Impact of P2X7 Purinoceptors on Goblet Cell Function: Implications for Dry Eye.

By providing ~70% of the eye's refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells. Here, we review progress in elucidating the roles of ion channels in mediating this important exocytotic response. Much is now known about the modulatory impact of ATP-sensitive potassium channels, nonspecific cation channels and voltage-gated calcium channels. Recently, we discovered that during unremitting extracellular hyperosmolarity, P2X7 receptor/channels also become activated and markedly impair goblet cell viability. However, our understanding of possible adaptive benefits of this P2X7 activation remains limited. In the present study, we utilized high-temporal resolution membrane capacitance measurements to monitor the exocytotic activity of single goblet cells located in freshly excised rat conjunctiva. We now report that activation of P2X7 purinoceptors boosts neural-evoked exocytosis and accelerates replenishment of mucin-filled granules after exocytotic depletion. Thus, P2X7 activation exerts a yin-yang effect on conjunctival goblet cells: the high-gain benefit of enhancing the supply of tear-stabilizing mucin is implemented at the high-risk of endangering goblet cell survival.

P2X receptor agonist enhances tumor-specific CTL responses through CD70+ DC-mediated Th17 induction.

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αß-methylene ATP (αß-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αß-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αß-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αß-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αß-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αß-ATP against E.G7-OVA. Collectively, αß-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction.

Distributive differences of P2Xs between the forelimb and hind limb of adjuvant arthritis rats and intervention by Notopterygh rhizoma et radix.

CONTEXT: Notopterygium incisum Ting ex H. T. Chang (Umbelliferae) (NI) specializes in treatment of upper limb rheumatoid arthritis (RA), but the exact mechanism is unclear. P2Xs are useful targets for inflammatory pain therapy. It led us to hypothesize that NI may preferentially act on particular P2Xs and these receptors may be unevenly distributed in the upper/lower limb. OBJECTIVE: To investigate P2Xs distribution in the upper/lower limb and NI's targets in upper limb RA. MATERIALS AND METHODS: The SD rats were randomized into 11 groups of 10 animals each. Eight experimental groups were established by the injection of 0.1 mL FCA into the plantar surface of rat paw. Three control groups suffered the same volume of saline. The articular cavities were then taken on the seventh day to detect P2Xs expression. NI (3 g/kg) and prednisone (10 mg/kg) were respectively given by oral gavage once daily for 14 d. The swelling degree and P2Xs were evaluated individually. RESULTS: In normal rats, the expressions of P2X3 and P2X6 in forelimb were markedly higher than that of in hind limb (P < 0.05). After induced by FCA, P2X1, P2X3, P2X4, P2X5 and P2X7 were increased significantly (P < 0.01). The biggest difference was P2X3. In NI treatment rats, swelling degree of the 7th/14th day in forelimb was 68.24%/38.89%, whereas that of in hind limb was 88.72%/79.92%. P2X3 mRNA and protein expression was significantly reduced as contrasted with the control group (P < 0.05). CONCLUSIONS: P2X3 receptor was predominantly expressed in the forelimb RA rat. NI relieved the FCA-induced RA by inhibiting upper limb's P2X3 receptor.

Natural compounds acting at P2 receptors alleviate peripheral neuropathy.

Neuropathic pain is generally resistant to currently available treatments, and it is often a consequence of nerve injury due to surgery, diabetes or infection. Myocardial ischemic nociceptive signaling increases the sympathoexcitatory reflex to aggravate myocardial injury. Elucidation of the pathogenetic factors might provide a target for optimal treatment. Abundant evidence in the literature suggests that P2X and P2Y receptors play important roles in signal transmission. Traditional Chinese medicines, such as emodin, puerarin and resveratrol, antagonize nociceptive transmission mediated by purinergic 2 (P2) receptors in primary afferent neurons. This review summarizes recently published data on P2 receptor-mediated neuropathic pain and myocardial ischemia in dorsal root ganglia (DRG), superior cervical ganglia (SCG) and stellate ganglia (SG), with a special focus on the beneficial role of natural compounds.

P2X receptors and acupuncture analgesia.

Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how the field is likely to move forward in the future.

The therapeutic potential of purinergic signalling.

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y12 receptor antagonists for stroke and thrombosis, P2Y2 receptor agonists for dry eye, and A1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles.

Extracellular ATP and adenosine: The Yin and Yang in immune responses?

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses.

Glycosides from edible sea cucumbers stimulate macrophages via purinergic receptors.

Since ancient times, edible sea cucumbers have been considered a jewel of the seabed and used in Asian folk medicine for stimulation of resistance against different diseases. However, the power of this sea food has not been established on a molecular level. A particular group of triterpene glycosides was found to be characteristic metabolites of the animals, responsible for this biological action. Using one of them, cucumarioside A2-2 (CA2-2) from the edible Cucumaria japonica species as an example as well as inhibitory analysis, patch-clamp on single macrophages, small interfering RNA technique, immunoblotting, SPR analysis, computer modeling and other methods, we demonstrate low doses of CA2-2 specifically to interact with P2X receptors (predominantly P2X4) on membranes of mature macrophages, enhancing the reversible ATP-dependent Ca2+ intake and recovering Ca2+ transport at inactivation of these receptors. As result, interaction of glycosides of this type with P2X receptors leads to activation of cellular immunity.

Acupuncture-Induced Analgesia: A Neurobiological Basis in Purinergic Signaling.

Chronic pain is a debilitating and rather common health problem. The present shortage in analgesic drugs with a favorable spectrum but without remarkable side effects furthered the search for alternative therapeutic manipulations. Increasing evidence from both basic and clinical research on acupuncture, a main alternative therapy of traditional Chinese medicine, suggests that chronic pain is sensitive to acupuncture procedures. Clarification of the underlying mechanisms is a challenge of great theoretical and practical significance. The seminal hypothesis of Geoffrey Burnstock and the astounding findings of Maiken Nedergaard on the involvement of purinergic signaling in the beneficial effects of acupuncture fertilized the field and led to an intensification of research on acupurines. In this review, we will summarize the state-of-the-art situation and try to forecast how the field is likely to develop in the future.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Chronic arsenic exposure in nanomolar concentrations compromises wound response and intercellular signaling in airway epithelial cells.

Paracrine ATP signaling in the lung epithelium participates in a variety of innate immune functions, including mucociliary clearance, bactericide production, and as an initiating signal in wound repair. We evaluated the effects of chronic low-dose arsenic relevant to U.S. drinking water standards (i.e., 10 ppb [130nM]) on airway epithelial cells. Immortalized human bronchial epithelial cells (16HBE14o-) were exposed to 0, 130, or 330nM arsenic (as Na-arsenite) for 4-5 weeks and examined for wound repair efficiency and ATP-mediated Ca(2+) signaling. We found that chronic arsenic exposure at these low doses slows wound repair and reduces ATP-mediated Ca(2+) signaling. We further show that arsenic compromises ATP-mediated Ca(2+) signaling by altering both Ca(2+) release from intracellular stores (via metabotropic P2Y receptors) and Ca(2+) influx mechanisms (via ionotropic P2X receptors). To better model the effects of arsenic on ATP-mediated Ca(2+) signaling under conditions of natural exposure, we cultured tracheal epithelial cells obtained from mice exposed to control or 50 ppb Na-arsenite supplemented drinking water for 4 weeks. Tracheal epithelial cells from arsenic-exposed mice displayed reduced ATP-mediated Ca(2+) signaling dynamics similar to our in vitro chronic exposure. Our findings demonstrate that chronic arsenic exposure at levels that are commonly found in drinking water (i.e., 10-50 ppb) alters cellular mechanisms critical to airway innate immunity.

Perivagal antagonist treatment in rats selectively blocks the reflex and afferent responses of vagal lung C fibers to intravenous agonists.

The terminals of vagal lung C fibers (VLCFs) express various types of pharmacological receptors that are important to the elicitation of airway reflexes and the development of airway hypersensitivity. We investigated the blockade of the reflex and afferent responses of VLCFs to intravenous injections of agonists using perivagal treatment with antagonists (PAT) targeting the transient receptor potential vanilloid 1, P2X, and 5-HT(3) receptors in anesthetized rats. Blockading these responses via perivagal capsaicin treatment (PCT), which blocks the neural conduction of C fibers, was also studied. We used capsaicin, α,ß-methylene-ATP, and phenylbiguanide as the agonists, and capsazepine, iso-pyridoxalphosphate-6-azophenyl-2',5'-disulfonate, and tropisetron as the antagonists of transient receptor potential vanilloid 1, P2X, and 5-HT(3) receptors, respectively. We found that each of the PATs abolished the VLCF-mediated reflex apnea evoked by the corresponding agonist, while having no effect on the response to other agonists. Perivagal vehicle treatment failed to produce any such blockade. These blockades had partially recovered at 3 h after removal of the PATs. In contrast, PCT abolished the reflex apneic response to all three agonists. Both PATs and PCT did not affect the myelinated afferent-mediated apneic response to lung inflation. Consistently, our electrophysiological studies revealed that each of the PATs prevented the VLCF responses to the corresponding agonist, but not to any other agonist. PCT inevitably prevented the VLCF responses to all three agonists. Thus these PATs selectively blocked the stimulatory action of corresponding agonists on the VLCF terminals via mechanisms that are distinct from those of PCT. PAT may become a novel intervention for studying the pharmacological modulation of VLCFs.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: a microdialysis study.

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.

Purinergic modulation of neuronal activity in developing auditory brainstem.

In the developing nervous system, spontaneous neuronal activity arises independently of experience or any environmental input. This activity may play a major role in axonal pathfinding, refinement of topographic maps, dendritic morphogenesis, and the segregation of axonal terminal arbors. In the auditory system, endogenously released ATP in the cochlea activates inner hair cells to trigger bursts of action potentials (APs), which are transferred to the central auditory system. Here we show the modulatory role of purinergic signaling beyond the cochlea, i.e., the developmentally regulated and cell-type-specific depolarizing effects on auditory brainstem neurons of Mongolian gerbil. We assessed the effects of P2X receptors (P2XRs) on neuronal excitability from prehearing to early stages of auditory signal processing. Our results demonstrate that in neurons expressing P2XRs, extracellular ATP can evoke APs in sync with Ca(2+) signals. In cochlear nucleus (CN) bushy cells, ATP increases spontaneous and also acoustically evoked activity in vivo, but these effects diminish with maturity. Moreover, ATP not only augmented glutamate-driven firing, but it also evoked APs in the absence of glutamatergic transmission. In vivo recordings also revealed that endogenously released ATP in the CN contributes to neuronal firing activity by facilitating AP generation and prolonging AP duration. Given the enhancing effect of ATP on AP firing and confinement of P2XRs to certain auditory brainstem nuclei, and to distinct neurons within these nuclei, it is conceivable that purinergic signaling plays a specific role in the development of neuronal brainstem circuits.

Peripheral inflammation upregulates P2X receptor expression in satellite glial cells of mouse trigeminal ganglia: a calcium imaging study.

Satellite glial cells (SGCs) in sensory ganglia are altered structurally and biochemically as a result of nerve injury. Whereas there is ample evidence that P2 purinergic receptors in central glial cells are altered after injury, there is very little information on similar changes in SGCs, although it is well established that SGCs are endowed with P2 receptors. Using calcium imaging, we characterized changes in P2 receptors in SGCs from mouse trigeminal ganglia in short-term cultures. Seven days after the induction of submandibular inflammation with complete Freund's adjuvant, there was a marked increase in the sensitivity of SGCs to ATP, with the threshold of activation decreasing from 5 µM to 10 nM. A similar observation was made in the intact trigeminal ganglion after infra-orbital nerve axotomy. Using pharmacological tools, we investigated the receptor mechanisms underlying these changes in cultured SGCs. We found that in control tissues response to ATP was mediated by P2Y (metabotropic) receptors, whereas after inflammation the response was mediated predominantly by P2X (ionotropic) receptors. As the contribution of P2X1,3,6 receptors was excluded, and the sensitivity to a P2X7 agonist did not change after inflammation, it appears that after inflammation the responses to ATP are largely due to P2X2 and/or 5 receptors, with a possible contribution of P2X4 receptors. We conclude that inflammation induced a large increase in the sensitivity of SGCs to ATP, which involved a switch from P2Y to P2X receptors. We propose that the over 100-fold augmented sensitivity of SGCs to ATP after injury may contribute to chronic pain states.

P2X receptors and modulation of pain transmission: focus on effects of drugs and compounds used in traditional Chinese medicine.

Adenosine triphosphate (ATP) is thought to play an important role in nociceptive transmission or pain signals. ATP is implicated in peripheral pain signaling by acting on P2X receptors. ATP can act on cell bodies of primary afferent fibers. Opening of P2X receptor channels and subsequent membrane depolarization are generally regarded as a key element for extracellular ATP to produce pain. Nociceptive neurons express homomeric P2X(3) as well as heteromeric P2X(2/3) receptors. Both types of channels can be expressed separately or together in individual neuron. Selective antagonists for P2X(3) and/or P2X(2/3) receptors may represent a novel series of useful analgesic drugs. In addition to P2X(3) and P2X(2/3) receptors, other subtypes of the P2X receptor family are also involved in the modulation of nocicpetive transmission (e.g. P2X(4), P2X(7) receptors). Chinese traditional medicine, i.e., tetramethylpyrazine (TMP), sodium ferulate (SF) and puerarin can antagonize the nociceptive or pain transmission mediated by P2X(3) and/or P2X(2/3) receptors in primary afferent neurons. P2X(3) and/or P2X(2/3) receptors are the pharmacological targets of TMP, SF and puerarin for the therapeutic treatment of pain. The myocardial ischemic injury enhanced the sensitization of sympathetic afferent neurons with increased intensity of P2X(3), P2X(2/3) immunoreactivity, protein and mRNA expression in SCG, SG or NG neurons. P2X(3), P2X(2/3) receptors antagonist A-317491 inhibited the transmission of cardiac nociceptive response. Myocardial ischemic nociceptive signaling via P2X(3) and P2X(2/3) receptors in the transmission of rat SCG, SG, NG neuronal circuits induces the sympathoexcitatory reflex to exaggerate myocardial tissue injury. Blocking the nociceptive transmission of SCG, SG, NG neuronal circuits mediated by P2X(3) and P2X(2/3) receptors may improve the cardiac dysfunction. P2X(3) and P2X(2/3) receptors in SCG, SG, NG neurons could be considered as new targets for treating myocardial ischemic injury and cardiac arrhythmia.

P2X and NMDA receptor involvement in temporomandibular joint-evoked reflex activity in rat jaw muscles.

We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of an N-methyl-d-aspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2'-(or-3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) or the selective NMDA antagonist (+/-)-d-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, alpha,beta-me-ATP injection into the TMJ (but not its systemic administration) following pre-injection of the vehicle significantly increased the magnitude and the duration of ipsilateral DIG and MASS EMG activity in a dose-dependent manner. The alpha,beta-me-ATP-evoked responses could be antagonized by pre-injection of TNP-ATP into the same TMJ site but contralateral TMJ injection of TNP-ATP proved ineffective. Furthermore, the alpha,beta-me-ATP-evoked responses could also be antagonized by APV injected into the same TMJ site but not by its systemic injection. These results indicate the interaction of peripheral purinergic as well as glutamatergic receptor mechanisms in the processing of TMJ nociceptive afferent inputs that evoke reflex activity in jaw muscles.

Adenosine sensory transduction pathways contribute to activation of the sensory irritation response to inspired irritant vapors.

The molecular mechanisms through which sensory irritants stimulate nasal trigeminal nerves are poorly understood. The current study was aimed at evaluating the potential contribution of purinergic sensory transduction pathways in this process. Aerosols of 4-36 mM adenosine 5'-triphosphate (ATP) and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of transient receptor potential vanilloid type-1 (TRPV1)-expressing C fibers greatly reduced, but did not abolish, the sensory irritation response to ATP aerosol and was without effect on the response to adenosine aerosol, indicating that ATP acts largely on capsaicin-sensitive (primarily C fibers) and adenosine acts on capsaicin-insensitive (primarily Adelta fibers) nerves. The response to adenosine was diminished by pretreatment with the broad-based adenosine receptor antagonist theophylline (20 mg/kg) and A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 mg/kg), providing evidence that adenosine stimulates capsaicin-insensitive nerves via the A1 receptor. The sensory irritation responses to 275 ppm styrene and 110 ppm acetic acid vapors were significantly reduced by theophylline pretreatment suggesting a role for adenosine signaling pathways in activation of the sensory irritant response by these vapors. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then nasal sensory nerve activation may be a reflection of irritant-induced alterations in airway cell integrity.

Sympathetic and parasympathetic component of bradycardia triggered by stimulation of NTS P2X receptors.

We have previously shown that activation of P2X purinoceptors in the subpostremal nucleus tractus solitarius (NTS) produces a rapid bradycardia and hypotension. This bradycardia could occur via sympathetic withdrawal, parasympathetic activation, or a combination of both mechanisms. Thus we investigated the relative roles of parasympathetic activation and sympathetic withdrawal in mediating this bradycardia in chloralose-urethane anesthetized male Sprague-Dawley rats. Microinjections of the selective P2X purinoceptor agonist alpha,beta-methylene ATP (25 pmol/50 nl and 100 pmol/50 nl) were made into the subpostremal NTS in control animals, after atenolol (2 mg/kg i.v.), a beta1-selective antagonist, and after atropine methyl bromide (2 mg/kg i.v.), a muscarinic receptor antagonist. The bradycardia observed with activation of P2X receptors at the low dose of the agonist is mediated almost entirely by sympathetic withdrawal. After beta1-adrenergic blockade, the bradycardia was reduced to just -5.1 +/- 0.5 versus -28.8 +/- 5.1 beats/min in intact animals. Muscarinic blockade did not produce any significant change in the bradycardic response at the low dose. At the high dose, both beta1-adrenergic blockade and muscarinic blockade attenuated the bradycardia similarly, -37.4 +/- 6.4 and -40.6 +/- 3.7 beats/min, respectively, compared with -88.0 +/- 11 beats/min in control animals. Double blockade of both beta1-adrenergic and muscarinic receptors virtually abolished the response (-2.5 +/- 0.8 beats/min). We conclude that the relative contributions of parasympathetic activation and sympathetic withdrawal are dependent on the extent of P2X receptor activation.

Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain.

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on carrageenan-induced TMJ inflammatory hyperalgesia. Application of alpha,beta-meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co-application of lidocaine N-ethyl bromide quaternary salt, QX-314, (2%) or of the P2 receptor antagonist PPADS. Co-application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.