Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes

 In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, ”Myristyl- (Myr-) FRCRCFa”. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35–37°C. The Na-Ca exchange current (I _Na-Ca), L-type calcium current (I _Ca,L), inward rectifier potassium current (I _K1) and delayed rectifier potassium current (I _K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, I _Na-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of –40 mV, between +80 and –120 mV (ramp velocity 0.1 V s^–1). In untreated cells, I _Na-Ca at +60 mV was 7.1±0.6 pA/pF and at –100 mV was –2.7±0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 μM Myr-FRCRCFa, I _Na-Ca was reduced to 4.2±0.3 pA/pF at +60 mV and –1.5±0.2 pA/pF at –100 mV (P<0.02; n=7). After incubation with 20 μM Myr-FRCRCFa for 1 h, I _Na-Ca at both potentials was further reduced (2.3±0.8 pA/pF at +60 mV; –0.9±0.3 pA/pF at –100 mV; P<0.008 compared with control; n=4). Under selective recording conditions for I _Ca,L, there was little difference in I _Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the I _Ca,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. I _K1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I _K, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of I _Na-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an I _Na-Ca blocker. I _Ca,L, I _K1 and I _K were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger. Received: 6 February 1997 / Received after revision: 8 April 1998 / Accepted: 9 April 1998     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

NH4 + conductance in Xenopus laevis oocytes

 Superfusing Xenopus laevis oocytes with NH₄Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of –70 mV. In paired experiments (n=22), the NH₄Cl-induced peak current was –293±94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to –523±196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH₄Cl-induced inward current, membrane conductance at –70 mV doubled and the zero-current potential changed from +3.3±9.4 mV to –22.0±8.0 mV (n=22) in the presence of NH₄Cl during exposure to a hypoosmolar solution. In the absence of NH₄Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K⁺ channel. Membrane conductance in the presence of NH₄Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH₄ ⁺ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K⁺ channel. Received: 20 July 1998 / Received after revision and accepted: 19 October 1998     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

Modulation of the transient outward K+ current by inhibition of endothelin-A receptors in normal and hypertrophied rat hearts

Inhibition of endothelin-A (ET_A) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ET_A-receptor inhibition on the development of regional alterations of the transient outward K⁺ current (I _to) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague–Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ET_A-receptor antagonist darusentan (LU135252, 35 mg [kg body weight]⁻¹ day⁻¹) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight (p < 0.001) and caused a significant reduction in I _to (at +40 mV) in epicardial myocytes (19.5 ± 1.2 pA pF⁻¹, n = 32 vs 23.2 ± 1.2 pA pF⁻¹, n = 35, p < 0.05). Darusentan further reduced I _to in AS (15.4 ± 1.3 pA pF⁻¹, n = 37, p < 0.05) and sham-operated animals (19.8 ± 1.6 pA pF⁻¹, n = 48, ns.). The effects of AS and darusentan on I _to were significant and independent as tested by two-way analysis of variance. I _to was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I _to in the epicardial region of the left ventricle but that ET_A-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy.     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Low-amiloride-affinity Na+ channel in the epithelial cells isolated from the endolymphatic sac of guinea-pigs

 By using the whole-cell patch-clamp technique, an amiloride-sensitive Na⁺-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was –41.7±8.4 mV (n = 22). Application of amiloride at a concentration of 20 μM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9±6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4±8.3 mV (n = 22). At holding potential of –70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 μM and 50 μM, with an inhibitory constant (K _i) of 1.3±0.4 μM (n = 7) and 1.5±0.3 μM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na⁺. Superfusion with 20 μM amiloride induced a significant reduction of the inward current, shifted the reversal potential from –39.4±8.8 mV to –60.4±10.5 mV (n = 12), and decreased the inward conductance from 5.0±1.3 nS to 3.7±1.5 nS (n = 12). The permeability ratio of Na⁺ over K⁺, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na⁺ channel localized in the ES epithelial cells may be involved in uptake of Na⁺ in the ES. Received: 29 May 1996 / Received after revision: 1 August 1996 / Accepted: 2 August 1996     

Role of a Ca2+-activated K+ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells

 Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (V _m) was –41±2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) V _m became significantly less negative: –13±2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of V _m. Depolarizing voltage steps up to +60 mV from holding potentials of –60 mV resulted in large (1–10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above –20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca²⁺-activated potassium channels (BK_Ca), diminished the total current recorded at +60 mV by 45±14% (P<0.05, n=5) and 50±6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BK_Ca channels present in HSV smooth muscle cells contribute to the maintenance of the V _m and sustain a significant portion of the total voltage-activated, outward current. Finally, BK_Ca channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity. Received: 3 April 1998 / Received after revision: 23 September 1998 / Accepted: 13 October 1998     

Repetitive firing and oscillatory activity of pyramidal-like bursting neurons in the rat subiculum

 Electrophysiological characterization of neurons within the rat subiculum was carried out with intracellular recordings in an in vitro slice preparation. Subicular neurons responded to threshold pulses of depolarizing current delivered at a resting membrane potential (RMP) of –65.7±5.8 mV (mean±SD, n=85) with an initial burst of three to five fast action potentials that rode on a depolarizing envelope and was terminated by an afterhyperpolarization (burst AHP) (duration 113±35 ms; peak amplitude 2.7±0.6 mV, n=10). Tonic firing replaced the bursting mode at membrane potential less negative than –55 mV. Suprathreshold depolarizing pulses evoked at RMP both an initial burst and successive tonic firing. Intracellular staining with biocytin showed morphological features typical of pyramidal cells (n=8). The relationship between frequency of repetitive firing and injected current (f–I) revealed that the burst firing frequency (250–300 Hz) was only slightly influenced by the amount of injected current. By contrast, the f–I curve of the tonic firing phase depended upon current intensity: it displayed an initial segment that increased at first linearly and then turned into a plateau for both the early and the late inter-spike intervals. The frequency of the tonic firing declined only slightly with time, thus suggesting a lack of adaptation. During tonic firing, each single action potential was followed by a fast AHP and a depolarizing afterpotential. Termination of repetitive firing was followed by an AHP (spike-train AHP; duration 223±101 ms, peak amplitude 5.6±2.4 mV, n=17). Fast spike-train and burst AHPs were reduced by bath application of the Ca²⁺-channel blockers Co²⁺(2 mM) and Cd²⁺(1 mM) (n=8), thus suggesting the participation of Ca²⁺-dependent K⁺ conductances in these AHPs. Subicular bursting neurons generated persistent, subthreshold voltage oscillations at 5.3±1 Hz (n=20) during steady depolarization positive to –60 mV; at values positive to –55 mV, the oscillatory activity could trigger clusters of single action potentials with a periodicity of 0.9–2 Hz. Oscillations were not prevented by application of excitatory amino acid receptor and GABA_A receptor antagonists (n=5), Ca²⁺-channel blockers (n=5), or Cs⁺ (3 mM; n=4), but were abolished by the Na⁺-channel blocker tetrodotoxin (1 μM; n=6). Our findings demonstrate that pyramidal-like subicular neurons generate both bursting and non-adapting tonic firing, depending upon their membrane potential. These neurons also display oscillatory activity in the range of theta frequency that depends on the activation of a voltage-gated Na⁺ conductance. These electrophysiological properties may play a role in the process of signals arising from the hippocampal formation before being funnelled towards other limbic structures. Received: 20 May 1996 / Accepted: 6 November 1996     

Cystic fibrosis and non-cystic-fibrosis human nasal epithelium show analogous Na+ absorption and reversible block by phenamil

 Transepithelial short-circuit current (I _SC), potential (V _T) and resistance (R _T) of confluent monolayers of human nasal epithelium cultured from patients with and without cystic fibrosis (CF) were measured. In our Ussing chamber experiments with monolayers derived from non-CF and CF patients neither I _SC (non-CF: 14.1 ± 1.0 μA/cm², n = 77; CF: 16.7 ± 1.5 μA/cm², n = 42), nor R _T (non-CF: 288 ± 15 Ω· cm²; CF: 325 ± 20 Ω· cm²) showed any significant differences, only V _T showed moderate but significant different values (non-CF: –3.6 ± 0.4 mV; CF: –5.6 ± 0.7 mV, respectively). Total I _SC in CF cells was nearly completely inhibited by amiloride (92 ± 9.6%), while in non-CF tissue amiloride-insensitive conductances mediated a considerable amount of the I _SC (36.3 ± 6.1%), indicating a lower activity of amiloride-sensitive Na⁺ conductances in non-CF cells. In both tissues the amiloride-sensitive I _SC could also be blocked by the amiloride analogues benzamil, phenamil and 5-(N-ethyl-N-isopropyl)2’,4’-amiloride (EIPA) with different affinities. However, amiloride had a significant lower affinity in CF tissue (half-maximal blocker concentration, K _1/2 = 586 ± 59 nM) compared with non-CF tissue (K _1/2 = 294 ± 22 nM). Astonishingly, phenamil, a blocker which irreversibly blocks all epithelial Na⁺ channels hitherto described, inhibited the Na⁺ conductances of human nasal epithelium in a completely reversible way, but nevertheless with high affinity (non-CF: K _1/2 = 12.5 ± 1.2 nM; CF: K _1/2 = 17.1 ± 1.1 nM). Even in high doses none of these blockers had any effect on intracellular Ca²⁺ concentration as measured with Fura-2. From these findings, we conclude that the epithelial Na⁺ conductances of human CF nasal epithelium show modified regulation or are functionally different from those of other tissues. Received: 19 February 1996 / Received after revision: 10 December 1996 / Accepted: 7 January 1997     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

 The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (I _CCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. I _CCh was induced by carbachol (CCh, 50 μM) at a holding potential of –30 mV or –60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited I _CCh concentration dependently in a reversible manner (53 ± 8.6% at 1 μM, mean ± SE, n = 11). In addition, amplitudes of I _CCh were only 37 ± 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca²⁺ channel current. The peak value of Ba²⁺ current at 0 mV was reduced to 35 ± 7.4% (n = 9) by 3 μM of ML-7. As I _CCh is known to have an intracellular Ca²⁺ dependence, we tried to exclude the possibility that ML-7 inhibited I _CCh indirectly via suppression of Ca²⁺ current and the similar inhibitory effects of ML-7 on I _CChwere confirmed under the following conditions: (1) clamp of membrane potential at –60 mV; (2) clamp of intracellular [Ca²⁺] to 1 μM by 10 mM BAPTA; (3) pre-inhibition of Ca²⁺ channel by verapamil. Different from the effects on I _CCh, ML-7 barely inhibited the same cationic current induced by guanosine 5’-O-(3-thiotriphosphate) (GTP[γS], 0.2 mM) in the pipette solution. These results suggest that a Ca²⁺/calmodulin-MLCK-dependent pathway can modulate the activation of I _CCh in guinea-pig gastric antral myocytes. Received: 21 January 1997 / Received after revision and accepted: 2 April 1997     

Na+–Ca2+ exchange current from rabbit isolated atrioventricular nodal and ventricular myocytes compared using action potential and ramp waveforms

We measured and compared Na–Ca exchanger current (I_Na–Ca) from rabbit isolated ventricular and atrioventricular (AV) nodal myocytes, using action potential (AP) and ramp voltage commands. Whole cell patch‐clamp recordings were made at 35–37 °C; I_Na–Ca was measured as 5 m M nickel (Ni)‐ sensitive current with major interfering voltage and calcium‐activated currents blocked. In ventricular cells a 2‐s descending ramp elicited I_Na–Ca showing outward rectification and a reversal potential (E_rev) of –13.1 ± 1.2 mV (n = 12; mean ± SEM). With a ventricular AP as the voltage command, the profile of I_Na–Ca followed the applied waveform closely. The current–voltage relation during AP repolarization was almost linear and showed an E_rev of –38.3 ± 5.3 mV (n = 6). As I_Na–Ca depended on the applied voltage waveform, comparisons between the two cell types utilized the same command waveform (a series of AV nodal APs). In ventricular myocytes this elicited I_Na–Ca that reversed near –38 mV and was inwardly directed during the pacemaker potential. This command was also applied to AV node cells; mean I_Na–Ca density at all voltages encompassed by the AP (–70 to +30 mV) did not differ significantly from that in ventricular myocytes (P > 0.05, ANOVA ). This finding was confirmed using brief (250 ms) voltage ramp protocols (P > 0.1 ANOVA ). These data represent the first direct measurements of AV nodal I_Na–Ca and suggest that the exchanger may be functionally expressed to similar levels in the two cell types. They may also suggest a possible role for I_Na–Ca during the pacemaker potential in AV node as inward I_Na–Ca was observed over the pacemaker potential range even with bulk internal Ca buffered to a low level.     

Losartan antagonism of angiotensin-II-induced potassium secretion across rat colon

 The effect of angiotensin II (ANG II) on potassium transport across the short-circuited rat distal colon was investigated using ⁸⁶Rb⁺ as a tracer for unidirectional K⁺ fluxes. The addition of high concentrations of ANG II (≥10^–6 M) to the serosal bathing solution had no effect on the mucosal to serosal flux of Rb⁺, but significantly increased the serosal to mucosal flux and abolished the basal net absorptive Rb⁺ flux. These ANG-II-induced alterations in Rb⁺ transport were blocked by the AT₁ receptor antagonist losartan and its metabolite EXP3174, which is also known to have AT₁ receptor antagonistic activity. In contrast, an AT₂ receptor antagonist, PD123319, did not prevent the alterations in colonic Rb⁺ transport induced by ANG II in vitro. At lower bath concentrations (10^–7 M to 10^–10 M ), ANG II had no measurable effects on Rb⁺ transport across this tissue but ANG II did have a bimodal effect on short-circuit current (I _sc), indicating additional effects on the electrogenic transport of other ions. Dose-dependent reductions in I _sc were observed between 10^–7 M (↓ΔI _sc=1.96±0.49 μEq·cm^–2·h^–1, n=6) and 10^–10 M (↓ΔI _sc=0.16±0.19 μEq·cm^–2·h^–1, n=7) ANG II, whereas I _sc was increased at the higher concentrations (↑ΔI _sc= 3.36±0.52 μEq·cm^–2·h^–1, n=7, at 10^–4 M). The ANG-II-induced increases and decreases in I _sc were both blocked by losartan but not by PD123319. These studies are the first to demonstrate an effect of ANG II on K⁺ transport across rat colon that is independent of aldosterone and mediated by AT₁ receptors. Received: 13 March 1998 Received after revision and accepted: 26 May 1998     

Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias

 Previously it has been shown that the Na⁺2Cl^–K⁺ cotransporter accepts NH₄ ⁺ at its K⁺ binding site. This property can be used to estimate its transport rates by adding NH₄ ⁺ to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na⁺2Cl^–K⁺ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH₄ ⁺ to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH₃ uptake. This was followed by an acidification, corresponding to NH₄ ⁺ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl^– solution (6 mmol/l, low Cl^–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH₄ ⁺ uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba²⁺ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl^– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl^– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH₄ ⁺ uptake rate was increased furosemide (500 μmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na⁺2Cl^–K⁺ cotransporter probably independently: (1) lowering of cytosolic Cl^– concentration; (2) cell shrinkage; and (3) activation by cAMP. Received: 3 March 1998 / Received after revision: 22 April 1998 / Accepted: 27 April 1998     

Time course and voltage dependence of expressed HERG current compared with native ”rapid” delayed rectifier K current during the cardiac ventricular action potential

 It is widely believed that HERG (human ether-a-go-go-related gene) encodes the major subunit of the cardiac ”rapid” delayed rectifier K channel. The aims of the present study were threefold: (1) to record directly the time course and voltage dependence of expressed HERG current in a mammalian cell line, during an imposed ventricular action potential (AP); (2) to compare this with native rapid delayed rectifier current (I _Kr) elicited by applying an AP command to isolated guinea-pig ventricular myocytes; (3) to provide mechanistic information regarding the profile of HERG/I _Kr during the AP. We used the AP clamp technique and conventional whole-cell patch-clamp recordings at 32–34°C. HERG was transiently expressed in Chinese hamster ovary (CHO) cells. There was an outward current in transfected CHO cells, which developed progressively during the AP plateau and slow repolarisation phase. The instantaneous current-voltage (I-V) relation for both leak-subtracted HERG current (n=10) and E-4031-sensitive current (n=6) during AP repolarisation was maximal between –30 mV and –40 mV. The conductance-voltage (G-V) relation was maximal at potentials between –60 and –75 mV. A similar voltage dependence for HERG current was observed during a descending ramp from +60 to –80 mV (n=5), but not during either an ascending ramp (n=5), or a reversed AP waveform (n=8). These data suggest that instantaneous HERG current during the AP does not depend on the instantaneous command voltage alone, but upon the previous voltages during the applied waveform. The time course of activation of HERG current at potentials near the AP plateau was rapid. Tail currents recorded on premature repolarisation at different time points in the AP showed directly that HERG also activates rapidly during the AP. The I-V profiles of fully activated HERG and of current during the AP were very similar. I _Kr from guinea-pig ventricular myocytes was measured as E-4031-sensitive current during the AP clamp command. The current had a similar I-V and G-V profile to HERG current in CHO cells. These data indicate that HERG current and native I _Kr are similar during an applied AP waveform. Activation of HERG is rapid during the AP. However, due to rapid inactivation relatively little current flows until the potential becomes less positive than 0 mV. The removal of inactivation then allows more current to flow, giving rise to the distinct instantaneous I-V profile during the AP. The correlation between the voltage dependence of HERG during the AP and the fully activated I-V relation indicates that the contribution of HERG/I _Kr to AP repolarisation is more significantly determined by the open-channel I-V relation, than the precise activation time course of the current. Received: 20 April 1998 / Received after revision: 8 July 1998 / Accepted: 13 July 1998     

Estimation of outward currents in isolated human atrial myocytes using inactivation time course analysis

 The aim was to investigate outward currents in single, isolated, human, atrial myocytes and to determine the relative contribution of individual current components to the total outward current. Currents were recorded using the whole-cell patch-clamp technique at 36–37°C. Individual outward current components were estimated from recordings of total outward current using a mathematical procedure based on the inactivation time course of the respective currents. This method allows estimation of outward currents without the use of drugs or conditioning voltage-clamp protocols to suppress individual current components. A rapidly activating and partially inactivating total outward current was recorded when myocytes were voltage clamped at potentials positive to –20 mV (peak current density 24.0±0.97 pA/pF at +40 mV; n=107 cells, 33 patients). This total outward current comprised three overlapping currents: a rapidly inactivating, transient, outward current (I _to1) a slowly and partially inactivating current (ultrarapid delayed rectifier, I _Kur) and a third current component which most probably reflects a non selective cation current (not characterized). The average current densities at +40 mV were 8.92±0.44 pA/pF for I _to1 and 15.1±0.72 pA/pF for I _Kur (n=107 cells). Recovery from inactivation was bi-exponential for both currents and was faster for I _to1. A slowly activating delayed rectifier current (I _K) was not found. The current densities of peak I _to1 and I _Kur varied strongly between individual myocytes, even in those from the same patient. The ratio I _Kur/I _to1 was 0.5–6.9 with a mean of 1.98±0.11 (n=107 cells), suggesting that I _Kur is the main repolarizing current. The amplitudes of the total outward current, I _to1 and I _Kur, and the ratio of the latter two were independent of patient age (16–87 years). Received: 2 February 1998 / Received after revision: 12 March 1998 / Accepted: 31 March 1998     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

 The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (P_i) and H⁺ ([MgADP], [Pi] and [H⁺], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k₁ [5.6±0.1 s^–1 for barnacle, n=30; 26.3±0.7 s^–1 for frog, n=14 (mean±SEM)] and k₂ [0.6±0.1 s^–1 in barnacle, n=30; 10.4±1.0 s^–1 in frog, n=14 (mean±SEM)], at 10°C. Decreasing the pH by 0.5 pH units did not significantly affect k₁ for barnacle relaxation [5.6±0.1 s^–1 (mean±SEM), n=15] compared to the decrease in k₁ of 40% seen in frog. Use of the Ca²⁺-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa₅₀ value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in k_off from the labelled protein [322±32 s^–1 at pH 7.0 and 381±24 s^–1 (mean±SEM) at pH 6.6]. Increasing [MgADP] by 20 μM (50 μM added ADP) from control values of 50 μM in frog decreased k₁ to 12.3±0.4 s^–1 (mean±SEM, n=8), and at 400 μM MgADP, k₁=9.6±0.1 s^–1 (mean±SEM, n=12). In barnacle, 500 μM MgADP had a much smaller effect on k₁ (4.0±0.9 s^–1, mean±SEM, n=8). Increasing the free [P_i] from the contaminant level of 0.36 mM to 1.9 mM slowed k₁ by ≈15% in barnacle [4.8±0.8 s^–1, mean±SEM, n=7], compared to a ≈30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation. Received: 18 May 1998 / Received after revision and accepted: / 1 September 1998     

Developmental change in the voltage-dependence of the pacemaker current, if, in rat ventricle cells

 Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current i_f. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (–113 ± 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (–72 ± 2 mV; n=6) or cultured cells (–70 ± 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of i_f outside the physiological range. Received: 8 August 1996 / Received after revision and accepted: 29 October 1996