Dopamine regulation of amiloride-sensitive sodium channels in lung cells

Dopamine increases lung fluid clearance. This is partly due to activation of basolateral Na-K-ATPase. However, activation of Na-K-ATPase by itself is unlikely to produce large changes in transepithelial transport. Therefore, we examined apical and basolateral dopamine's effect on apical, highly selective sodium channels [epithelial sodium channels (ENaC)] in monolayers of an alveolar type 2 cell line (L2). Dopamine increased channel open probability (P(o)) without changing the unitary current. The D(1) receptor blocker SCH-23390 blocked the dopamine effect, but the D(2) receptor blocker sulpiride did not. The dopamine-mediated increase in ENaC activity was not a secondary effect of dopamine stimulation of Na-K-ATPase, since ouabain applied to the basolateral surface to block the activity of Na-K-ATPase did not alter dopamine-mediated ENaC activity. Protein kinase A (PKA) was not responsible for dopamine's effect since a PKA inhibitor, H89, did not reduce dopamine's effect. However, cpt-2-O-Me-cAMP, which selectively binds and activates EPAC (exchange protein activated by cAMP) but not PKA, increased ENaC P(o). An Src inhibitor, PP2, and the phosphatidylinositol-3-kinase inhibitor, LY-294002, blocked dopamine's effect on ENaC. In addition, an MEK blocker, U0126, an inhibitor of phospholipase A(2), and a protein phosphatase inhibitor also blocked the effect of dopamine on ENaC P(o). Finally, since the cAMP-EPAC-Rap1 pathway also activates DARPP32 (32-kDa dopamine response protein phosphatase), we confirmed that dopamine phosphorylates DARPP32, and okadaic acid, which blocks phosphatases (DARPP32), also blocks dopamine's effect. In summary, dopamine increases ENaC activity by a cAMP-mediated alternative signaling pathway involving EPAC and Rap1, signaling molecules usually associated with growth-factor-activated receptors.     

Biophysical and molecular properties of annexin-formed channels

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.     

Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.     

Bretylium opens mucosal amiloride-sensitive sodium channels

Addition of the quanternary ammonium compound, bretylium, to the outer surface of a frog skin leads to an increase in the potential difference and in the short circuit current across the skin. Bretylium does not have any effect when applied to the inside face of the frog skin. The effect of bretylium is dependent upon the presence of sodium ions in the outer medium; it is depressed when sodium is replaced by choline or potassium but not when lithium substitutes for sodium. The bretylium effect is blocked by the specific sodium channel blocker, amiloride. It is proposed that bretylium opens mucosal, amiloride-sensitive sodium channels.     

Bretylium opens mucosal amiloride-sensitive sodium channels

Addition of the quanternary ammonium compound, bretylium, to the outer surface of a frog skin leads to an increase in the potential difference and in the short circuit current across the skin. Bretylium does not have any effect when applied to the inside face of the frog skin. The effect of bretylium is dependent upon the presence of sodium ions in the outer medium; it is depressed when sodium is replaced by choline or potassium but not when lithium substitutes for sodium. The bretylium effect is blocked by the specific sodium channel blocker, amiloride. It is proposed that bretylium opens mucosal, amiloride-sensitive sodium channels.     

Beta-adrenergic regulation of amiloride-sensitive lung sodium channels

We investigated the mechanism by which cAMP increases sodium transport in lung epithelial cells. Alveolar type II (ATII) cells have two types of amiloride-sensitive, cation channels: a nonselective cation channel (NSC) and a highly selective channel (HSC). Exposure of ATII cells to cAMP, beta-adrenergic agonists, or other agents that increase adenylyl cyclase activity increased activity of both channel types, albeit by different mechanisms. NSC open probability (P(o)) increased severalfold when exposed to terbutaline, isoproterenol, forskolin, or cAMP analogs without any change in NSC number. In contrast, terbutaline increased HSC number with no significant change in HSC P(o). For both channels, the effect of terbutaline was blocked by propranolol and H-89, suggesting a protein kinase A (PKA) requirement for beta-adrenergic-induced changes in channel activity. Terbutaline increased cAMP levels in ATII cells, but intracellular calcium also increased. Calcium sequestration with BAPTA blocked beta-adrenergic-induced increases in NSC P(o) but did not alter HSC activity. These observations suggest that beta-adrenergic stimulation increases intracellular cAMP and activates PKA. PKA increases HSC number and increases intracellular calcium. The increase in calcium increases NSC P(o). Thus increased cAMP levels are likely to increase lung sodium transport regardless of which channel type is present.     

Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations

Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-microm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2+/-2.5 and 22+/-3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 microM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31+/-0.19 to 0.60+/-0.21 (P<0.001). The D1 receptor antagonist SCH-23390 (10 microM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.     

Activation of large conductance sodium channels upon expression of amiloride-sensitive sodium channel in Sf9 insect cells.

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na(+) reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of alpha-ENaC, beta-ENaC, and gamma-ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of alpha-rENaC and alphabetagamma-rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the alpha-rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in alphabetagamma-rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

alpha -Adrenergic receptors regulate human lymphocyte amiloride-sensitive sodium channels

Two independentsignal transduction pathways regulate lymphocyte amiloride-sensitivesodium channels (ASSCs), one utilizing cAMP as a second messenger andthe other utilizing a GTP-binding protein. This implies that two plasmamembrane receptors play a role in the regulation of lymphocyte ASSCs.In this study, we tested the hypothesis that₁- and₂-adrenergic receptorsindependently regulate lymphocyte ASSCs via the two previouslyidentified second messengers. Direct measurements indicated thatnorepinephrine increased lymphocyte cAMP and activated ASSCs. The₂-specific inhibitor,yohimbine, blocked this activation, thereby linking ₂-adrenergic receptors to ASSCregulation via cAMP. The₁-specific ligand, terazosin,acted as an agonist and activated lymphocyte ASSCs but inhibited ASSCcurrent that had been preactivated by norepinephrine or8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on thelymphocyte whole cell ASSC currents preactivated by treatment withpertussis toxin. This finding indirectly links ₁-adrenergic receptors tolymphocyte ASSC regulation via GTP-binding proteins. Terazosin had nodirect inhibitory or stimulatory effects on ,,-endothelialsodium channels reconstituted into planar lipid bilayers and expressedin Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both₁- and₂-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins andcAMP, respectively.

     

Biophysical properties of epithelial water channels

The biophysical models describing the structure of water pores or channels have evolved, during the last forty years, from a pure 'black box' approach to a molecular based proposal. The initial 'sieving pore' in which water and other molecules were moving together was replaced by a more restrictive model, where water is moving alone in a 'single file' mode. Aquaporins discovery and cloning [G.M. Preston, T.P. Carroll, W.B. Guggino, P. Agre, Science 256 (1992) 365] leaded to the 'hour-glass model' and other alternative proposals, combining information coming from molecular biology experiments and two dimensional crystallography. Concerning water transfers in epithelial barriers the problem is quite complex, because there are at least two alternative pathways: paracellular and transcellular and three different driving forces: hydrostatic pressure, osmotic pressure or 'transport coupled' movements. In the case of ADH-sensitive epithelia it is more or less accepted that regulated water channels (AQP2), that can be inserted in the apical membrane, coexist with basolateral resident water channels (AQP3). The mechanism underlying the so-called 'transport associated water transfer' is still controversial. From the classical standing gradient model to the ion-water co-transport, different hypothesis are under consideration. Coming back to hormonal regulations, other than the well-known regulation by neuro-hypophysis peptides, a steroid second messenger, progesterone, has been recently proposed [P. Ford, G. Amodeo, C. Capurro, C. Ibarra, R. Dorr, P. Ripoche, M. Parisi, Am. J. Physiol. 270 (1996) F880].     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

Immunocytochemical localization of amiloride-sensitive sodium channels in the lower intestine of the hen

We have used polyclonal antibodies generated against purified bovine renal amiloride-sensitive Na⁺ channels to localize amiloride-sensitive Na⁺ channels within the lower intestine (colon and coprodeum) of the hen. These antibodies cross-reacted with two polypeptides exhibiting M_r's of 235 and 150 kDa on immunoblots of detergent-solubilized apical membrane fractions from both the colon and coprodeum. The apparent molecular masses of theses polypeptides are in agreement with the M_r's of 2 of the subunits of the renal high amiloride-affintiy Na⁺ channel, namely the and the (=amiloride binding) subunits. The cellular distribution of Na⁺ channels was determined by immunoperoxidase and indirect immunofluorescence cytochemical techniques. The apical (luminal) membrane and cytoplasm of villar principal cells in both colon and coprodeum exhibited immunoreactivity, whereas goblet cells were nagative. Both principal and goblet cells of the crypts were also negative. We conclude that the amiloride-sensitive Na⁺ channels are localized to the principal cells of the intestinal villi and that these cells are responsible for intestinal Na⁺ absorption.     

Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.     

Functional properties of sodium channels in cholesterol-depleted K562 cells

In the present paper, functional properties of nonvoltage-gated sodium channels in K562 cells were studied after cholesterol depletion, i.e., under conditions of the destruction of microdomains (rafts). For cholesterol depletion, cells were incubated with methyl-beta-cyclodextrin (MbCD), an oligosaccharide that selectively binds sterols. Single currents through sodium channels were recorded in cell-attached and inside-out experiments using the patch-clamp technique. After incubation with MbCD (2.5 or 5 mM), the activation of sodium channels in response to cytochalasin B or D was observed in both native cells and membrane fragments. Biophysical characteristics of sodium channels in cholesterol-depleted K562 cells were close to those in control; unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels in the modified cells. These data imply that sodium channels in K562 cells are not associated with cholesterol-rich membrane microdomains. Possible mechanisms of the interaction of the plasma membrane and the cortical cytoskeleton are discussed.