Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.     

Pharmacological block of Ca2+-activated Cl− current in rat vascular smooth muscle cells in short-term primary culture

Ca²⁺-activated Cl^– currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl^– currents can be activated separately by Ca²⁺ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The effects of several Cl^– channel blockers and of spironolactone (a substance known to reduce internal Ca²⁺ loading) on both Cl^– and Ca²⁺ currents were examined. Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2-dicarboxylic acid (DPC) inhibited the Ca²⁺-activated Cl^– current (IC₅₀ values between 16.5 and 306 M) with no effects on the inward Ca²⁺ current and on internal Ca²⁺ loading (tested by measuring the Ca²⁺-activated K⁺ current). These results indicate that the inhibition of Cl^– current by these compounds is due to a direct interaction with the Cl^– channel. In contrast, spironolactone inhibited both K⁺ and Cl^– currents (IC₅₀=7.6 M) by reducing the amount of Ca²⁺ located in the internal stores, whereas the Cl^– current activated by Ca²⁺ current through T-type Ca²⁺ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl^– channels or intracellular Ca²⁺ stores.     

β-Adrenoceptor-mediated depolarization of the resting membrane in guinea-pig papillary muscles: changes in intracellular Na+, K+ and Cl− activities

Effects of -adrenergc stimulation on the membrane potential and intracellular Na⁺, K⁺ and Cl^– activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM–1 M produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect (pD₂) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 M)-induced depolarization was not blocked by tetrodotoxin (10 M), nifedipine (10 M), Cs⁺ (5 mM), Ba²⁺ (0.3 mM), amiloride (1 mM) or ouabain (10 M), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl^–-channel blocker. Intracellular K⁺ activity increased, whereas intracellular Na⁺ activity slightly decreased during the transient depolarization. Intracellular Cl^– activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl^– movement, rather than inward Na⁺ movement, may be involved in the -adrenoceptor-mediated membrane depolarization.     

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na(+)-K(+) pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na(+)-K(+) pump alpha(2) subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the alpha(2) subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na(+)-K(+) pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the alpha(1) subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the alpha(2), the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the alpha subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.     

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca²⁺ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl^– efflux, which could be strongly inhibited by the Cl^– channel blockers flufenamic acid and niflumic acid. Removal of Mg²⁺ or Ba²⁺ from the solutions had the same effects as removing Ca²⁺. The reversal potential of –12 mV also indicated that Cl^– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca²⁺ concentration of about 20 M showed that quite low doses of extracellular Ca²⁺ profoundly influence the electrical properties of the oocyte membrane.     

Impact of mechanical stress on ion transport in native lung epithelium (Xenopus laevis): short-term activation of Na+, Cl− and K+ channels

Epithelia, in general, and the lung epithelium, in particular, are exposed to mechanical forces, but little is known about their impact on pulmonary ion transport. In our present study, we employed transepithelial ion transport measurements on Xenopus lung preparations using custom-built Ussing chambers. Tissues were exposed to mechanical stress by increasing the water column (5 cm) at one side of the tissues. Apical exposure to hydrostatic pressure significantly decreased the short circuit current (I (SC): 24 +/- 1%, n = 152), slightly decreased the transepithelial resistance (R (T): 7 +/- 2%, n = 152), but increased the apical membrane capacitance (C (M): 16 +/- 6%, n = 9). The pressure-induced effect was sensitive to Na(+) (amiloride), Cl(-) (DIDS, NFA, NPPB) and K(+) channel blockers (Ba(2+), glibenclamide). Further on, it was accompanied by increased extracellular ATP levels. The results show that mechanical stress leads to an activation of Na(+), Cl(-), and K(+) conductances in a native pulmonary epithelium resulting in a net decrease of ion absorption. This could be of considerable interest, since an altered ion transport may contribute to pathophysiological conditions, e.g., the formation of pulmonary edema during artificial ventilation.     

Contraction-induced changes in skeletal muscle Na+,K+ pump mRNA expression – importance of exercise intensity and Ca2+-mediated signalling

Aim: To investigate if exercise intensity and Ca²⁺ signalling regulate Na⁺,K⁺ pump mRNA expression in skeletal muscle. Methods: The importance of exercise intensity was evaluated by having trained and untrained humans perform intense intermittent and prolonged exercise. The importance of Ca²⁺ signalling was investigated by electrical stimulation of rat soleus and extensor digitorum longus (EDL) muscles in combination with studies of cell cultures. Results: Intermittent cycling exercise at ∼85% of VO_2peak increased (P < 0.05) α1 and β1 mRNA expression ∼2-fold in untrained and trained subjects. In trained subjects, intermittent exercise at ∼70% of VO_2peak resulted in a less (P < 0.05) pronounced increase (∼1.4-fold; P < 0.05) for α1 and no change in β1 mRNA. Prolonged low intensity exercise increased (P < 0.05) mRNA expression of α1 ∼3.0-fold and α2 ∼1.8-fold in untrained but not in trained subjects. Electrical stimulation of rat soleus, but not EDL, muscle increased (P < 0.05) α1 mRNA expression, but not when combined with KN62 and cyclosporin A incubation. Ionomycin incubation of cultured primary rat skeletal muscle cells increased (P < 0.05) α1 and reduced (P < 0.001) α2 mRNA expression and these responses were abolished (P < 0.05) by co-incubation with cyclosporin A or KN62. Conclusion: (1) Exercise-induced increases in Na⁺,K⁺ pump α1 and β1 mRNA expression in trained subjects are more pronounced after high- than after moderate- and low-intensity exercise. (2) Both prolonged low and short-duration high-intensity exercise increase α1 mRNA expression in untrained subjects. (3) Ca²⁺_i regulates α1 mRNA expression in oxidative muscles via Ca²⁺/calmodulin-dependent protein kinase (CaMK) and calcineurin signalling pathways.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Thick ascending limb: the Na+:K+:2Cl− co-transporter, NKCC2, and the calcium-sensing receptor, CaSR

The thick ascending limb of Henle’s loop is a nephron segment that is vital to the formation of dilute and concentrated urine. This ability is accomplished by a consortium of functionally coupled proteins consisting of the apical Na⁺:K⁺:2Cl⁻ co-transporter, the K⁺ channel, and basolateral Cl⁻ channel that mediate electroneutral salt absorption. In thick ascending limbs, salt absorption is importantly regulated by the calcium-sensing receptor. Genetic or pharmacological disruption impairing the function of any of these proteins results in Bartter syndrome. The thick ascending limb is also an important site of Ca²⁺ and Mg²⁺ absorption. Calcium-sensing receptor activation inhibits cellular Ca²⁺ absorption induced by parathyroid hormone, as well as passive paracellular Ca²⁺ transport. The present review discusses these functions and their genetic and molecular regulation.

Small-conductance Cl− channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

The present study demonstrates the activation of Cl^– channels in HT₂₉ cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca²⁺, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast wholecell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with halfmaximal effects at 0.4 umol/l for ATP, 60 pmol/l for neurotensin and 0.8 mol/l for carbachol. The depolarization, which was caused by Cl^– conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295±32 pS to 1180±271 pS (ATP; 10 mol/l, n=21) and 192±37 pS to 443±128 pS (neurotensin; 1 nmol/l, n=8). The effects of the agonists could be mimicked by ionomycin (0.2 umol/l), suggesting that an increase in intracellular Ca²⁺ was responsible for the activation of Cl^– channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10±0.8 mV and the cell-attached conductance increased from 228±29 pS to 410±65 (n=26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments.5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) did not prevent the effects of Ca²⁺-increasing hormones and of hypotonic solutions. An increase in Cl^– conductance was also induced by 8-Br-cGMP (1 mmol/l) but not by heat-stable Escherichia coli toxin. In contrast to their conductance-increasing effects in CAN patches, the different agonists and cell swelling did not activate resolvable single channels in these cell-attached membranes. This indicates that the Cl^– channels involved have a single-channel conductance too small ( 4 pS, 150 Hz) to be resolved by our techniques.     

GTP γ causes contraction of skinned frog skeletal muscle via the DHP-sensitive Ca2+ channels of sealed T-tubules

We have investigated the involvement of G-proteins in excitation-contraction coupling of fast-twitch skeletal muscle, using a fibre preparation designed to retain intact T-tubules and sarcoplasmic reticulum. The nonhydrolysable analogue of guanosine triphosphate, GTP S (50–500 M) caused a strong, transient isometric contraction in this preparation. Reduction of ethylene-bis(oxonitrilo) tetraacetete (EGTA) in the sealed T-tubules from 5 mM to 0.1 mM lowered the threshold to GTP S and removal of sodium reversibly raised it. The dihydropyridine (DHP) calcium channel antagonists nicardipine and nifedipine allowed a first contraction and then blocked subsequent GTP S action. The phenylalkylamine methoxyverapamil (D-600) did likewise, reversibly, at 10° C. The guanosine diphosphate analogue, GDP S, and procaine reversibly blocked the action of GTP S pertussis toxin also blocked it. Photolytic release of 40–100 M GTP S within 0.1 s from S-caged GTP S caused contraction after a latent period of 0.3–20 s. We conclude that GTP S can activate contraction in frog skeletal muscle via a route requiring both the integrity of the T-tubular DHP-sensitive calcium channel (DHPr) and the presence of sodium in the sealed T-tubules. We propose that in this preparation GTP S activates a G-protein, which in turn activates the DHPr as a calcium channel and releases stored calcium from within the sealed T-tubule. Implications of these results for the excitation-contraction coupling mechanism in skeletal muscle are discussed.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

Increase in intracellular Cl− concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells

In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl^– secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl^–]_i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP_V163S. Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl^–]_i (0–100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 µM) and forskolin (2 µM) increased [Cl^–]_i by 9.6±1.5 mM (n=35). Similarly, ATP (100 µM) increased [Cl^–]_i transiently by 9.5±2.2 mM (n=17). The increase in [Cl^–]_i was reduced by the Na⁺/K⁺/2 Cl^–-cortransporter-1 (NKCC1) blocker azosemide (100 µM), the CFTR blocker SP-303 (50 µM), the blocker of Ca²⁺-activated Cl^– channels DIDS (100 µM) or the ENaC blocker amiloride (10 µM). Changes in YFP_V163S fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl^–]_i following stimulation with secretagogues, which could participate in the inhibition of ENaC.     

Modulation of Ca2+ release through ryanodine receptors in vascular smooth muscle by protein kinase Cα

The role of protein kinase C (PKC) in Ca²⁺ release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca²⁺]_i transients. Application of the PKC inhibitor calphostin C caused [Ca²⁺]_i transients which were not blocked by nifedipine or by removing extracellular Ca²⁺ but were abolished after inhibition of the SR Ca²⁺–ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca²⁺]_i but no further [Ca²⁺]_i increase was seen with subsequent application of caffeine. Selective inhibition of PKCα with safingol blocked caffeine-induced [Ca²⁺]_i transients, but the PKCε inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCα, caffeine-induced [Ca²⁺]_i transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCα in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCα indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca²⁺-dependent PKCα isoform.     

Modification of wild-type and batrachotoxin-resistant muscle µ1 Na+ channels by veratridine

Biochemical evidence indicates that veratridine (VTD) and batrachotoxin (BTX) share a common binding site in Na+ channels. Under whole-cell voltage-clamp conditions, we examined this single receptor hypothesis by studying the VTD phenotype in BTX-resistant muscle Na+ channels, microl-I433K, N434K, L437K, F1579K, and N1584K. Derived from point mutations at segments D1-S6 and D4-S6, these mutant Na+ channels are resistant to 5 microM BTX when expressed in human embryonic kidney cells. In contrast to the wild-type phenotype, VTD at 200 microM elicits little or no maintained current during a test pulse at +50 mV, and little or no "tail" current after the test pulse in all BTX-resistant mutant channels. Paradoxically, VTD retains its ability to inhibit the peak Na+ current in BTX-resistant mutant Na+ channels. To explain these mutant phenotypes, we propose a two-step binding reaction scheme. An initial VTD-binding interaction with the Na+ channel results in the inhibition of peak current amplitude, and a second binding reaction results in the trapping of VTD within the D1-S6 and D4-S6 domain interface. The failure of BTX-resistant mutant Na+ channels to trap VTD suggests that segments of D1-S6 and D4-S6 form a common receptor for VTD and BTX.     

Effect of GABAergic and adrenergic agents on activity of Na+/K+ pump and Cl−-cotransport in somatic muscle cells of earthworm Lumbricus Terrestris

GABA, baclofen, epinephrine, and norepinephrine hyperpolarized the membrane of earthworm somatic cells. This effect was prevented by furosemide, removal of Cl⁻ from the medium, or activation of Na⁺/K⁺ pump by 3-fold increase external potassium concentration. It was hypothesized that GABA, baclofen, epinephrine, and norepinephrine stimulate Na⁺/K⁺ transport via specific receptor inputs, but their effect on resting potential can be realized only under conditions of working Cl⁻ symport. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 572–574, May, 2006