Mechanisms of the inhibition of epithelial Na+ channels by CFTR and purinergic stimulation

The epithelial Na+ channel ENaC is inhibited when the cystic fibrosis transmembrane conductance regulator (CFTR) coexpressed in the same cell is activated by the cyclic adenosine monophosphate (cAMP)-dependent pathway. Regulation of ENaC by CFTR has been studied in detail in epithelial tissues from intestine and trachea and is also detected in renal cells. In the kidney, regulation of other membrane conductances might be the predominant function of CFTR. A similar inhibition of ENaC takes place when luminal purinergic receptors a re activated by 5 ' -adenosine triphosphate (ATP) or uridine triphosphate (UTP). Because both stimulation of purinergic receptors and activation of CFTR induce a Cl- conductance, it is likely that Cl- ions control ENaC activity.

Effects of purinergic stimulation, CFTR and osmotic stress on amiloride-sensitive Na+ transport in epithelia and Xenopus oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl-secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl(-)dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in chocytes even after exposure to hypertonic or bypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

Autocrine loop in the purinergic control of airway surface liquid volume : monitoring with a novel side-view imaging technique

La Fibrose Kystique (FK) est une maladie dégénérative qui entraine une dégénération des poumons dû au problème de clairance mucociliaire (CMC). Le volume de surface liquide (SL) couvrant les cellules pulmonaires est essentiel à la clairance de mucus et au combat contre les infections. Les nucléotides extracellulaires jouent un rôle important dans la CMC des voies aériennes, en modifiant le volume de la SL pulmonaire. Cependant, les mécanismes du relâchement de l’ATP et de leurs déplacements à travers la SL, restent inconnus. Des études ultérieures démontrent que l’exocytose d’ATP mécano-sensible et Ca2+-dépendant, dans les cellules A549, est amplifié par les actions synergétiques autocrine/paracrine des cellules avoisinantes. Nous avions comme but de confirmer la présence de la boucle purinergique dans plusieurs modèles de cellules épithéliales et de développer un système nous permettant d’observer directement la SL. Nous avons démontrés que la boucle purinergique est fonctionnelle dans les modèles de cellules épithéliales examinés, mis appart les cellules Calu-3. L’utilisation de modulateur de la signalisation purinergique nous a permis d’observer que le relâchement d’ATP ainsi que l’augmentation du [Ca2+]i suivant un stress hypotonique, sont modulés par le biais de cette boucle purinergique et des récepteurs P2Y. De plus, nous avons développé un système de microscopie qui permet d’observer les changements de volume de SL en temps réel. Notre système permet de contrôler la température et l’humidité de l’environnement où se trouvent les cellules, reproduisant l’environnement pulmonaire humain. Nous avons démontré que notre système peut identifier même les petits changements de volume de SL.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

Efeito da estimulação purinérgica sobre a produção de melatonina em macrófagos da linhagem RAW 264.7

A melatonina é um hormônio produzido de forma rítmica e no período de escuro pela glândula pineal bem como de forma não rítmica por diversos tecidos e células imunocompetentes. É sintetizada pela acetilação e metilação da serotonina pela ação das enzimas arilalquilamina N-acetiltransferase (AA-NAT) e acetilserotonina -O-metiltransferase (ASMT) que levam à formação de N-acetilserotonina (NAS) e melatonina (MEL), respectivamente. Nos últimos anos temos demonstrado que síntese de melatonina pela pineal pode ser negativamente modulada por mediadores inflamatórios e pelo ATP que atua como co-transmissor juntamente com a noradrenalina liberada no terminal nervoso simpático que a inerva. Perifericamente, contudo, estes mediadores inflamatórios apresentam um efeito contrário induzindo a produção de melatonina em células imunocompetentes. Estas observações levaram à criação da hipótese de um eixo imune-pineal. Esse trabalho teve como objetivo verificar o efeito do ATP sobre produção de melatonina em macrófagos da linhagem RAW 264.7 Os dados desse trabalho mostram que o ATP é capaz de induzir de maneira dose dependente a produção de melatonina em macrófagos através da modulação das enzimas AA-NAT e ASMT. Foi demostrado também que esse efeito é mediado pelo receptor P2X7 e que a melatonina produzida age autocrina e paracrinamente aumentando a fagocitose de particulas de zimosan. Com isso, podemos concluir que o ATP é um ativador endógeno do eixo imune-pineal

Glutamatergic and purinergic mechanisms on respiratory modulation in the caudal NTS of awake rats

In the present study we evaluated the role of ionotropic glutamate receptors and purinergic P2 receptors in the caudal commissural NTS (cNTS) on the modulation of the baseline respiratory frequency (fR), and on the tachypneic response to chemoreflex activation in awake rats. The selective antagonism of ionotropic glutamate receptors with kynurenic acid (2 nmol/50 nl) in the cNTS produced a significant increase in the baseline fR but no changes in the tachypneic response to chemoreflex activation. The selective antagonism of purinergic P2 receptors by PPADS (0.25 nmol/50 nl) in the cNTS produced no changes in the baseline fR or in the tachypneic response to chemoreflex activation. The data indicate that glutamate acting on ionotropic receptors in the cNTS plays a inhibitory role on the modulation of the baseline fR but had no effect on the tachypneic response to chemoreflex activation, while ATP acting on P2 receptors in the cNTS plays no major role in the modulation of the baseline fR or in the tachypneic response to chemoreflex activation. We suggest that neurotransmitters other than L-glutamate and ATP are involved in the processing of the tachypneic response of the chemoreflex at the cNTS level. (C) 2008 Elsevier B.V. All rights reserved.

Purinergic receptors in ocular inflammation

Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly 'tuned,' can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P(1),P(4)-diadenosine tetraphosphate (Ap4A), and P(1),P(5)-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particular A2A adenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3 receptor, selective agonists like N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods

BACKGROUND/AIMS ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.

Transcutaneous Tibial Nerve Stimulation In The Treatment Of Lower Urinary Tract Symptoms And Its Impact On Health-related Quality Of Life In Patients With Parkinson Disease: A Randomized Controlled Trial.

A randomized controlled trial study was performed to evaluate the efficacy of transcutaneous tibial nerve stimulation (TTNS) and sham TTNS, in patients with Parkinson disease (PD) with lower urinary tract symptoms (LUTS). Randomized controlled trial. Thirteen patients with a diagnosis of PD and bothersome LUTS were randomly allocated to one of the following groups: Group I: TTNS group (n = 8) and group II: Sham group (n = 5). Both groups attended twice a week during 5 weeks; each session lasted 30 minutes. Eight patients received TTNS treatment and 5 subjects allocated to group II were managed with sham surface electrodes that delivered no electrical stimulation. Assessments were performed before and after the treatment; they included a 3-day bladder diary, Overactive Bladder Questionnaire (OAB-V8), and the International Consultation on Incontinence Quality of Life Questionnaire Short Form (ICIQ-SF), and urodynamic evaluation. Following 5 weeks of treatment, patients allocated to TTNS demonstrated statistically significant reductions in the number of urgency episodes (P = .004) and reductions in nocturia episodes (P < .01). Participants allocated to active treatment also showed better results after treatment in the OAB-V8 and ICIQ-SF scores (P < .01, respectively). Urodynamic testing revealed that patients in the active treatment group showed improvements in intravesical volume at strong desire to void (P < .05) and volume at urgency (P < .01) when compared to subjects in the sham treatment group. These findings suggest that TTNS is effective in the treatment of LUTS in patients with PD, reducing urgency and nocturia episodes and improving urodynamic parameters as well as symptom scores measured by the OAB-V8 and health-related quality-of-life scores measured by the ICIQ-SF.

Application Of Tactile/kinesthetic Stimulation In Preterm Infants: A Systematic Review.

To verify the methods used by the clinical trials that assessed the effect of tactile/kinesthetic stimulation on weight gain in preterm infants and highlight the similarities and differences among such studies. This review collected studies from two databases, PEDro and PubMed, in July of 2014, in addition to bibliographies. Two researchers assessed the relevant titles independently, and then chose which studies to read in full and include in this review by consensus. Clinical trials that studied tactile stimulation or massage therapy whether or not associated with kinesthetic stimulation of preterm infants; that assessed weight gain after the intervention; that had a control group and were composed in English, Portuguese, or Spanish were included. A total of 520 titles were found and 108 were selected for manuscript reading. Repeated studies were excluded, resulting in 40 different studies. Of these, 31 met all the inclusion criteria. There were many differences in the application of tactile/kinesthetic stimulation techniques among studies, which hindered the accurate reproduction of the procedure. Also, many studies did not describe the adverse events that occurred during stimulation, the course of action taken when such events occurred, and their effect on the outcome. These studies made a relevant contribution towards indicating tactile/kinesthetic stimulation as a promising tool. Nevertheless, there was no standard for application among them. Future studies should raise the level of methodological rigor and describe the adverse events. This may permit other researchers to be more aware of expected outcomes, and a standard technique could be established.

Altered Urinary Sodium Excretion Response After Central Cholinergic And Adrenergic Stimulation Of Adult Spontaneously Hypertensive Rats.

In this study, we hypothesized that blunting of the natriuresis response to intracerebroventricularly (i.c.v.) microinjected cholinergic and adrenergic agonists is involved in the development of hypertension in spontaneously hypertensive rats (SHR). We evaluated the effect of i.c.v. injection of cholinergic and noradrenergic agonists, at increasing concentrations, and of muscarinic cholinergic and α1 and α2-adrenoceptor antagonists on blood pressure and urinary sodium handling in SHR, compared with age-matched Wistar Kyoto rats (WR). We confirmed that CCh and NE microinjected into the lateral ventricle (LV) of conscious rats leads to enhanced natriuresis. This response was associated with increased proximal and post-proximal sodium excretion accompanied by an unchanged rate of glomerular filtration. We showed that cholinergic-induced natriuresis in WR and SHR was attenuated by previous i.c.v. administration of atropine and was significantly lower in the hypertensive strain than in WR. In both groups the natriuretic effect of injection of noradrenaline into the LV was abolished by previous local injection of an α1-adrenoceptor antagonist (prazosin). Conversely, LV α2-adrenoceptor antagonist (yohimbine) administration potentiated the action of noradrenaline. The LV yohimbine pretreatment normalized urinary sodium excretion in SHR compared with age-matched WR. In conclusion, these are, as far as we are aware, the first results showing the importance of interaction of central cholinergic and/or noradrenergic receptors in the pathogenesis of spontaneous hypertension. These experiments also provide good evidence of the existence of a central adrenergic mechanism consisting of α1 and α2-adrenoceptors which works antagonistically on regulation of renal sodium excretion.

Scanning electron microscopic study of the in situ effect of salivary stimulation on erosion and abrasion in human and bovine enamel

This in situ study investigated, using scanning electron microscopy, the effect of stimulated saliva on the enamel surface of bovine and human substrates submitted to erosion followed by brushing abrasion immediately or after one hour. During 2 experimental 7-day crossover phases, 9 previously selected volunteers wore intraoral palatal devices, with 12 enamel specimens (6 human and 6 bovine). In the first phase, the volunteers immersed the device for 5 minutes in 150 ml of a cola drink, 4 times a day (8h00, 12h00, 16h00 and 20h00). Immediately after the immersions, no treatment was performed in 4 specimens (ERO), 4 other specimens were immediately brushed (0 min) using a fluoride dentifrice and the device was replaced into the mouth. After 60 min, the other 4 specimens were brushed. In the second phase, the procedures were repeated but, after the immersions, the volunteers stimulated the salivary flow rate by chewing a sugar-free gum for 30 min. Enamel superficial alterations of all specimens were then evaluated using a scanning electron microscope. Enamel prism core dissolution was seen on the surfaces submitted to erosion, while on those submitted to erosion and to abrasion (both at 0 and 60 min) a more homogeneous enamel surface was observed, probably due to the removal of the altered superficial prism layer. For all the other variables - enamel substrate and salivary stimulation -, the microscopic pattern of the enamel specimens was similar.