Gasotransmitters: novel regulators of ion channels and transporters

More than 25 years ago, it was a big surprise for physiologists that nitric oxide (NO) was identified as the endothelium derived relaxing factor which is responsible for endothelium-induced smooth muscle relaxation (Ignarro et al., 1987). Until then, small gaseous molecules were simply regarded as byproducts of cellular metabolism which were unlikely to be of any physiological relevance. The discovery that NO was synthesized by specific enzymes (NO-synthases), upon stimulation by specific, physiologically relevant stimuli (e.g., acetylcholine stimulation of endothelial cells), as well as the fact that it acted on specific cellular targets (e.g., soluble guanylate cyclase), set the course for numerous studies which investigated the physiological roles of gaseous signaling molecules—in other words, gasotransmitters (Wang, 2002)

Why Do We have to Move Fluid to be Able to Breathe?

The ability to breathe air represents a fundamental step in vertebrate evolution that was accompanied by several anatomical and physiological adaptations. The morphology of the air-blood barrier is highly conserved within air-breathing vertebrates. It is formed by three different plies, which are represented by the alveolar epithelium, the basal lamina, and the endothelial layer. Besides these conserved morphological elements, another common feature of vertebrate lungs is that they contain a certain amount of fluid that covers the alveolar epithelium. The volume and composition of the alveolar fluid is regulated by transepithelial ion transport mechanisms expressed in alveolar epithelial cells. These transport mechanisms have been reviewed extensively. Therefore, the present review focuses on the properties and functional significance of the alveolar fluid. How does the fluid enter the alveoli? What is the fate of the fluid in the alveoli? What is the function of the alveolar fluid in the lungs? The review highlights the importance of the alveolar fluid, its volume and its composition. Maintenance of the fluid volume and composition within certain limits is critical to facilitate gas exchange. We propose that the alveolar fluid is an essential element of the air-blood barrier. Therefore, it is appropriate to refer to this barrier as being formed by four plies, namely (1) the thin fluid layer covering the apical membrane of the epithelial cells, (2) the epithelial cell layer, (3) the basal membrane, and (4) the endothelial cells

Amiloride-Sensitive Sodium Channels and Pulmonary Edema

The development of pulmonary edema can be considered as a combination of alveolar flooding via increased fluid filtration, impaired alveolar-capillary barrier integrity, and disturbed resolution due to decreased alveolar fluid clearance. An important mechanism regulating alveolar fluid clearance is sodium transport across the alveolar epithelium. Transepithelial sodium transport is largely dependent on the activity of sodium channels in alveolar epithelial cells. This paper describes how sodium channels contribute to alveolar fluid clearance under physiological conditions and how deregulation of sodium channel activity might contribute to the pathogenesis of lung diseases associated with pulmonary edema. Furthermore, sodium channels as putative molecular targets for the treatment of pulmonary edema are discussed

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration

Background: Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. Methods: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results: Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential

Laminar shear stress modulates the activity of heterologously expressed P2X4 receptors

AbstractP2X4 receptors are involved in mechanotransduction processes, but it is unknown whether or not P2X4 receptors form mechanosensitive ion channels. This study questioned, whether laminar shear stress (LSS) can modulate P2X4 receptor activity. Mouse P2X4 receptor was cloned and heterologously expressed in Xenopus laevis oocytes. In two-electrode-voltage-clamp experiments the application of ATP (100μM) produced a transient inward current that was decreased by about 50% upon a second ATP application, corresponding to the desensitization behavior of P2X4 receptors. In P2X4 expressing oocytes LSS (shear forces of ~5.1dynes/cm2) did not produce any effect. However, LSS modulated the response of P2X4 to ATP. With LSS (~5.1dynes/cm2) the desensitization of the current due to the second ATP application was diminished. Ivermectin (IVM), a compound which stabilizes the open state of P2X4 receptors, mimicked the effect of LSS (~5.1dynes/cm2), since there was no additional effect of LSS after pre-incubation with IVM detected. This indicates that LSS like IVM stabilizes the open state of the receptor, although the particular mechanism remains unknown. These data demonstrate that LSS modulates the activity of P2X4 receptors by eliminating the desensitization of the receptors in response to ATP probably by stabilizing the open state of the channel

Nitric Oxide Inhibits Highly Selective Sodium Channels and the Na+/K+-ATPase in H441 Cells

Nitric oxide (NO) is an important regulator of Na+ reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate and diethylammonium(Z)-1-(N, N-diethylamino) diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na+ channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na+/K+-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na+/K+-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na+/K+-ATPase a subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na+ reabsorption by interfering with the activity of highly selective Na+ channels and the Na+/K+-ATPase