Transient expression of thyrotropin releasing hormone peptide and mRNA in the rat hippocampus following global cerebral ischemia/reperfusion injury.

INTRODUCTION: The role of extra-hypothalamic thyrotropin-releasing hormone (TRH) has been investigated by pharmacological studies using TRH or its analogues and found to produce a wide array of effects in the central nervous system. METHODS: Immunofluorescence, In situ labeling of DNA (TUNEL), in situ hybridization chain reaction and quantitative real-time polymerase chain reaction were used in this study. RESULTS: We found that the granular cells of the dentate gyrus expressed transiently a significant amount of TRH-like immunoreactivity and TRH mRNA during the 6-24 h period following global cerebral ischemia/reperfusion injury. TUNEL showed that apoptosis of neurons in the CA1 region occurred from 48 h and almost disappeared at 7 days. TRH administration 30 min before or 24 h after the injury could partially inhibit neuronal loss, and improve the survival of neurons in the CA1 region. CONCLUSION: These data suggest that endogenous TRH expressed transiently in the dentate gyrus of the hippocampus may play an important role in the survival of neurons during the early stage of ischemia/reperfusion injury and that delayed application of TRH still produced neuroprotection. This delayed application of TRH has a promising therapeutic significance for clinical situations.

Expression of P2X receptors in the rat anterior pituitary.

In this study, the distribution patterns of P2X1 to P2X7 receptors in the anterior pituitary cells of rat were studied with single-, double-, and triple-labeling immunofluorescence, combined method of immunofluorescence and in situ hybridization, and Western blot. The results showed that the expression level of the P2X4 receptor protein was highest, followed by P2X5, P2X3, P2X2, P2X6, and P2X7 receptor proteins, but no P2X1 receptor protein was detected. Strong P2X4 receptor-immunoreactivity was detected in almost all the anterior pituitary cells. Different combinations of P2X receptors were detected in each individual cell type of the rat anterior pituitary. Gonadotrophs express P2X4, P2X5, and P2X6 receptors. Corticotrophs express P2X3 and P2X4 receptors. Folliculo-stellate cells express P2X2 and P2X4 receptors, and somatotrophs, lactotrophs, and thyrotrophs express only P2X4 receptors. The macrophages with Iba-1-ir expressed P2X7 receptors. The possible functions of these P2X receptors in each individual cell type of the rat anterior pituitary are discussed.

The potential of P2X7 receptors as a therapeutic target, including inflammation and tumour progression.

Seven P2X ion channel nucleotide receptor subtypes have been cloned and characterised. P2X7 receptors (P2X7R) are unusual in that there are extra amino acids in the intracellular C terminus. Low concentrations of ATP open cation channels sometimes leading to cell proliferation, whereas high concentrations of ATP open large pores that release inflammatory cytokines and can lead to apoptotic cell death. Since many diseases involve inflammation and immune responses, and the P2X7R regulates inflammation, there has been recent interest in the pathophysiological roles of P2X7R and the potential of P2X7R antagonists to treat a variety of diseases. These include neurodegenerative diseases, psychiatric disorders, epilepsy and a number of diseases of peripheral organs, including the cardiovascular, airways, kidney, liver, bladder, skin and musculoskeletal. The potential of P2X7R drugs to treat tumour progression is discussed.

Cell culture: complications due to mechanical release of ATP and activation of purinoceptors.

There is abundant evidence that ATP (adenosine 5'-triphosphate) is released from a variety of cultured cells in response to mechanical stimulation. The release mechanism involved appears to be a combination of vesicular exocytosis and connexin and pannexin hemichannels. Purinergic receptors on cultured cells mediate both short-term purinergic signalling of secretion and long-term (trophic) signalling such as proliferation, migration, differentiation and apoptosis. We aim in this review to bring to the attention of non-purinergic researchers using tissue culture that the release of ATP in response to mechanical stress evoked by the unavoidable movement of the cells acting on functional purinergic receptors on the culture cells is likely to complicate the interpretation of their data.

A subpopulation of gonadotropin-releasing hormone neurons in the adult mouse forebrain is γ-Aminobutyric acidergic.

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in reproductive function. GnRH is released in distinct pulses that are regulated by neurotransmitters or neuromodulators. With immunohistochemistry and GAD67-GFP knockin mice, this study shows for the first time that a subset of GnRH neurons in the forebrain of adult mouse is γ-aminobutyric acid (GABA)-ergic. There is a gender difference in the percentage of GnRH neurons expressing GAD67-GFP in female vs. male mice. The percentage of GnRH neurons expressing GAD67-GFP decreased after castration of female mice and increased to the normal female level after estradiol treatment. The percentage of GnRH neurons expressing GAD67-GFP did not change significantly in intact, castrated, or castration + testosterone propionate-treated male mice. During the female estrous cycle, the percentage of GnRH neurons expressing GAD67-GFP was higher during the estrous stage than during the diestrous stage. During sexual maturation of postnatal development, GnRH neurons did not express GAD67-GFP until postnatal day (P) 15, and the gender differences were first detected at P30, which corresponds to the maturation stage. In conclusion, our data suggest that 1) a subset of GnRH neurons in mouse forebrain is GABA-ergic, 2) expression of GAD67-GFP in GnRH neurons is at least in part regulated by estrogen, and 3) GnRH neurons secrete GABA to regulate themselves.

Extracellular ATP signaling in equine digital blood vessels.

The functional distribution of ATP-activated P2 receptors is well characterized for many blood vessels, but not in the equine digital vasculature, which is a superficial vascular bed that displays thermoregulatory functions and has been implicated in ischemia-reperfusion injuries of the hoof. Isolated equine digital arteries (EDA) and veins (EDV) were submitted to isometric tension studies, whereby electric field stimulation (EFS) and concentration-response curves to exogenously applied agonists were constructed under low tone conditions. Additionally, immunofluorescent localization of P2X and P2Y receptor subtypes was performed. EFS-induced constriction was abolished by tetrodotoxin (1 µM, n=4). Endothelium denudation did not modify the EFS-induced constriction (n=3). The EFS-induced constriction in EDA was inhibited by phentolamine (67.7±1.8%, n=6; 10 µM), and by the non-selective P2 receptor antagonist suramin (46.2±1.3%, n=6; 10 µM). EFS-induced constriction in EDV was reduced by suramin (48.2±2.4%, n=6; 10 µM), the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (58.3±4.5%, n=6; 10 µM), and phentolamine (23.2±2.5%, n=6; 10 µM). Exogenous methoxamine and ATP mimicked EFS-induced constriction in EDA and EDV. Immunostaining for P2X1, P2X2 and P2X3, and, for P2X1 and P2X7 receptor subunits were observed in EDA and EDV smooth muscle and adventitia, respectively. ATP and noradrenaline are co-transmitters in sympathetic nerves supplying the equine digital vasculature, noradrenaline being the dominant agonist in EDA, and ATP in EDV. In conclusion, P2X receptors mediate vasoconstriction in EDA and EDV, although different P2X subunits are involved in these vessels. The physiological significance of this finding in relation to thermoregulatory functions and equine laminitis is discussed.

Purinergic signaling in healthy and diseased skin.

Adenosine 5'-triphosphate and adenosine receptors have been identified in adult and fetal keratinocytes, fibroblasts, melanocytes, mast cells, Langerhans cells, and Meissner's corpuscles, as well as in hair follicles, sweat glands, and smooth muscle and endothelial cells of skin vessels. Purinergic signaling is involved in skin pathology, including inflammation, wound healing, pain, psoriasis, scleroderma, warts, and skin cancer.

Electroconvulsive therapy: a novel hypothesis for the involvement of purinergic signalling.

It is proposed that ATP is released from both neurons and glia during electroconvulsive therapy (ECT) and that this leads to reduction of depressive behaviour via complex stimulation of neurons and glia directly via P2X and P2Y receptors and also via P1 receptors after extracellular breakdown of ATP to adenosine. In particular, A(1) adenosine receptors inhibit release of excitatory transmitters, and A(2A) and P2Y receptors may modulate the release of dopamine. Sequential ECT may lead to changes in purinoceptor expression in mesolimbic and mesocortical regions of the brain implicated in depression and other mood disorders. In particular, increased expression of P2X7 receptors on glial cells would lead to increased release of cytokines, chemokines and neurotrophins. In summary, we suggest that ATP release following ECT involves neurons, glial cells and neuron-glial interactions acting via both P2 and after breakdown to adenosine via P1 receptors. We suggest that ecto-nucleotidase inhibitors (increasing available amounts of ATP) and purinoceptor agonists may enhance the anti-depressive effect of ECT.

Purinergic contraction of the rat vas deferens in L-NAME-induced hypertension: effect of sildenafil.

Hypertension (HTN) is a risk factor for erectile dysfunction, but its effect on vas deferens (VD) contractility and the ejaculatory response has not been delineated. NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used for induction of nitric oxide (NO)-deficient HTN. Our aim was to evaluate the effects of L-NAME-induced HTN on rat VD contractility and to determine whether sildenafil affects VD contractility. A total of 36 male rats were divided into (1) control, (2) L-NAME-HTN, (3) sildenafil treated L-NAME-HTN groups. Group 2 was treated with L-NAME (40 mg kg(-1) per day) in drinking water for 4 weeks. Group 3 received sildenafil (1.5 mg kg(-1) per day, by oral gavage) concomitantly with L-NAME. The prostatic portion of the VD was subjected to electrical field stimulation (EFS, 1-20 Hz), and the P2X(1) agonist alpha,beta-methylene ATP (alpha,beta-meATP, 100 micromol L(-1)-1 micromol L(-1)) and the alpha1-adrenoceptor agonist phenylephrine (Phe, 100 micromol L(-1)-1 mmol L(-1)) were used to construct concentration-response curves. These experiments were repeated in the presence of P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 micromol L(-1)). VD contractions in response to EFS, alpha,beta-meATP and Phe were significantly enhanced by L-NAME. Sildenafil treatment in the L-NAME group improved the contractile response of VD to EFS (20 Hz). In the presence of PPADS, the enhanced contractile response of VD to EFS and alpha,beta-meATP in hypertensive rats was reversed. In the rat model of chronic NO depletion, the purinergic and adrenergic components and EFS affect VD contractility. The VD contractile response may be mediated more by the purinergic system than the adrenergic system, and sildenafil may alter the ejaculatory response in men with PE.

Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck.

BACKGROUND AND PURPOSE: As adenosine 5'-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck. EXPERIMENTAL APPROACH: Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37 degrees C and gassed with 95% O(2) and 5% CO2 for isometric force recordings. KEY RESULTS: EFS, in the presence of atropine, guanethidine and N(G)-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5'-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5'-triphosphate, uridine 5'-diphosphate and alpha,beta-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y(1) and P1 receptor blockers 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist's potency was: 5'-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N(6)-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A(2A) antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na(+) channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X(1) and P2X(3) receptors. CONCLUSIONS AND IMPLICATIONS: ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A(2A) receptors after breakdown to adenosine, and P2Y(1) receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency.

An in vivo model of melanoma: treatment with ATP.

Athymic mice, injected with A375 human melanoma cells, were treated daily with intraperitoneal injections of adenosine 5'-triphosphate (ATP). The tumour volume and animal weight were measured over the course of the experiment and the final tumour nodule weight was measured at the end of the experiment. Tumour volume decreased by nearly 50% by 7 weeks in treated mice. Weight loss in untreated animals was prevented by ATP. Histological examination of the excised tumour nodules showed necrosis in the ATP-treated tumours only. The presence of P2Y(1) and P2X(7) receptors, previously proposed as extracellular targets for melanoma treatment with ATP, were demonstrated in the excised specimens by immunohistochemistry. This paper provides further support for the use of ATP as a treatment for melanoma.

Hypoxia stimulates vesicular ATP release from rat osteoblasts.

Many neuronal and non-neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y(1) receptor, stimulate osteoclast formation and activity, whilst P2Y(2) receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation-dependent manner, with mature, bone-forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N-ethylmaleimide (1-1,000 microM) inhibited basal ATP release by up to 99%. The presence of granular ATP-filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O(2)) for up to 3 min increased ATP release from osteoblasts up to 2.5-fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 microM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N-ethylmaleimide (100 microM) attenuated the hypoxia-induced ATP release by up to 80%. Depletion of quinacrine-stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O(2) could influence local purinergic signaling and affect the balance between bone formation and bone resorption.

The purinergic component of human vas deferens contraction.

OBJECTIVE: To examine purinergic signaling in human vas deferens. DESIGN: To study contractile responses of the scrotal vas deferens. SETTING: Research department of a university teaching hospital. PATIENT(S): Undergoing vasectomy or orchidectomy (aged 27-88 years, n = 14). INTERVENTION(S): Vasectomy or orchidectomy. MAIN OUTCOME MEASURE(S): Strips of vas deferens were suspended in an organ bath and subjected to electrical stimulation to establish frequency-response curves. These stimulations were repeated in the presence of pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2 receptor antagonist), prazosin (adrenergic alpha1 antagonist), and tetrodotoxin. Concentration-response curves were constructed to noradrenaline and the P2X agonists ATP and alpha,beta-methylene ATP (alpha,beta-meATP). The P2X receptor subtype distribution was assessed by immunohistochemistry using specific antibodies. RESULT(S): The response at 32 Hz in the presence of PPADS was reduced by 40% and in the presence of prazosin by 80%. Noradrenaline caused concentration-dependent contractions (EC50 = 11.8 microM). Contractions to ATP and alpha,beta-meATP (EC50 = 6.27 microM) suggested that the functional receptor was P2X1 and/or P2X3. However, immunohistochemistry demonstrated P2X1, but not P2X3, receptor immunoreactivity on the smooth muscle cells. CONCLUSION(S): This study demonstrated that ATP is a co-transmitter with noradrenaline in the contraction of the human vas deferens predominantly acting through the P2X1 receptor.

Osteoblast responses to nucleotides increase during differentiation.

Accumulating evidence suggests that extracellular nucleotides, signaling through P2 receptors, play a role in modulating bone cell function. ATP and ADP stimulate osteoclastic resorption, while ATP and UTP are powerful inhibitors of bone formation by osteoblasts. We investigated changes in the expression of P2 receptors with cell differentiation in primary osteoblast cultures. Rat calvarial osteoblasts, cultured for up to 10 days, were loaded with the intracellular Ca(2+)-sensing fluorophore, Fluo-4 AM, and a fluorescence imaging plate reader was used to measure responses to nucleotide agonists. Peak responses occurred within 20 s and were evoked by ATP or UTP at concentrations as low as 2 microM. Osteoblast number doubled between day 4 and 10 of culture, but the peak intracellular Ca(2+) response to ATP or UTP increased up to 6-fold over the same period, indicating that osteoblast responsiveness to nucleotides increases as cell differentiation proceeds. The approximate order of potency for the most active nucleotide agonists at day 8 of culture was ATP > UTP and ATPgammaS > ADP > UDP, consistent with the expression of functional P2Y(2), P2X(2), P2Y(4), P2Y(1) and P2Y(6) receptors. Smaller responses were elicited by 2-MeSATP, Bz-ATP and alpha,beta-meATP, additionally suggesting the presence of functional P2X(1), P2X(3), P2X(5) and P2X(7) receptors. Expression of mRNA for the ATP- and UTP-selective P2Y(2) receptor increased strongly between day 6 and 15 in primary rat osteoblasts, whereas mRNAs for the P2Y(4) (also ATP/UTP selective) and P2Y(6) (UDP/UTP selective) receptors were highly expressed at intermediate time points. In contrast, mRNA for the cell-proliferation-associated P2X(5) receptor decreased to undetectable as osteoblasts matured, but mRNA for the cell-death-associated P2X(7) receptor was detected at all time points. Similar trends were evident using immunostaining and Western blotting for P2 receptors. Exposure to 10 muM ATP or UTP during days 10-14 of culture was sufficient to cause near-total blockade of the 'trabecular' bone nodules formed by osteoblasts; however, UDP and ADP were without effect. Our results show that there is a shift from P2X to P2Y expression during differentiation in culture, with mature osteoblasts preferentially expressing the P2Y(2) receptor and to a lesser extent P2Y(4) and P2Y(6) receptors. Taken together, these data suggest that the P2Y(2) receptor, and possibly the P2Y(4) receptor, could function as 'off-switches' for mineralized bone formation.

Postnatal development of P2 receptors in the murine gastrointestinal tract.

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADP>or=alpha,beta-meATP>or=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.

Changes in purinergic signalling in developing and ageing rat tail artery: importance for temperature control.

This study aimed to examine the expression and function of P2 receptors of the rat tail and mesenteric arteries during maturation and ageing (4, 6 and 12 weeks, 8 and 24 months). Functional studies and receptor expression by immunohistochemistry revealed a heterogeneous phenotype of P2 receptor subtypes depending on artery age. The purinergic component of nerve-mediated responses in the tail artery was greater in younger animals; similarly responses to ATP and alpha,beta-meATP and the expression of P2X1 receptors decreased with age. Contractile responses to 2-MeSADP decreased with age, and were absent at 8 and 24 months; P2Y1 receptor expression followed this pattern. UTP-induced contractions and P2Y2 receptor expression also decreased with age. The mesenteric artery contracted to UTP, responses at 4 and 6 weeks were larger than at other ages although P2Y2 receptor expression did not significantly differ with age. 2-MeSADP induced relaxation of the mesenteric artery, responses being greatest at 6 weeks and decreased thereafter, which was mimicked by the P2Y1 receptor immunostaining. We speculate that the dramatic changes in expression of P2 receptors in the rat tail artery, compared to the mesenteric artery, during development and ageing are related to the role of the tail artery in temperature regulation.

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule.

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.

Presence of the P2X(7) purinergic receptor on immune cells that invade the rat endometrium during oestrus.

Eosinophils, macrophages and other leucocytes invade the uterine endometrium during oestrus and play a role in the tissue remodeling and immune responses that occur prior to implantation of the fertilized ovum. Adenosine 5'-triphosphate (ATP) and its metabolites influence uterine function via ATP receptors. In this study, we investigated the presence and localisation of the P2X(7) nucleotide receptor in the cells that infiltrate the uterine endometrium of adult female rats during oestrus at the electron microscope level, using gold-silver pre-embedding immunocytochemical techniques. P2X(7) receptor expression was found in the cytoplasm and the cell membrane of eosinophils, macrophages and fibroblasts in the endometrium during oestrus. These results suggest that ATP-mediated responses may be important in uterine preparation and remodeling before implantation and that this may involve several types of cells. In particular, the presence of P2X(7) receptors on endometrial stromal cells may indicate their involvement in apoptosis and immune and inflammatory responses.

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP.

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2-/-) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3(Dbl-/-)), and compare these with previously characterized P2X3-/- mice. In patch-clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild-type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2-/- mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3(Dbl-/-) mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2-/- and P2X2/P2X3(Dbl-/-) mice had reduced pain-related behaviours in response to intraplantar injection of formalin. Significantly, P2X3-/-, P2X2-/-, and P2X2/P2X3(Dbl-/-) mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2-/- mice. Taken together, these data extend our findings for P2X3-/- mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder.

Impairment of the splenic immune system in P2X(2)/P2X(3) knockout mice.

The isolated spleens from male and female mice lacking P2X(2) and P2X(3) receptors (P2X(2)/P2X(3) knockout (KO) mice) and those from wild-type (WT) mice were investigated by flow cytometry, immunohistochemistry and functionally by organ-bath pharmacology. The spleens from the P2X(2)/P2X(3) KO mice weighed significantly more than the corresponding WT mice. Flow cytometry was used to isolate the mononuclear cells, which were then phenotyped. T-lymphocytes, B-lymphocytes and macrophages were identified and counted. It was found that the increase in size of the spleens from the KO animals corresponded to an increase in the numbers of mononuclear cells present and that all three cell types (T-lymphocytes, B-lymphocytes and macrophages) increased in much the same proportion as those from the WT animals. Immunohistochemical localisation of P2Y(1), P2Y(2) and P2X(1) receptors revealed their presence on the spleen capsule and trabeculae. P2X(1) receptors were also present on blood vessels. There was no difference in the expression of these receptors between the WT and P2X(2)/P2X(3) KO spleens. Functional studies revealed the presence of multiple P2 receptors inducing the contraction of the spleen capsule, from both WT and KO mice. There was no difference in the contractions induced by adenosine 5'-triphosphate (ATP), alpha,beta-methylene ATP, 2-methylthio ADP or uridine triphosphate from WT and KO mice. It is concluded that mice lacking both P2X(2) and P2X(3) receptors have enlarged spleens and that this is correlated with an increase in the number of immune cells, perhaps as a consequence of a compromised immune system and chronic infection.