1D‐adrenoceptor component to the peak response, but both peak and plateau contractions largely involve α1A‐adrenoceptors and purinoceptors. The α1A‐adrenoceptor and purinoceptor components largely involve calcium entry through L‐type channels, but the α1D‐adrenoceptor mediated component involves T‐type calcium channels (Seto et al., 2010). In conclusion, the α1D‐adrenoceptor mediated component to both low and high frequency stimulation is very short‐lived, occuring in the first second of stimulation at low and high frequencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in the Neuronal Control of the Urinary Bladder in a Model of Radiation Cystitis.

Currently, we have assessed the neuronal control of the urinary bladder in radiation cystitis and whether interstitial cells contribute to the condition. Fourteen days after bladder irradiation (20 Gy), rats were sedated and the urinary bladder was cut into two sagittal halves where electrical field stimulation (EFS; 5-20 Hz) was applied on the pelvic nerve afferents or stretch (80 mN) on one-half of the bladder, while contractions were registered on the contralateral half of the bladder in the absence and presence of increasing doses of imatinib (1-10 mg/kg; inhibitor of c-kit-positive interstitial cells), atropine (1 mg/kg; to block muscarinic M3 receptors), or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (2 mg/kg; P2X1 purinoceptor antagonist). Urinary bladders were also excised for organ bath experiments, Western blot, quantitative polymerase chain reaction, and immunohistochemistry. In vivo, EFS contractions were enhanced after irradiation, and imatinib (1-10 mg/mg) inhibited contractions by EFS and stretched dose-dependently in controls but not in irradiated bladders. In the irradiated bladder in vitro, atropine resistance was increased and imatinib (100 µM) inhibited contractions by EFS and agonists (ATP, methacholine) in irradiated bladders and controls. The urinary bladder expressions of P2X1 purinoceptors, muscarinic M3 receptor, choline acetyltransferase, c-kit, and the agonist of c-kit, stem cell factor, were not changed by irradiation. In conclusion, bladder irradiation affects several levels of neuronal control of the urinary bladder. Interstitial cells may contribute to some of the symptoms associated with radiation cystitis.

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

Inhibitory action of oxytocin on spontaneous contraction of rat distal colon by nitrergic mechanism: involvement of cyclic GMP and apamin-sensitive K+ channels.

The mechanisms underlying the inhibitory effects of oxytocin (OT) on colon tone are not totally understood. We explore the mechanisms of OT on spontaneous contractility in rat distal colon and identify the mediators involved in this action. In rat distal colon strips, mechanical activity was analysed and the production of nitric oxide (NO) in tissue loaded with the fluorochrome DAF-FM was visualized by confocal microscopy. OT receptor (OTR) expression was determined by Western blotting and immunofluorescence. In rat distal colon, OT produced a concentration-dependent reduction in the spontaneous contraction, which was abolished by the OTR antagonist atosiban, the neural blocker tetrodotoxin and the inhibitor of neuronal nitric oxide synthase (nNOS) NPLA. The inhibitory effects of OT were not affected by propranolol, atropine, the nicotinic cholinoceptor blocker hexamethonium, the vasoactive intestinal peptide receptor antagonist VIPHyb, the P2 purinoceptor antagonist PPADS, the adenosine A1 receptors antagonist DPCPX and the prostacyclin receptor antagonist Ro1138452. The soluble guanylyl cyclase (sGC) inhibitor ODQ and the small conductance Ca2+ -activated K+ (Ca K+ ) channels blocker apamin significantly reduced the relaxation induced by OT, nicotine, sodium nitroprusside and the sGC activator BAY 41-2272. The neural release of NO elicited by OT was prevented by NPLA, tetrodotoxin and atosiban. The presence of the OTR and its co-localization with nNOS was detected by immunohistochemistry and Western blotting experiments. These results demonstrate the NO release from enteric neurones induced by activation of OTR mediates distal colon relaxation. sGC and small conductance Ca K+ channels are involved in this relaxation.

Shear Stress Induces Transverse Ca2+ Waves via Autocrine Activation of P2X Purinoceptors in Rat Atrial Myocytes

Atrial myocytes are exposed to high shear stress during blood regurgitation and high intra-atrial pressure due to valve diseases and heart failure, since such disturbances disrupt endocardium. We have previously reported that shear stress induces two types of global Ca2+ waves in atrial myocytes, longitudinal and transverse Ca2+ waves (T-waves) (Biophys J 2012;102(3, Suppl 1);227a), and that the longitudinal wave is triggered by Ca2+ release via P2Y1 purinoceptor-inositol 1,4,5-trisphosphate receptor signaling (J Physiol 2015;593:5091-5109). Here, we investigated cellular mechanism for the generation of T-wave in atrial cells under shear stress. Shear stress of ∼16 dyn/cm2 was applied onto single myocytes using micro fluid-jet, and two-dimensional confocal Ca2+ imaging was performed. Shear stress-induced T-waves were observed repetitively under 3-4 min intervals between the stimuli, and occurred at ∼1 event per 10 s. They were eliminated by inhibition of the voltage-gated Na+ or Ca2+ channels, or ryanodine receptors, suggesting that the T-wave is mediated by action potential-triggered Ca2+ release. Blockades of key stretch signaling molecules, stretch-activated channel, Na+-Ca2+ exchange, and NADPH oxidase, did not suppress T-wave generation by shear. However, shear-induced T-wave generation was abolished by pre-incubation of cells with external ATP-metabolizing enzyme apyrase, the gap junction blocker carbenoxolone, or with P2X purinoceptor antagonist iso-PPADS. Inhibition of P2Y1 purinergic signaling that mediates the longitudinal Ca2+ wave under shear did not attenuate the occurrence of T-waves. Our data suggest that shear stress induces activation of P2X purinoceptors via gap junction-mediated ATP release, thereby triggering action potential with subsequent T-wave in atrial myocytes.

Effects of Allyl Isothiocyanate, Acetaminophen, and Dipyrone in the Guinea-Pig Ileum

Allyl isothiocyanate (AITC, mustard oil, 50-200 µmol/l), depending on specific dosages, inhibited the cholinergic twitch response in the longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum. AITC also induced short-lasting contractile responses, and decreases of the basal tone of the LMMP strip at low concentrations and increases at high concentrations. Hexamethonium, a blocker of nicotinic ganglionic transmission, was able to prevent the AITC-evoked inhibitory effect, an effect that was also observed with the opioid antagonist naloxone. The P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid and guanethidine had no significant influence on the inhibitory effect of AITC. Since AITC also reduced the electrical stimulation-induced myogenic smooth muscle contractions in the LMMP preparation, its contractile and relaxant actions can be regarded as neurogenic and myogenic in nature. The analgesics, acetaminophen (paracetamol, 100-500 µmol/l) and dipyrone (metamizole, 100-500 µmol/l), reduced both the cholinergic twitch and the myogenic contractions in the LMMP strip to the same extent; therefore, their action in the intestinal smooth muscle can be regarded as myogenic spasmolytic in nature.

Decompensated liver cirrhosis and neural regulation of mesenteric vascular tone in rats: role of sympathetic, nitrergic and sensory innervations.

We evaluated the possible alterations produced by liver cholestasis (LC), a model of decompensated liver cirrhosis in sympathetic, sensory and nitrergic nerve function in rat superior mesenteric arteries (SMA). The vasoconstrictor response to electrical field stimulation (EFS) was greater in LC animals. Alpha-adrenoceptor antagonist phentolamine and P2 purinoceptor antagonist suramin decreased this response in LC animals more than in control animals. Both non-specific nitric oxide synthase (NOS) L-NAME and calcitonin gene related peptide (CGRP) (8-37) increased the vasoconstrictor response to EFS more strongly in LC than in control segments. Vasomotor responses to noradrenaline (NA) or CGRP were greater in LC segments, while NO analogue DEA-NO induced a similar vasodilation in both experimental groups. The release of NA was not modified, while those of ATP, nitrite and CGRP were increased in segments from LC. Alpha 1 adrenoceptor, Rho kinase (ROCK) 1 and 2 and total myosin phosphatase (MYPT) expressions were not modified, while alpha 2B adrenoceptor, nNOS expression and nNOS and MYPT phosphorylation were increased by LC. Together, these alterations might counteract the increased splanchnic vasodilation observed in the last phases of decompensated liver cirrhosis.

The TRPA1 Activator Allyl Isothiocyanate (AITC) Contracts Human Jejunal Muscle: Pharmacological Analysis.

The contractile effect of AITC (300 μM) on human jejunal longitudinal strips was inhibited by the TRPA1 antagonist HC 030031 and atropine or scopolamine, but was insensitive to tetrodotoxin, purinoceptor antagonists or capsaicin desensitization. It is concluded that TRPA1 activation stimulates a cholinergic mechanism in a tetrodotoxin-resistant manner.

P2Y purinoceptors mediate ATP-induced changes in intracellular calcium and amylase release in acinar cells of mouse parotid glands.

Adenosine 5'-triphosphate (ATP) can act as an extracellular signal that regulates various cellular functions. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in intracellular Ca(2+) ([Ca(2+)]i) and amylase secretion in mouse parotid glands. ATP induced a steep increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) or the use of Ca(2+) channel blockers slightly inhibited this increase. Inhibition of PLCγ by U73122 and of IP3 by xestospongin C did not completely block this increase. The purinoceptor antagonists suramin and reactive blue-2 strongly inhibited the ATP-induced changes in [Ca(2+)]i. 2-MeSATP induced a strong increase in [Ca(2+)]i, while Bz-ATP induced a small [Ca(2+)]i increase, and UTP and α,β-MeATP had no effect. The potency order of ATP analogs (2-MeSATP > ATP >> UTP) suggested that P2Y1 and P2Y12 play a significant role in the cellular response to ATP. RT-PCR revealed that P2X2,4,7 and P2Y1,2,10,12,14 were expressed in acinar cells. Ca(2+)-dependent exocytotic secretion of amylase was detected in parotid glands. These findings indicated that ATP activates P2Y receptors more than P2X receptors at low concentrations. Thus, P2Y receptors were found to be the main receptors involved in Ca(2+)-related cell homeostasis and amylase secretion in mouse parotid glands.

Three Gaseous Neurotransmitters, Nitric oxide, Carbon Monoxide, and Hydrogen Sulfide, Are Involved in the Neurogenic Relaxation Responses of the Porcine Internal Anal Sphincter.

Background/AimsThe internal anal sphincter (IAS) plays an important role in maintaining continence and a number of neurotransmitters are known to regulate IAS tone. The aim of this study was to determine the relative importance of the neurotransmitters involved in the relaxant and contractile responses of the porcine IAS.MethodsResponses of isolated strips of IAS to electrical field stimulation (EFS) were obtained in the absence and presence of inhibitors of neurotransmitter systems.ResultsContractile responses of the sphincter to EFS were unaffected by the muscarinic receptor antagonist, atropine (1 μM), but were almost completely abolished by the adrenergic neuron blocker guanethidine (10 μM). Contractile responses were also reduced (by 45% at 5 Hz, P < 0.01) following desensitisation of purinergic receptors with α,β-methylene-ATP (10 μM). In the presence of guanethidine, atropine, and α,β-methylene-ATP, the remaining relaxatory responses to EFS were examined. These responses were not altered by the cyclooxygenase inhibitor, indomethacin (5 μM), the vasoactive intestinal polypeptide receptor antagonist, [d-p-Cl-Phe6,Leu17]-vasoactive intestinal peptide (PheLeu-VIP; 100 nM), or the purinoceptor antagonists, 8-phenyltheophyline (P1 receptors) or suramin (P2 receptors). However, relaxation responses were reduced by Nω-nitro-L-arginine (L-NNA; 100 μM), an inhibitor of nitric oxide synthesis (40–50% reduction), zinc protoprophyrin IX (10 μM), an inhibitor of carbon monoxide synthesis (20–40% reduction), and also propargylglycine (30 μM) and aminooxyacetic acid (30 μM), inhibitors of hydrogen sulphide synthesis (15–20% reduction).ConclusionsStimulation of IAS efferent nerves releases excitatory and inhibitory neurotransmitters: noradrenaline is the predominant contractile transmitter with a smaller component from ATP, whilst 3 gases mediate relaxation responses to EFS, with the combined contributions being nitric oxide > carbon monoxide > hydrogen sulfide.

ATP is not involved in α1-adrenoceptor-mediated vasoconstriction in resistance arteries

Recent publications suggest that α1-adrenoceptor stimulation by exogenous agonists such as phenylephrine in resistance arteries cause contraction through the release of ATP from within the vascular smooth muscle cells. This ATP exits the cell through pannexin-1 channels to act back “autocrine-like” on P2 receptors on the smooth muscle that cause the contraction. In this work we directly test this hypothesis by using a selective P2X1 purinoceptor antagonist NF449 (1–10µM) against phenylephrine and ATP concentration–response curves in small mesenteric arteries of the rat and thoracodorsal arteries of the mouse. We show that NF449 is a simple competitive antagonist of ATP with a pKB of 6.43 and 6.41 in rat and mouse arteries, respectively, but did not antagonise phenylephrine concentration–response curves. This work cautions against the growing overstated role of the reputed pannexin-1/ATP release axis following α1-adrenoceptor activation in small resistance arteries.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

Functional properties of submucosal venules in the rat stomach.

Venules in the stomach may have intrinsic properties for maintaining active microcirculation drainage even during gastric filling. Properties of spontaneous and nerve-mediated activity of submucosal venules in the rat stomach were investigated. Changes in vasodiameter and intracellular Ca(2+) in venular smooth muscle cells (SMCs) were monitored by video tracking and Fluo-8 Ca(2+) imaging, respectively. Venular SMCs developed synchronous spontaneous Ca(2+) transients and corresponding rhythmic constrictions of the venules. Nominally Ca(2+)-free solution or an L-type Ca(2+) channel blocker (1 μM nifedipine) disrupted the Ca(2+) transient synchrony and abolished spontaneous constrictions. Spontaneous constrictions were also prevented by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase (10 μM cyclopiazonic acid (CPA)), IP3 receptors (100 μM 2-APB) or Ca(2+)-activated Cl(-) channels (100 μM niflumic acid). Transmural nerve stimulation (TNS) induced a long-lasting venular constriction that was abolished by α-adrenoceptor antagonist (1 μM phentolamine), while TNS evoked a sympathetic transient constriction of arterioles that was abolished by a combination of phentolamine and a P2 purinoceptor antagonist (10 μM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS)). Consistently, P2X1 purinoceptor immunoreactivity was detected in arteriolar but not venular SMCs. Primary afferent nerve stimulation (300 nM capsaicin) caused a venular dilatation by releasing calcitonin gene-related peptide. Thus, Ca(2+) release from the sarcoplasmic reticulum may play a fundamental role in the generation of spontaneous Ca(2+) transients, while electrical coupling amongst venular SMCs via L-type Ca(2+) channel activation appears to be critical for Ca(2+) transient synchrony as well as spontaneous contractions. Sympathetic venular constrictions appear to be exclusively mediated by noradrenaline due to the lack of P2X1 receptor in venular SMCs.

Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels

Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions. Hemichannels were activated by removing extracellular Ca(2+) . The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release. Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca(2+) without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury. Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations.