Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

Phytoestrogen-Based Hormonal Replacement Therapy Could Benefit Women Suffering Late-Onset Asthma.

It has been observed that plasmatic concentrations of estrogens, progesterone, or both correlate with symptoms in asthmatic women. Fluctuations in female sex steroid concentrations during menstrual periods are closely related to asthma symptoms, while menopause induces severe physiological changes that might require hormonal replacement therapy (HRT), that could influence asthma symptoms in these women. Late-onset asthma (LOA) has been categorized as a specific asthmatic phenotype that includes menopausal women and novel research regarding therapeutic alternatives that might provide relief to asthmatic women suffering LOA warrants more thorough and comprehensive analysis. Therefore, the present review proposes phytoestrogens as a promising HRT that might provide these females with relief for both their menopause and asthma symptoms. Besides their well-recognized anti-inflammatory and antioxidant capacities, phytoestrogens activate estrogen receptors and promote mild hormone-like responses that benefit postmenopausal women, particularly asthmatics, constituting therefore a very attractive potential therapy largely due to their low toxicity and scarce side effects.

Estrogenic Modulation of Ionic Channels, Pumps and Exchangers in Airway Smooth Muscle.

To preserve ionic homeostasis (primarily Ca2+, K+, Na+, and Cl-), in the airway smooth muscle (ASM) numerous transporters (channels, exchangers, and pumps) regulate the influx and efflux of these ions. Many of intracellular processes depend on continuous ionic permeation, including exocytosis, contraction, metabolism, transcription, fecundation, proliferation, and apoptosis. These mechanisms are precisely regulated, for instance, through hormonal activity. The lipophilic nature of steroidal hormones allows their free transit into the cell where, in most cases, they occupy their cognate receptor to generate genomic actions. In the sense, estrogens can stimulate development, proliferation, migration, and survival of target cells, including in lung physiology. Non-genomic actions on the other hand do not imply estrogen's intracellular receptor occupation, nor do they initiate transcription and are mostly immediate to the stimulus. Among estrogen's non genomic responses regulation of calcium homeostasis and contraction and relaxation processes play paramount roles in ASM. On the other hand, disruption of calcium homeostasis has been closely associated with some ASM pathological mechanism. Thus, this paper intends to summarize the effects of estrogen on ionic handling proteins in ASM. The considerable diversity, range and power of estrogens regulates ionic homeostasis through genomic and non-genomic mechanisms.

Possible Beneficial Actions of Caffeine in SARS-CoV-2.

The COVID-19 pandemic has established an unparalleled necessity to rapidly find effective treatments for the illness; unfortunately, no specific treatment has been found yet. As this is a new emerging chaotic situation, already existing drugs have been suggested to ameliorate the infection of SARS-CoV-2. The consumption of caffeine has been suggested primarily because it improves exercise performance, reduces fatigue, and increases wakefulness and awareness. Caffeine has been proven to be an effective anti-inflammatory and immunomodulator. In airway smooth muscle, it has bronchodilator effects mainly due to its activity as a phosphodiesterase inhibitor and adenosine receptor antagonist. In addition, a recent published document has suggested the potential antiviral activity of this drug using in silico molecular dynamics and molecular docking; in this regard, caffeine might block the viral entrance into host cells by inhibiting the formation of a receptor-binding domain and the angiotensin-converting enzyme complex and, additionally, might reduce viral replication by the inhibition of the activity of 3-chymotrypsin-like proteases. Here, we discuss how caffeine through certain mechanisms of action could be beneficial in SARS-CoV-2. Nevertheless, further studies are required for validation through in vitro and in vivo models.

Sex Hormones and Lung Inflammation.

Inflammation is a characteristic marker in numerous lung disorders. Several immune cells, such as macrophages, dendritic cells, eosinophils, as well as T and B lymphocytes, synthetize and release cytokines involved in the inflammatory process. Gender differences in the incidence and severity of inflammatory lung ailments including asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), lung cancer (LC), and infectious related illnesses have been reported. Moreover, the effects of sex hormones on both androgens and estrogens, such as testosterone (TES) and 17ß-estradiol (E2), driving characteristic inflammatory patterns in those lung inflammatory diseases have been investigated. In general, androgens seem to display anti-inflammatory actions, whereas estrogens produce pro-inflammatory effects. For instance, androgens regulate negatively inflammation in asthma by targeting type 2 innate lymphoid cells (ILC2s) and T-helper (Th)-2 cells to attenuate interleukin (IL)-17A-mediated responses and leukotriene (LT) biosynthesis pathway. Estrogens may promote neutrophilic inflammation in subjects with asthma and COPD. Moreover, the activation of estrogen receptors might induce tumorigenesis. In this chapter, we summarize the most recent advances in the functional roles and associated signaling pathways of inflammatory cellular responses in asthma, COPD, PF, LC, and newly occurring COVID-19 disease. We also meticulously deliberate the influence of sex steroids on the development and progress of these common and severe lung diseases.

Functional expression of P2X1, P2X4 and P2X7 purinergic receptors in human monocyte-derived macrophages.

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA. In whole-cell patch clamp recordings of human MDMs, rapid application of ATP (0.01 mM) evoked fast current activation and two different desensitization kinetics: 1. a rapid desensitizing current antagonized by PPADS (1 µM), reminiscent of the P2X1 receptor's current; 2. a slow desensitizing current, insensitive to PPADS but potentiated by ivermectin (3 µM), similar to the P2X4 receptor's current. Application of 5 mM ATP induced three current modalities: 1. slow current activation with no desensitization, similar to the P2X7 receptor current, present in 69% of human macrophages and antagonized by A-804598 (0.1 µM); 2. fast current activation and fast desensitization, present in 15% of human MDMs; 3. fast activation current followed by biphasic desensitization, observed in 15% of human MDMs. Both rapid and biphasic desensitization kinetics resemble those observed for the recombinant human P2X1 receptor expressed in oocytes. These data demonstrate, for the first time, the co-expression of P2X1, P2X4, and P2X7 transcripts and confirm the presence of functional P2X1, P2X4, and P2X7 receptors in human macrophages.

COVID-19 y su asociación con los inhibidores de la enzima convertidora de angiotensina y los antagonistas de los receptores para angiotensina II / COVID-19 and its Association with Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Antagonists

Resumen En la pandemia de COVID-19 que se ha presentado en México y en el mundo, y que ya ha infectado alrededor de 7 millones de personas, las comorbilidades que se han asociado a esta enfermedad en orden de importancia son: hipertensión, diabetes mellitus, obesidad, enfermedad pulmonar obstructiva crónica, enfermedad cardiovascular, insuficiencia renal crónica, tabaquismo e inmunosupresión, y la hipertensión arterial es un rasgo característico en todas ellas. La enzima convertidora de angiotensina-2 (ACE2), es el receptor funcional para el SARS-CoV-2. Este virus, tiene una proteína llamada espiga (proteína S) que reconoce a la ACE2 como su receptor para ingresar a las células. La ACE2 es una proteína de la membrana plasmática y se encuentra expresada en las células alveolares tipo I y II, células epiteliales, fibroblastos, células endoteliales y macrófagos. El tratamiento con inhibidores de la enzima convertidora de angiotensina (ACEi) o con antagonistas del receptor a angiotensina II (ARBs) aumentan notablemente la expresión de ACE2. Por lo tanto, en pacientes con estas patologías y tratados con estos medicamentos, se podría incrementar el riesgo de desarrollar la COVID-19 en forma severa y fatal. Cabe destacar que los pacientes con mayor mortalidad por la COVID-19 en México son los que presentan hipertensión, diabetes mellitus, obesidad y los mayores de 65 años. Por todo lo anterior, podríamos sugerir que, durante la etapa crítica de la pandemia por SARS-CoV-2, a los pacientes, particularmente en personas de edad avanzada, con hipertensión o con diabetes mellitus y obesidad que cursan con hipertensión y si su tratamiento es con ACEi o con ARBs, se les debería modificar a medicamentos alternativos como los bloqueadores de los canales de Ca2+ tipo L (amlodipino), que hasta el momento no han sido asociados con la ACE2.

Abstract Worldwide, over 7 million people have been infected due to the pandemic of COVID-19. The comorbidities associated to this disease are: hypertension, diabetes mellitus, obesity, obstructive pulmonary disease (COPD), cardiovascular disease, chronic renal failure, smoking, immunosuppression, and hypertension. Angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV-2. This virus has an S protein that recognizes ACE2 as its receptor to enter the cell. ACE2 is a plasmatic protein expressed in alveolar cells type I, II, fibroblasts, endothelial cells and macrophages. Treatment with inhibitors of the angiotensin-converting enzyme (ACEi) or the receptor antagonist for angiotensin II (ARBs) notably increase the expression of ACE2. Therefore, in patients with these pathologies and treated with these medicines, the risk of developing the COVID-19 in a severe and fatal way could be increased. In Mexico, the major mortality due to COVID-19 is related to hypertension, diabetes mellitus, obesity and being over 65 years of age. Therefore, we suggest that during the SARS-CoV-2 pandemic, patients with hypertension treated with ACEi or ARBs, should receive alternative treatments such as L-type Ca2+ channel blockers (amlodipine) that have not been associated with ACE2 until now.

Sex steroids effects on guinea pig airway smooth muscle tone and intracellular Ca2+ basal levels.

Testosterone (TES), other androgens and female sex steroids induce non-genomic rapid relaxing effects in airway smooth muscle (ASM). In guinea pig ASM, basal tension was relaxed by dehydroepiandrosterone (DHEA) and TES; 17ß-estradiol (E2) had a small effect. Blockers of L-type voltage dependent Ca2+ channel (L-VDCC, D-600) and store operated Ca2+ channel (SOC, 2-APB) also relaxed the basal tone. In tracheal myocytes, DHEA and TES diminished intracellular basal Ca2+ concentrations (b[Ca2+]i) as D-600+2-APB but to a higher extend. TES after D-600+2APB or Pyr3, a blocker of canonical transient receptor potential 3 (TRPC3), further decreased b[Ca2+]i rendering this response equal to TES alone. With indomethacin, the b[Ca2+]i decrease induced by the blockade of L-VDCC and TRPC3 was not changed by the addition of TES. PGE2 or forskolin addition after D600+2-APB, decreased b[Ca2+]i resembling TES response. An adenylate cyclase inhibitor followed by D-600+2-APB lowered b[Ca2+]i, TES showed no further effect. Carbachol-induced [Ca2+]i increment was reduced by TES or DHEA. 17ß-estradiol diminished KCl-induced contraction and, in tracheal myocytes, the voltage-dependent inward Ca2+ current. CONCLUSION: DHEA and TES diminish ASM tone and b[Ca2+]i by blocking L-VDCC and probably a constitutively active TRPC3, and by PGE2 synthesis. E2 lowers ASM basal tone by blocking only L-VDCC.

Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms.

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.Currents through homomeric channels had significantly different Hill coefficients. P2XR are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible when both P2X2 isoforms are coexpressed, the heteromeric channels might be formed by: i) 2(P2X2-1a)+1(P2X2-2bm); or ii) 1(P2X2-1a)+2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different ATP sensitivities. Thus, co-expressing both P2X2 isoforms, two oocyte populations were distinguished based on their sensitivity to ATP and Hill coefficients. For the first population (P2X2-1a like), the ATP EC50 and the Hill coefficient were not different than those of homomeric P2X2-1a channels similarly, for the second population (P2X2-2bm like), these variables were also not different than for those of homomeric P2X2-2bm channels. Various findings indicate that homomeric channel expression is not responsible for such differences. Our observations indicate that two heteromeric channels can be assembled from two P2X2 receptor isoforms. Our data support a current model, according to which, ATP activation of two subunits can open P2X2 channel. However, PPADS appears to bind to all three subunits in order to inhibit ATP effects on P2X2 receptors.

ATP releases ATP or other nucleotides from human peripheral blood leukocytes through purinergic P2 receptors.

AIMS: Almost every eukaryotic cell releases ATP under certain conditions. The idea that ATP induces the release of ATP has been scantly investigated. METHODS: We explored this possibility by assessing the rate of exogenous ATP breakdown (measured by phosphates production) by human peripheral blood leukocytes. The role of P2Y and P2X receptors was evaluated pharmacologically, by patch clamp, or by flow cytometry. KEY FINDINGS: In mononuclear and/or polymorphonuclear cells, ATP increased phosphates formation in a time- and concentration-dependent manner. Uncoupling of P2Y receptors with N-ethylmaleimide and antagonism of P2Y and P2X receptors through suramin reduced phosphate formation after 500µM ATP, suggesting that part of the phosphate production was due to activation of P2 receptors, with subsequent release of ATP or other nucleotides. Similar results were obtained with UTP and ATPγS. Gadolinium (connexins inhibitor) also significantly reduced the ATP-induced phosphate production. Blockade of P2X receptors with SKF 96365 or NF023 did not modify the phosphate production. In monocytes, 500µM ATP induced inward currents suggestive of P2X1 activation, but higher concentrations (1-5mM) induced inward currents suggestive of P2X7 activation. We discarded a role of adenosine in the ATP-evoked nucleotides release. Flow cytometry identified that almost all mononuclear and polymorphonuclear cells expressed P2Y1,2,4,6,11 receptors. SIGNIFICANCE: 500µM ATP induced the release of ATP or other nucleotides through activation of P2Y2,4,6,11 receptors in human leukocytes, and probably via P2X receptors at higher concentrations. This ATP-induced nucleotides release constitutes a potential mechanism leading to amplification of ATP signaling.

17ß-Aminoestrogens induce guinea pig airway smooth muscle hyperresponsiveness through L-type Ca(2+) channels activation.

Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17ß-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17ß-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca(2+) concentration [Ca(2+)]i, pentolame also showed an increase in [Ca(2+)]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca(2+)]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca(2+) currents. Thus, 17ß-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca(2+) channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17ß-aminoestrogens studied.

P2X4 subunits are part of P2X native channels in murine myenteric neurons.

The aim of the present study was to investigate if P2X4 receptors are expressed in murine myenteric neurons and if these receptors contribute to form functional channels in the neuronal membrane by using molecular and electrophysiological techniques. The whole-cell recording technique was used to measure membrane currents induced by ATP (I(ATP)) in myenteric neurons. Compared with recombinant P2X4 receptor-channels (reported by others in a previous study), native myenteric P2X receptors have a relative lower sensitivity for ATP (EC50=102 µM) and α,ß methylene ATP (not effect at 30 or 100 µM). BzATP was a weak agonist for native P2X receptors. KN-62 had no effect on myenteric P2X channels whereas PPADS (IC50=0.54 µM) or suramin (IC50=134 µM) were more potent antagonists than on P2X4 homomeric channels. I(ATP) were potentiated by ivermectin (effect that is specific on P2X4 receptors) and zinc. Western blotting shows the presence of P2X4 protein and RT-PCR the corresponding mRNA transcript in the small intestine. Immunoreactivity for P2X4 receptors was found in most myenteric neurons in culture. Single-cell RT-PCR shows the presence of P2X4 mRNA in 90% of myenteric neurons. Our results indicate that P2X4 receptors are expressed in the majority of myenteric neurons, contribute to the membrane currents activated by ATP, and because most properties of I(ATP) does not correspond to P2X4 homomeric channels it is proposed that P2X4 are forming heteromeric channels in these neurons. P2X4 subunits have a widespread distribution within the myenteric plexus and would be expected to play an important role in cell signaling.

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway.

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons.

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I(ATP)) in guinea pig myenteric neurons. I(ATP) was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01-10mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01-0.3mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC(50) values of these phases were 38 and 1759 µM, respectively. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01-1mM), and less potent (EC(50) 142 µM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1mM) and for BzATP-induced currents (0.1mM; I(BzATP)) but a significant lower rectification was noticed when lsP2X receptors were activated (5mM). Brilliant blue G (BBG) at a concentration of 0.3 µM (known to inhibit only P2X7 receptors) reduced I(ATP) when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I(BzATP). However, hsP2X receptors and I(BzATP) were both inhibited by concentrations ≥ 1 µM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,ß-methylene-ATP was inactive from 1 to 100 µM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors.

In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.

Functional interactions between nicotinic and P2X receptors in celiac ganglia neurons.

Here we characterized the cross-inhibitory interactions between nicotinic and P2X receptors of celiac neurons from the guinea pig by recording whole-cell currents induced by 1mM ACh (I(ACh)), 1mM ATP (I(ATP)) and by the simultaneous application of both agonists (I(ACh)(+ATP)). I(ACh) and I(ATP) were inhibited by hexamethonium (nicotinic channel blocker) and PPADS (P2X receptor antagonist), respectively. The amplitude of I(ACh)(+ATP) was equal to the current induced by the most effective agonist, indicating a current occlusion. Various observations indicate that I(ACh)(+ATP) is carried out through both nicotinic (nACh) and P2X channels: i) I(ACh)(+ATP) desensitisation kinetics were in between that of I(ACh) and I(ATP); ii) application of ATP+ACh, decreased I(ACh) and I(ATP), whereas no cross-desensitisation was observed between nACh and P2X receptors; iii) ATP did not affect I(ACh) in the presence of PPADS or after P2X receptor desensitisation; and iv) ACh did not affect I(ATP) when nACh channels were blocked with hexamethonium or after nACh receptor desensitisation. Current occlusion is not mediated by activation of metabotropic receptors as it is: i) voltage dependent (was not observed at + 5 mV); ii) present at low temperature (10 degrees C) and after inhibition of protein kinase activity (with staurosporine); and iii) absent at 30 microM ATP and 30 microM ACh (concentrations that should activate metabotropic receptors). In conclusion, current occlusion described here is similar to the previously reported myenteric neurons. This occlusion is likely the result of allosteric interactions between these receptors.