Endothelin-1 inhibitsl-type Ca currents enhanced by isoproterenol in guinea-pig ventricular myocytes

To investigate the action of Endothelin-1 (ET-1) on L-type Ca currents (I _Ca,L) in guinea-pig ventricular cells, whole-cell currents were recorded at 36-37° C in enzymatically isolated myocytes. ET-1 ( 10 nM) suppressed the basal I_ca,L to 79 ± 8% of control at 20 nM. Bath application of isoproterenol (ISO; 10 nM) enhanced I_Ca,L to 192±28% with about a – 10-mV shift of its relationship with membrane potential. ET-1 concentration dependently inhibited this ISO-enhanced I_Ca,L with a half-maximally inhibitory concentration (IC₅₀) of 168 pM. The inhibitory actions of ET-1 were antagonised by BQ-123 (300 nM), cyclo(d-Asp-l-Pro-d-Val-l-Leu-d-Trp), a specific ETA receptor antagonist. Histamine-enhancedI _Ca,L was also suppressed by ET-1, butI _Ca,L potentiated by internal adenosine 3,5-cyclic monophosphate (cAMP) was unaffected. Preincubation of myocytes with pertussis toxin (PTX, at 5 g/ml for >60 min at 36° C) completely occluded the ET-1 action. Thus, stimulation of ET_A receptors by subnanomolar ET-1 inhibitsI _Ca,L via PTX-sensitive G-proteins.     

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (I _Ca,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal I _Ca,L at concentrations in the range 0.05–10 M, without previous -adrenergic stimulation. The ACh-induced reduction of I _Ca,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal I _Ca,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on I _Ca,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Calcium transients which accompany the activation of sodium current in rat ventricular myocytes at 37°C: a trigger role for reverse Na-Ca exchange activated by membrane potential?

We investigated the role of the fast sodium current (I _Na) in triggering Ca release from the sarcoplasmic reticulum (SR), using adult rat left ventricular myocytes, loaded with Fura-2 to measure intracellular Ca (Ca_i), which were whole-cell patch-clamped at 35–37°C. Before each test pulse, a series of 400-ms conditioning pulses to +10 mV were applied to establish a constant level of SR Ca load. Pulses were applied every 15 s. A test pulse from –80 mV to –50 mV elicited a rapidI _Na and a phasic Ca_i transient. When the solution perfusing a myocyte was rapidly switched for 15 s before a test pulse to one containing the L-type Ca channel blocker nifedipine (20 M), the test pulse still activatedI _Na and a phasic Ca_i transient, the amplitude of which was not significantly different from control (P>0.05;t-test). When a rapid switch to 20 M nifedipine plus 30 M tetrodotoxin (TTX) was made 15 s before a test pulse, bothI _Na and the Ca_i transient were completely abolished (n=6). When a switch was made to Na-free (Li) solution, which contained 20 M nifedipine to block L-type Ca current,I _Ca,L, there was no significant difference in the Ca_i transient amplitude from that of control (P>0.05;n=6). Brief depolarising test pulses (–80 mV to +20 mV, 10 ms duration) to simulate membrane potential escape also elicited a Ca_i transient which attained 90.0% (±2.8%;n=7) of the Ca_i transient activated by a conditioning pulse to +10 mV. The Ca_i transient with a brief pulse was not significantly affected by application of 20 M nifedipine (P>0.05), but adding TTX with nifedipine reduced the Ca_i transient amplitude to 76.9% (±6.8%;P<0.02;n=8). In four cells, the Ca_i transient remaining in the presence of nifedipine plus TTX was abolished by adding 5 mM Ni. These data are consistent with voltage escape during activation ofI _Na leading to a trigger Ca entry via a mechanism other than L-type Ca channels or subsarcolemmal Na accumulation with reverse Na-Ca exchange. The block by Ni of the Ca_i transient suggests that a brief membrane potential escape might directly activate reverse mode Na-Ca exchange to trigger SR release, and this mechanism would seem to account largely for the Ca_i transient which accompaniesI _Na in rat myocytes, under these experimental recording conditions.     

A combined study of sodium current and T-type calcium current in isolated cardiac cells

Sodium currents (I _Na) and T-type calcium currents (I _Ca,T) of isolated guinea-pig ventricular myocytes were recorded using the whole-cell voltage-clamp technique. Separation of the two currents was obtained by using the difference current method in the presence and absence of 2 mM extracellular Na (Na_o). Time to peak and the time constant of inactivation of. I_Na were about 5 times faster than that of I _Ca,T (test potential –30 mV). and I _Ca,T had an activation range positive to –50 mV, were inactivated at –50 mV, and their current/voltage relationships peaked at –22.3±1.8 mV (n=18) and –29.3±0.5 mV (n=18) respectively, with a reversal potential of +40.3±4 mV (n=18) and +30±10 mV (n=18), respectively [2 mM Na_o; 5.4 mM extracellular Ca (Ca_o)]. I _Na was blocked by 30 M tetrodotoxin (TTX), 500 M lidocaine, partly inhibited by 1 mM amiloride, but not affected by 100 M nickel (Ni). I _Ca,T was neither affected by 30 M TTX nor 500 M lidocaine, but blocked by 100 M Ni, 1 mM amiloride, 10 M R 56865 and use-dependently reduced by 5 M flunarizine. Adenosine (500 M) affected neither I _Na nor I _Ca,T, whereas 1 M isoprenaline did not affect I _Ca,T, but slightly increased I _Na. Our results demonstrate that the characteristics of I _Ca,T are not affected by the concomitant activation of I _Na, and vice versa. We conclude that I _Ca,T are not Ca currents through Na channels.     

Noise analysis of cAMP-stimulated Na current in frog colon

The effects of oxytocin and cAMP on the electrogenic Na⁺-transport in the short-circuited epithelium of the frog colon (Rana esculenta, Rana temporaria) were investigated. Oxytocin (100 mU · ml^–1) elevated the shortcircuit current (I _sc) transiently by 70% whereas cAMP (1 mmol · l^–1) elicited a comparable sustained response. The mechanism of the natriferic action of cAMP was studied by analysing current fluctuations through apical Na⁺-channels induced by amiloride or CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamid). The noise data were used to calculate Na⁺-channel density (M) and single apical Na⁺-current (i _Na).i _Na-Values obtained with amiloride and CDPC were 1.0±0.1 pA (n=5) and 1.1±0.2 pA (n=6) respectively and unaffected by cAMP. On the other hand, cAMP caused a significant increase in M from 0.23±0.08 m^–2 (n=5) to 0.49±0.17 m^–2 (n=5) in the amiloride experiments. In our studies with CDPC we obtained smaller values for M in control (0.12±0.04 m^–2;n=6) as well as during cAMP treatment (0.19±0.06 m^–2;n=6). However, the cAMP-induced increase in M was also significant. We conclude that cAMP stimulates Na⁺-transport across the frog colon by activating silent apical Na⁺-channels. Thus, the mechanism of regulation of colonic Na-transport in frogs differs considerably from that in other vertebrates as mammals and birds.     

Potassium currents in rat colonic smooth muscle cells and changes during development and aging

In a previous study on freshly isolated single smooth muscle cells from the circular layer of the rat distal colon, we reported that the L-type Ca²⁺ current density increased during development and gradually declined with further aging [ZI Xiong, N Sperelakis, N Noffsinger, C Fenoglio-Preiser (1993) Am J Physiol 265: C617–C625]. Since K⁺ current plays a key role in controlling excitability of the cells and hence the motility of the colon, in the present study the voltage-gated K⁺ channel currents, (I _K) were investigated using the whole-cell voltage-clamp technique in colonic myocytes from rats of different ages. A Ca²⁺-sensitive K⁺ current [I _K(Ca)] and two kinds of Ca²⁺-insensitive outward K⁺ currents were identified and characterized. I _K(Ca) was recorded at potentials more positive than –40 mV in Ca²⁺-containing bath solution, and was blocked by Ca²⁺ channel antagonists and tetraethylammonium ion (TEA⁺). After removing Ca²⁺ from the bath solution and using a high ethylenebis(oxonitrilo)tetraacetate (EGTA, 4 mM) concentration in the pipette, two types of Ca²⁺-insensitive I _K were recorded. The first and faster component was usually activated at potentials more positive than –50 mV, and was more sensitive to 4-aminopyridine (4-AP). In contrast, the second and slower (delayed) component was activated at potentials more positive than –30 mV, and was more sensitive to TEA. The total density of the Ca²⁺-insensitive I _K component decreased dramatically during the neonatal period: from 32.2±3.2 pA/pF in 3-day-old rats to 17.8±2.6 pA/pF in 40-day-old rats; there was no further decline during aging (up to 480 days). This total I _K decline was due primarily to a decline in the 4-AP-sensitive component; the TEA-sensitive component remained unchanged at all ages. The possible physiological implications of the decline in density of the 4-AP-sensitive I _K component during neonatal development are discussed.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

Effects of calcitonin gene-related peptide on membrane currents in mammalian cardiac myocytes

We examined the effects of calcitonin gene-related peptide (CGRP) on the membrane currents of single atrial and ventricular cells of guinea pig heart. The tightseal whole-cell voltage-clamp technique was used. In atrial cells, like isoproterenol, CGRP increased the L-type Ca channel current (I _Ca.L) in a concentration-dependent manner. Human CGRP-(8-37), a putative CGRP receptor antagonist, completely abolished the CGRP-induced increase of I _Ca.L. Although the effects of CRGP were similar to those of isoproterenol, propranolol, a -adrenergic receptor antagonist, did not affect the CGRP-induced increase of I _Ca.L. After I _Ca.L had been maximally activated by isoproterenol (2 M) or intracellular cyclic adenosine 5-monophosphate (100 M), CGRP failed to increase I _Ca.L. Acetylcholine antagonized the effects of CGRP on I _Ca.L. Unlike the effects on atrial cells, CGRP had no significant effects on the membrane currents of ventricular myocytes. Thes results indicate that CGRP increases I _Ca.L via adenylate cyclase activation by binding to specific membrane receptors in cardiac atrial myocytes. Furthermore, CGRP receptors are expressed in atrial cells but probably not in ventricular cells.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Currents carried by sodium ions through transient calcium channels in clonal GH3 pituitary cells

Summary The transient or T-type Ca channel currents of voltage-clamped clonal (GH₃) pituitary cells were studied after blockade of Na and K channels. Removal of Ca ions from the bathing medium abolished the Ca currents and allowed Na ions to convey current through the transient channels. These Na currents (I_Na,t) were activated at threshold potentials of ca. –85 mV, reached peak amplitudes (up to 2,000 pA) at –60 mV, and were 50% inactivated at holding membrane potentials of –110 to –120 mV. The time constant for inactivation of the maximal I_Na,t was 12±2.4 msec (compared with 22.1±0.85 msec for maximal I_{Ca, t}). The I_Na,t was insensitive to tetrodotoxin (1 M) and to nimodipine (200 nM) but was abolished by 100 M external [Ca²⁺]. It is suggested that high affinity Ca binding sites in the channel modulate the ion selectivity and the current flow through T-type Ca channels.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Structure and mechanics of growing arterial microvessels from hypertrophied urinary bladder in the rat

Rat bladder hypertrophy, induced by a partial ligation of the urethra, was used to study the accompanying changes of microvascular smooth muscle mechanics, pharmacology and morphology. A segment of a microarterial vessel to the bladder was taken from a defined anatomical location and studied in a wire myograph in vitro at the length for maximal isometric force development (L _max). After 10 days of ligation, bladder hypertrophy resulted in a microvascular growth response compared to non-operated controls which was characterized by (i) an increase of the calculated diameter at L _max from 134±5 m to 222±19 m; (ii) an increase of the media thickness from 22.4±1.9 m to 32.2±3.0 m; (iii) an increase of the active tension from 1.42±0.28 mN/mm to 3.06±0.33 mN/mm; (iv) no change of the wall/lumen ratio (from 0.83±0.10 to 0.79±0.15). Normalized length/force relations (active, passive and total) did not differ significantly between microarteries from control and hypertrophic bladders. Microvascular smooth muscle growth was also associated with a decreased sensitivity to K⁺-induced depolarization and an increased sensitivity to ₁-adrenergic stimulation. No differences were noted regarding the Ca²⁺ sensitivity of force during K⁺-induced depolarization. The results suggest that microvascular growth (1) is immediately and positively influenced by the organ growth; (2) results in a functional resetting of the microvascular segments towards larger diameters without gross morphological or mechanical alterations; and (3) is accompanied by pharmacological alterations of the smooth muscle reactivity.     

Electrical properties and electrolyte transport in bovine tracheal epithelium: Effects of ion substitutions,transport inhibitors and histamine

The active transport of Na⁺ and Cl^– across the tracheal epithelium of the cow was investigated in vitro, using the short-circuit technique, by means of ion substitutions, transport inhibitors and by measuring²²Na and³⁶Cl fluxes. Under short-circuit conditions, short-circuit current (i _o) was 168±5A cm^–2 (mean±SEM,n=30), i.e. 6.2±0.2 Eq h^–1cm^–2 and resistance (R) was 248±10cm². Net Na⁺ flux toward the submucosa (J _{Na net} ^L-S ) and net Cl^– flux toward the lumen (J _{Cl net} ^S-L ) were of the same magnitude, i.e. 2.7±0.2 and 2.9±0.2 Eq h^–1 cm^–2, respectively. The permeability coefficients were 3.6·10^–6 forP _Na and 7.8·10^–6 cm s^–1 forP _Cl. Under open-circuit conditions, the transepithelial electrical potential difference () was 43±2 mV (lumen negative,n=20).J _{Na net} ^L-S andJ _{Cl net} ^S-L were close to zero.Bilateral substitution of Cl^– with SO ₄ ^2– or isethionate, or administration of furosemide 5·10^–3M or bumetanide 10^–4M in the submucosal bathing medium produced a 40 to 50% decrease ini ₀; furosemide abolishedJ _{Cl net} ^S-L . Bilateral substitution of Na⁺ with choline or Mg²⁺, or addition of ouabain 10^–4M to the submucosal bath abolishedi ₀;J _{Na net} ^L-S andJ _{Cl net} ^S-L were suppressed by ouabain. Amiloride 10^–4M in the luminal bath reducedi ₀ by 23% and diminishedJ _{Na net} ^L-S by 80%. Histamine 10^–4M, added to the submucosal bathing medium, reducedJ _{Na net} ^L-S and increasedJ _{Cl net} ^S-L , under short-circuit conditions. In open-circuit conditions, histamine had little effect on ion fluxes. This substance had no effect on the electrical properties, as shown previously.These results are consistent with the model proposed by Silva et al. [20] for a Cl^–-secreting, Na⁺-reabsorbing epithelium.Supported by the Swiss National Foundation (SNF), grant no. 3.588-0.79     

The Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP production

The role of glycolysis and oxidative phosphorylation in providing the ATP for the cardiac Na⁺/K⁺ pump was studied in cardioballs from sheep Purkinje fibres. As an indicator of the pump activity, the pump current I _p was measured at -20mV and 30–33° C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose I _p reached a maximum within 8 min and declined to 50% of this value within 27 min after gaining access to the cell interior. Perfusion with an ATP- and glucose-free medium barely enhanced the I _p decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 M or 20 M) moderately accelerated the effect of the ATP- and glucose-free pipette solution. Addition of 2 mM iodoacetic acid (an inhibitor of glycolysis) to the latter medium further enhanced the I _p decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of I _p to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast I _p decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 M CCCP still hastened the fast I _p decrease slightly. This effect of complete metabolic inhibition was reversed by switching to an inhibitor-free pipette solution containing 15 mM ATP. It is concluded that the Na⁺/K⁺ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP synthesis.This work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe Konzell)     

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig)

The effect of arginine-vasopressin (AVP, 0.1 M) on elementary Ca²⁺ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl₂ as the charge carrier. At a constant potential of –30 mV, bath applied AVP increased the channel openness (NP _o) by a factor of 4.7±3.0 (mean±SD, n=9), the effect resulted from an increase in the frequency of opening (factor 2.5±0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (_o1=0.8±0.4 ms), a double exponential distribution was obtained during AVP (_o1=0.8±0.5 ms, _o2=7.5±0.7 ms). The Ca²⁺ agonist BAYk8644 (1 M) changed the open time distribution similarly to AVP (_o1=1.0±0.5 ms, _o2=9±2.8 ms). With 1 M BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0±1 (n=6). The results are compatible with the idea that AVP can change the gating of L-type Ca²⁺ channels from mode 1 to mode 2.     

The thyroid cell monolayer in culture

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (V _ab), short circuit current (I _sc) and transepithelial resistance were respectively 12±1 mV (apical side negative), 3.8±0.2 A cm^–2 and 3250±214 cm² (mean±SEM,n=75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (K _m=0.2 M) and by basal ouabain (K _1/2=0.3 M). Amphotericin B (5–25 g/ml) added to the apical bath increasedI _sc suggesting an apical rate-limiting step. Step by step replacement of choline by Na⁺ in a Na⁺-free medium resulted in a progressive increase inV _ab andI _sc with half maximal effect at 20±1 mM Na⁺. Thyrotropin (TSH) increasedI _sc andV _ab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 U/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na⁺, K⁺-ATPase as the electromotive force.     

Modulation of manganese currents by 1, 4-dihydropyridines,isoproterenol and forskolin in rabbit ventricular cells

Although often used as a Ca²⁺ channel blocker, Mn²⁺, in fact, permeates through Ca²⁺ channels. Under Na⁺-free conditions, depolarizing pulses evoked slowly-decaying Mn²⁺ currents (I_Mn). Maximal I_Mn densities in the presence of 5 and 20 mM Mn²⁺ were 0.42±0.12 pA/pF (mean±SEM, n=17) and 1.23±0.10 pA/pF (n=40), respectively. At 5 mM, the ratio of maximal amplitude of I_Mn to that of the Ca²⁺ current (I_Ca) was 0.079±0.009 (n=8). I_Mn elicited from a holding potential of –50 mV was depressed by nitrendipine (1 µM) by ~70%. Nitrendipine (0.3 µM) shifted the steady-state inactivation curve to more negative potentials and shifted the potential for half-maximal inactivation (E_0.5) from 1.3 to –8.8 mV and also decreased the time constant of decay of I_Mn at 20 mV from 986.2 to 167.9 ms. BAY K 8644 (1 µM), isoproterenol (10 µM) and forskolin (10 µM) all increased I_Mn and shifted the current/voltage (I/V) relationship to more negative potentials. The small, slowly-inactivating I_Mn is thus modulated by dihydropyridine Ca²⁺ channel modulators and cyclic AMP-mediated phosphorylation in a manner similar to other L-type Ca²⁺ channel currents. L-type Ca²⁺ channels are involved in the regulation of intracellular [Mn] in ventricular myocytes.     

Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells

Intracellular adenosine triphosphate (ATP)-inhibited K⁺ currents (I _{K, ATP} ) in canine coronary artery smooth muscle cells were characterized in the wholecell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATP_i) showed little detectable whole-cell current at potentials more negative than –30 mV. However, cells dialysed with ATP_i-free solutions developed a time- and voltage-independent current which reached a maximum of 132±25 pA at –40 mV about 10 min following patch rupture. After run-up, the current showed little run-down. Concentration-dependent inhibition by ATP_i yielded an inhibition constant (K _i of 350 M and a Hill coefficient of 2.3. In ATP_i-free solutions, the large current at –40 mV was reduced by glibenclamide with aK _i of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATP_i solutions, the small current at –40 mV was increased by P-1075 from 8±2 pA to 143±33 pA, with a dissociation constant (K _d) of 0.16 M and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATP_i depletion or external application of the channel opener P-1075 was similar with slope conductances of 81±10 pS/pF and 76±13 pS/pF respectively in the potential range of –90 to –40 mV. External Ca²⁺ had no effect on this current. Finally, in 1 mM ATP_i, 20 and 50 M adenosine increased the current slope conductance by 36±15% and 73±10% respectively between –90 to –40 mV. TheI _{K, ATP} although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.     

Conductance of the slow inward channel in the rabbit sinoatrial node

The elementary conductance of the slow inward current channel in the rabbit sinoatrial node was measured by analysis of the current fluctuations. The preparations were voltage-clamped to –30 mV, where d and f of the slow inward current (I_si) intersect. In the presence of Ba, which increases I_si and decreases outward currents, a small steadystate component of I_si could be detected.The fluctuations of the current in 10 mM Ba were smaller due to the block of the outward channels. They were further reduced after the inhibition of I_si by the Ca channel blocker D600. The spectral power density distribution of the current fluctuations originating from I_si could be fitted at frequencies <30 Hz with a single Lorentzian which was attributed to the inactivation process. The corner frequency was 5.28 ±1.16 Hz (n=10), corresponding to an average open time of the single channel of about 30 msec at –30mV. The single channel conductance was determined to 6.50±3.15 pS (S.D.,n=10).This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, Membranforschung, project G