pA pF-1 Research Articles

ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors

Effects of extracellular adenosine tri-phosphate (ATP) on ionic currents were investigated using the perforated-patch whole-cell recording technique on isolated terminals of the Hypothalamic Neurohypophysial System (HNS). ATP induced a current response in 70% of these isolated terminals. This inwardly-rectifying, inactivating current had an apparent reversal near 0 mV and was dose-dependent on ATP with an EC50=9.6+/-1.0 microM. In addition, current amplitudes measured at maximal ATP concentrations and optimum holding potentials had a current density of 70.8 pA pF(-1) and were greatly inhibited by suramin and PPADS. Different purinergic receptor agonists were tested, with the following efficacy: ATP > or = 2-methylthioATP > ATP-gamma-S > Bz-Bz-ATP > alpha,beta-methylene-ATP > beta,gamma-methylene-ATP. However, UTP and ADP were ineffective. These data suggest the involvement of a P2X purinergic receptor in the ATP-induced responses. Immunocytochemical labeling in vasopressinergic terminals indicates the existence of P2X(2,3,4, and 7), but not P2X6 receptors. Additionally, P2X(2 and 3) were not found in terminals which labeled for oxytocin. In summary, the EC50, decay, inactivation, and pharmacology indicate that a functional mixture of P2X(2 and 3) homomeric receptors mediate the majority of the ATP responses in vasopressinergic HNS terminals. We speculate that the characteristics of these types of receptors reflect the function of co-released ATP in the terminal compartment of these and other CNS neurons.

The protein kinase inhibitor, staurosporine, inhibits L-type Ca 2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca 2+ channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I–V relationship of LTCC (from −16.8 ± 2.55 to −3.74 ± 1.22 pA pF −1 at 0 mV). The level of L-type Ca 2+ current ( I Ca,L) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I Ca,L in a dose-dependent manner with a K d value of 61.98 ± 6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 μM) and chelerythrine (3 μM), and PKA inhibitor, PKA-IP (5 μM), did not show any inhibitory effect on I Ca,L. Additional application of ST in the presence of isoproterenol (1 μM), a selective β-adrenoreceptor agonist, reduced peak I Ca,L (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.

Ionic conductances in sustentacular cells of the mouse olfactory epithelium

The electrical properties of sustentacular cells (SCs) in the olfactory epithelium (OE) were investigated in tissue slices taken from neonatal mice (P0-P4). Conventional whole-cell recordings were obtained from SCs and also from olfactory receptor neurones (ORNs) in situ. SCs had a larger apparent cell capacitance (C(cell)) (18.6 +/- 0.5 pF) than ORNs (4.4 +/- 0.4 pF) and a lower apparent membrane resistance (R(m)) (160 +/- 11 MOmega versus 664 +/- 195 MOmega, respectively). When corrected for a seal resistance of 1 GOmega, these mean R(m) values were increased to 190 MOmega and 2 GOmega in SCs and ORNs, respectively. SCs generated a TTX (1 microm)-resistant voltage-activated Na(+) current (I(Na)) that had a peak density at -38 mV of -44 pA pF(-1) and supported action potential firing. Peak current density of I(Na) in neurones was 510 +/- 96 pA pF(-1). The outward K(+) current in SCs was composed (> 70%) of a TEA (2 mm)-sensitive component that was mediated by the opening of large-conductance (237 +/- 10 pS; BK) channels. The resting leak conductance (g(L)) of SCs was permeable to monovalent cations and anions and was largely inhibited by substitution of external Na(+) with NMDG and by internal F(-) with gluconate. g(L) deactivated up to 50% at potentials negative of -70 mV and was inhibited by 18beta-glycyrrhetinic acid (20 mum). SCs were identified using fluorescent dyes (Lucifer Yellow and Alexa Fluor 488) in the whole-cell patch pipette-filling solution. Our findings indicate that SCs in the OE of neonates are electrically excitable and are distinguishable from neurones by a having a resting g(L).

Functional diversity of electrogenic Na+–HCO3− cotransport in ventricular myocytes from rat, rabbit and guinea pig

The Na(+)-HCO(3)(-) cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO(3)(-) : 1Na(+); coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO(3)(-) : 1Na(+); N= 2). We have used voltage-clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady-state pH(i) while hyperpolarization decreased it, effects seen only in CO(2)/HCO(3)(-)-buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pH(i) changes were rat > guinea pig approximately rabbit. Tonic depolarization (-140 mV to -0 mV) accelerated NBC-mediated pH(i) recovery from an intracellular acid load. At 0 mV, NBC-mediated outward current at resting pH(i) was +0.52 +/- 0.05 pA pF(-1) (rat, n= 5), +0.26 +/- 0.05 pA pF(-1) (guinea pig, n= 5) and +0.10 +/- 0.03 pA pF(-1) (rabbit, n= 9), with reversal potentials near -100 mV, consistent with N= 2. The above results indicate a functionally active voltage-sensitive NBCe in these species. Voltage-clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC-mediated pH(i)-recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC-mediated pH(i) recovery was associated with a rise of [Na(+)](i) at a rate approximately 25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.

Mechanisms underlying enhanced cardiac excitation contraction coupling observed in the senescent sheep myocardium

Ageing related stiffening of the vascular system is believed to be in part responsible for a number of clinical outcomes including hypertension and heart failure. In the present study, we sought to determine whether there are alterations in cardiac excitation contraction coupling that may help compensate for the increased vessel stiffness. Experiments were performed on single cardiac myocytes isolated from young (18 months) and aged (>8 years) sheep. Intracellular Ca 2+ concentration, action potentials, L-type Ca 2+ currents and SR Ca 2+ content were measured at 23 °C. With ageing, cell capacitance increased by 26% indicating cellular hypertrophy. Action potential duration (APD90) (590 ± 21 vs. 726 ± 36 ms), Ca 2+ transient amplitude (112 ± 15 vs. 202 ± 25 nmol l –1) and fractional cell shortening (by 37%) also increased in the aged hearts (all values P < 0.05). The larger Ca 2+ transient amplitude observed under current clamp conditions was maintained under voltage clamp control; however, SR Ca 2+ content was identical. Both the peak L-type Ca 2+ current (2.8 ± 0.3 vs. 4.9 ± 0.5 pA pF –1) and integrated Ca 2+ entry (5.1 ± 0.7 vs. 7.9 ± 0.8 μmol l –1, all P < 0.01) were greater in aged cells. In this study we show that in the ageing ovine myocardium, the amplitude of the systolic Ca 2+ transient is increased. The larger Ca 2+ transients cannot simply be explained by changes in APD and we suggest that the greater inward L-type Ca 2+ current provides a more effective trigger for calcium-induced-calcium release from the SR whilst maintaining a stable SR Ca 2+ content. These changes in cardiac excitation contraction coupling may help maintain cardiac output in the face of increased great vessel stiffness.

SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes.

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K(+) channels (K(ATP)). To investigate the protective role of K(ATP) channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K(ATP) currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 +/- 1.22; and A-fragment 0.94 +/- 0.07 pA pF(-1), n= 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 +/- 0.85 pA pF(-1), n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca(2+) loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca(2+) and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca(2+) loading, was abolished by the A-fragment. Our data are consistent with a role for K(ATP) channels in causing action potential failure and reduced Ca(2+) loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K(ATP) channel, but we also discuss the possibility of mitochondrial effects.

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle.

We investigated the effects of pressure overload hypertrophy on inward sodium (I Na) and calcium currents (I Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I Ca density was unchanged whereas I Na density after stepping from -80 to -30 mV was decreased by 30% (-9.0 +/- 1.16 pA pF(-1) in control and -6.31 +/- 0.67 pA pF(-1) in hypertrophy, p < 0.05, n = 6). Steady-state activation/inactivation variables of I Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca2+ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na+ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I Na density so that net Na+ influx remains unaltered. Changes in the fast I Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle.

Characterization of a hyperpolarization-activated time-dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium.

Cardiomyocytes from the pulmonary vein sleeves (PVs) are known to play an important role in atrial fibrillation. PVs have been shown to exhibit time-dependent hyperpolarization-induced inward currents of uncertain nature. We observed a time-dependent K(+) current upon hyperpolarization of PV and left atrial (LA) cardiomyocytes (I(KH)) and characterized its biophysical and pharmacological properties. The activation time constant was weakly voltage dependent, ranging from 386 +/- 14 to 427 +/- 37 ms between -120 and -90 mV, and the half-activation voltage averaged -93 +/- 4 mV. I(KH) was larger in PV than LA cells (e.g. at -120 mV: -2.8 +/- 0.3 versus-1.9 +/- 0.2 pA pF(-1), respectively, P < 0.01). The reversal potential was approximately -84 mV with 5.4 mm[K(+)](o) and changed by 55.7 +/- 2.4 mV per decade [K(+)](o) change. I(KH) was exquisitely Ba(2+) sensitive, with a 50% inhibitory concentration (IC(50)) of 2.0 +/- 0.3 microm (versus 76.0 +/- 17.9 microm for instantaneous inward-rectifier current, P < 0.01), and showed similar Cs(+) sensitivity to instantaneous current. I(KH) was potently blocked by tertiapin-Q, a selective Kir3-subunit channel blocker (IC(50) 10.0 +/- 2.1 nm), was unaffected by atropine and was significantly increased by isoproterenol (isoprenaline), carbachol and the non-hydrolysable guanosine triphosphate analogue GTPgammaS. I(KH) activation by carbachol required GTP in the pipette and was prevented by pertussis toxin pretreatment. Tertiapin-Q delayed repolarization in atropine-exposed multicellular atrial preparations studied with standard microelectrodes (action potential duration pre- versus post-tertiapin-Q: 190.4 +/- 4.3 versus 234.2 +/- 9.9 ms, PV; 202.6 +/- 2.6 versus 242.7 +/- 6.2 ms, LA; 2 Hz, P < 0.05 each). Seven-day atrial tachypacing significantly increased I(KH) (e.g. at -120 mV in PV: from -2.8 +/- 0.3 to -4.5 +/- 0.5 pA pF(-1), P < 0.01). We conclude that I(KH) is a time-dependent, hyperpolarization-activated K(+) current that likely involves Kir3 subunits and appears to play a significant role in atrial physiology.

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes.

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.

Mice overexpressing the cardiac sodium-calcium exchanger: defects in excitation-contraction coupling.

Homozygous overexpression of the cardiac Na(+)-Ca(2+) exchanger causes cardiac hypertrophy and increases susceptibility to heart failure in response to stress. We studied the functional effects of homozygous overexpression of the exchanger at the cellular level in isolated mouse ventricular myocytes. Compared with patch-clamped myocytes from wild-type animals, non-failing myocytes from homozygous transgenic mice exhibited increased cell capacitance (from 208 +/- 16 pF to 260 +/- 15 pF, P < 0.05). Intracellular Ca(2+) oscillations were readily elicited in homozygous transgenic animals during depolarizations to +80 mV, consistent with rapid Ca(2+) overload caused by reverse Na(+)-Ca(2+) exchange. After normalization to cell capacitance, transgenic myocytes had significant increases in Na(+)-Ca(2+) exchange activity (318%) and peak L-type Ca(2+) current (8.2 +/- 0.7 pA pF(-1) at 0 mV test potential) compared to wild-type (5.8 +/- 0.9 pA pF(-1) at 0 mV, P < 0.02). The peak Ca(2+) current amplitude and its rate of inactivation could be modulated by rapid reversible block of the exchanger. Thus, we describe an unexpected direct influence of Na(+)-Ca(2+) exchange activity on the L-type Ca(2+) channel. Despite intact sarcoplasmic reticular Ca(2+) content and larger peak L-type Ca(2+) currents, homozygous transgenic animals exhibited smaller Ca(2+) transients (Delta[Ca(2+)](i)= 466 +/- 48 nm in transgenics versus 892 +/- 104 nm in wild-type, P < 0.0005) and substantially reduced gain of excitation-contraction coupling. These alterations in excitation-contraction coupling may underlie the tendency for these animals to develop heart failure following haemodynamic stress.

Cav1.3 (alpha1D) Ca2+ currents in neonatal outer hair cells of mice.

Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.

NADH supplementation decreases pinacidil-primed I K ATP in ventricular cardiomyocytes by increasing intracellular ATP.

1 The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current I(K(ATP)) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. 2 Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36-37 degrees C. Adenine nucleotides were determined by HPLC. 3 Under physiological conditions (4.3 mM ATP in the pipette solution, ATP(i)) I(K(ATP)) did not contribute to basal electrical activity. 4 The ATP-dependent potassium (K((ATP))) channel opener pinacidil activated I(K(ATP)) dependent on [ATP](i) showing a significantly more pronounced activation at lower (1 mM) [ATP](i). 5 Supplementation of cardiomyocytes with 300 micro g ml(-1) NADH (4-6 h) resulted in a significantly reduced I(K(ATP)) activation by pinacidil compared to control cells. The current density was 13.8+/-3.78 (n=6) versus 28.9+/-3.38 pA pF(-1) (n=19; P<0.05). 6 Equimolar amounts of the related compounds nicotinamide and NAD(+) did not achieve a similar effect like NADH. 7 Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6+/-1.88 nmol mg(-1) protein versus control: 35.4+/-2.57 nmol mg(-1) protein, P<0.000005). 8 These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of I(K(ATP)) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. 9 Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP.

Molecular determinants of cAMP-mediated regulation of the Na+-Ca2+ exchanger expressed in human cell lines

The cardiac Na+–Ca2+ exchanger (NCX1) is one of the major sarcolemmal Ca2+ transporters of cardiomyocytes. Structure–function studies suggest that β-adrenergic inhibition of NCX1, as reported for frog, but not mammalian hearts, may be associated with a unique splice variant of frog cardiac NCX1 where insertion of an extra exon completes the coding of a nucleotide binding P-loop. To test the involvement of the P-loop in cAMP-mediated regulation of NCX1 we used four stably transfected human cell lines (a previously established line of baby hamster kidney (BHK) cells and three new lines of human embryonic kidney (HEK) cells) expressing: (1) wild-type dog NCX1 (dog NCX1); (2) wild-type frog NCX1 (frog NCX1); (3) chimeric frog-dog NCX1 incorporating the completed P-loop from the frog NCX1 into the dog NCX1 sequence (frog/dog NCX1); and (4) a mutated frog NCX1 where a putative protein kinase A (PKA) site was disrupted by substitution of a single serine residue with glycine (S374G frog NCX1). Structural expression of these NCX1 constructs was confirmed using Western blot analysis of extracted proteins and immunofluorescence imaging. The NCX1-generated current (INa–Ca) was reliably measured in cells expressing dog (2.0 ± 0.15 pA pF−1), frog (0.6 ± 0.1 pA pF−1) and frog/dog (0.6 ± 0.1 pA pF−1) NCX1, but less so in those expressing S374G frog NCX1 (0.3 ± 0.1 pA pF−1). Addition of 100 μm 8-bromoadenosine 3′,5′ cyclic monophosphate (8-Br-cAMP) suppressed INa–Ca of frog and frog/dog NCX1 by 60–80 %. The suppression of INa–Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca2+ (Ca12+)-transients, activated by rapid withdrawal of Na+, were also downregulated in the frog and frog/dog NCX1 and to a smaller and transient extent in S374G frog NCX1. Our findings suggest that the suppressive effect of β-adrenergic agonists requires the presence of the P-loop domain of the frog NCX1, and provide evidence that the putative PKA site, present in both dog and frog NCX1, might also be critical in the cAMP-mediated regulation of the exchanger.

Molecular determinants of cAMP-mediated regulation of the Na+-Ca2+ exchanger expressed in human cell lines.

The cardiac Na+-Ca2+ exchanger (NCX1) is one of the major sarcolemmal Ca2+ transporters of cardiomyocytes. Structure-function studies suggest that beta-adrenergic inhibition of NCX1, as reported for frog, but not mammalian hearts, may be associated with a unique splice variant of frog cardiac NCX1 where insertion of an extra exon completes the coding of a nucleotide binding P-loop. To test the involvement of the P-loop in cAMP-mediated regulation of NCX1 we used four stably transfected human cell lines (a previously established line of baby hamster kidney (BHK) cells and three new lines of human embryonic kidney (HEK) cells) expressing: (1) wild-type dog NCX1 (dog NCX1); (2) wild-type frog NCX1 (frog NCX1); (3) chimeric frog-dog NCX1 incorporating the completed P-loop from the frog NCX1 into the dog NCX1 sequence (frog/dog NCX1); and (4) a mutated frog NCX1 where a putative protein kinase A (PKA) site was disrupted by substitution of a single serine residue with glycine (S374G frog NCX1). Structural expression of these NCX1 constructs was confirmed using Western blot analysis of extracted proteins and immunofluorescence imaging. The NCX1-generated current (INa-Ca) was reliably measured in cells expressing dog (2.0 +/- 0.15 pA pF-1), frog (0.6 +/- 0.1 pA pF-1) and frog/dog (0.6 +/- 0.1 pA pF-1) NCX1, but less so in those expressing S374G frog NCX1 (0.3 +/- 0.1 pA pF-1). Addition of 100 microM 8-bromoadenosine 3',5' cyclic monophosphate (8-Br-cAMP) suppressed INa-Ca of frog and frog/dog NCX1 by 60-80 %. The suppression of INa-Ca was smaller and transient in cells expressing S374G frog NCX1, and absent in cells expressing dog NCX1. Intracellular Ca2+ (Ca2+i)-transients, activated by rapid withdrawal of Na+, were also downregulated in the frog and frog/dog NCX1 and to a smaller and transient extent in S374G frog NCX1. Our findings suggest that the suppressive effect of beta-adrenergic agonists requires the presence of the P-loop domain of the frog NCX1, and provide evidence that the putative PKA site, present in both dog and frog NCX1, might also be critical in the cAMP-mediated regulation of the exchanger.

An antisense oligonucleotide against H1 inhibits the classical sodium current but not ICa(TTX) in rat ventricular cells.

ICa(TTX) is a sodium current component, functionally distinct from the main body of sodium current, seen in cardiac and other cells. To determine if ICa(TTX) channels are a separate isoform from the classical cardiac sodium channels, we exposed rat ventricular cells in primary culture to an antisense oligonucleotide (AON) directed against rH1 (rNav1.5): 5'-CTCCTCATACCCTCT-3'. The homologous human sequence has been identified (and confirmed by us on HEK 293 cells) as effective against hH1 expressed heterologously. Scrambled sequence (5'-CCCCCCTTATCTACT-3') controls were also included. The AON (10 microM; day 2 of exposure) reduced the classical sodium current by 69.6 % compared to untreated and 60.8 % compared to scrambled sequence (10 microM; day 2 of exposure) controls (mean +/- S.E.M. maximum peak inward current density of -8.23 +/- 0.60 pA pF-1, 18 cells, for untreated; -6.37 +/- 0.79 pA pF-1, 16 cells, for scrambled sequence; and -2.50 +/- 0.31 pA pF-1, 18 cells, for AON-treated cells). The two control groups are not significantly different from each other, but are both significantly different from the AON-treated group (P < 0.001). The inhibition was specific for sodium channels, with no significant AON effect on the L-type calcium current. This confirms that H1 generates the classical cardiac sodium current. This same AON at the same concentration and time of exposure had no significant effect on ICa(TTX) (mean of -4.72 +/- 0.55, 15 cells; -5.47 +/- 0.53, 13 cells; and -5.04 +/- 0.63 pA pF-1, 15 cells, for untreated controls, scrambled controls and AON treated, respectively). Hence, ICa(TTX), which is functionally distinct from the classical cardiac sodium current, is encoded by a distinct gene.

Calcium channel heterogeneity in canine left ventricular myocytes

Regional variations in the electrophysiological properties of myocytes across the left ventricular wall play an important role in both the normal physiology of the heart and the genesis of arrhythmias. To investigate the possible contributions of calcium channels to transmural electrical heterogeneity, whole-cell patch-clamp recordings were made from isolated canine epicardial and endocardial left ventricular myocytes. Two major differences in Ca2+ channel properties were found between epi- and endocardial cells. First, the L-type Ca2+ current was larger in endocardial than in epicardial myocytes. The average peak current density at +10 mV in endocardial myocytes was 3.4 ± 0.2 pA pF−1, and was 45 % higher than that in epicardium (2.3 ± 0.1 pA pF−1). The kinetic properties of the L-type current in epi- and endocardial cells were not significantly different. Second, a low-threshold, rapidly activating and inactivating Ca2+ current that resembled the T-type current was present in all endocardial myocytes but was small or absent in epicardial myocytes. This T-like current had an average peak density of 0.5 pA pF−1 at −40 mV in endocardial cells. In most endocardial cells the T-like Ca2+ current comprised two components: a Ni2+-sensitive T-type current and a tetrodotoxin-sensitive Ca2+ current. We conclude that there are considerable regional variations in the density and properties of Ca2+ channels across the canine left ventricular wall. These variations may contribute to the overall transmural electrical heterogeneity.

Calcium channel heterogeneity in canine left ventricular myocytes.

Regional variations in the electrophysiological properties of myocytes across the left ventricular wall play an important role in both the normal physiology of the heart and the genesis of arrhythmias. To investigate the possible contributions of calcium channels to transmural electrical heterogeneity, whole-cell patch-clamp recordings were made from isolated canine epicardial and endocardial left ventricular myocytes. Two major differences in Ca2+ channel properties were found between epi- and endocardial cells. First, the L-type Ca2+ current was larger in endocardial than in epicardial myocytes. The average peak current density at +10 mV in endocardial myocytes was 3.4 +/- 0.2 pA pF-1, and was 45 % higher than that in epicardium (2.3 +/- 0.1 pA pF-1). The kinetic properties of the L-type current in epi- and endocardial cells were not significantly different. Second, a low-threshold, rapidly activating and inactivating Ca2+ current that resembled the T-type current was present in all endocardial myocytes but was small or absent in epicardial myocytes. This T-like current had an average peak density of 0.5 pA pF-1 at -40 mV in endocardial cells. In most endocardial cells the T-like Ca2+ current comprised two components: a Ni2+-sensitive T-type current and a tetrodotoxin-sensitive Ca2+ current. We conclude that there are considerable regional variations in the density and properties of Ca2+ channels across the canine left ventricular wall. These variations may contribute to the overall transmural electrical heterogeneity.

Effect of androgen deficiency on mouse ventricular repolarization

We previously demonstrated that female mouse ventricles have longer action potential durations (APDs) than males. This delayed repolarization results from a lower current density of the ultrarapid delayed rectifier K(+) current (I(K,ur)) and a lower expression level of its underlying K(+) channel (Kv1.5). To evaluate whether this sex difference could be attributable to the action of male sex hormones, we studied the effect of androgen deficiency on ventricular repolarization. We compared cardiac electrophysiological properties in castrated (orchiectomized; ORC) and control (CTL) male mice. Q-Tc intervals as well as APDs measured at 20 %, 50 % and 90 % of repolarization were all significantly longer in ORC than in CTL. The current density of I(K,ur) was significantly lower in ORC than in CTL (at +50 mV, ORC: 29 +/- 4 pA pF(-1), n = 25; CTL: 48 +/- 5 pA pF(-1), n = 17; P = 0.006). In contrast, all the other K(+) currents present in mouse ventricular myocytes were comparable between ORC and CTL. Moreover, results of Western blot analysis showed a lower expression level of Kv1.5 protein in ORC but no difference between the two groups for the other K(+) channels studied. This study demonstrates that androgen deficiency leads to a reduction in the density of I(K,ur) and Kv1.5 in mouse ventricle, and consequently, to prolongation of APD and Q-Tc interval. In conclusion, these findings strongly suggest that male sex hormones contribute to the sex difference that we previously reported in cardiac repolarization in adult mouse heart.

Osmotic Shrinkage of Human Cervical Cancer Cells Induces an Extracellular Cl−-dependent Nonselective Cation Channel, Which Requires p38 MAPK

This study is to integrate a functional role of nonselective cation (NSC) channels into a model of volume regulation on osmotic shrinkage for human cervical cancer cells. Application of a hypertonic solution (400 mosm kg(-1)) induced cell shrinkage, which was accompanied by a 7-fold increase of inward currents at -80 mV from -4.1 +/- 0.4 pA pF(-1) to -29 +/- 1.1 pA pF(-1) (n = 36, p < 0.001). There is a good correlation of channel activity and cell volume changes. Replacement of bath Na(+) by K(+), Cs(+), Li(+), or Rb(+) did not affect the stimulated inward current significantly, but replacement by Ca(2+), Ba(2+), or the impermeable cation N-methyl-d-glucamine abolished the inward current; this demonstrates that the shrinkage-induced currents discriminate poorly between monovalent cations but are not carried by divalent cations. Replacement of extracellular Cl(-) by gluconate abolished the shrinkage-induced currents in a concentration-dependent manner without changing the reversal potential. Gadolinium (Gd(3+)) inhibited the stimulated current, whereas bumetanide and amiloride had no inhibitory effect. Cell shrinkage triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of MAP/extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (MEK1/2), and p38 kinase. Interference with p38 MAPK by either the specific inhibitor (SB202190), or a dominant-negative mutant profoundly suppressed the activation of the shrinkage-induced NSC channels. In contrast, the regulatory mechanism of shrinkage-induced NSC channels was independent of the volume-responsive MEK1/2 signaling pathway. More importantly, the cell volume response to hypertonicity was inhibited significantly in p38 dominant-negative mutant or by SB202190. Therefore, p38 MAPK is critically involved in the activation of a shrinkage-induced NSC channel, which plays an important role in the volume regulation of human cervical cancer cells.

Characterization of the A-type potassium current in murine gastric antrum.

A-type currents are rapidly inactivating potassium currents that operate at subthreshold potentials. A-type currents have not been reported to occur in the phasic muscles of the stomach. We used conventional voltage-clamp techniques to identify and characterize A-type currents in myocytes isolated from the murine antrum. A-type currents were robust in these cells, with peak current densities averaging 30 pA pF(-1) at 0 mV. These currents underwent rapid inactivation with a time constant of 83 ms at 0 mV. Recovery from inactivation at -80 mV was rapid, with a time constant of 252 ms. The A-type current was blocked by 4-aminopyridine (4-AP) and was inhibited by flecainide, with an IC(50) of 35 microM. The voltage for half-activation was -26 mV, while the voltage of half-inactivation was -65 mV. There was significant activation and incomplete inactivation at potentials positive to -60 mV, which is suggestive of sustained current availability in this voltage range. Under current-clamp conditions, exposure to 4-AP or flecainide depolarized the membrane potential by 7-10 mV. In intact antral tissue preparations, flecainide depolarized the membrane potential between slow waves by 5 mV; changes in slow waves were not evident. The effect of flecainide was not abolished by inhibiting enteric neurotransmission or by blocking delayed rectifier and ATP-sensitive K(+) currents. Transcripts encoding Kv4 channels were detected in isolated antral myocytes by RT-PCR. Immunocytochemistry revealed intense Kv4.2- and Kv4.3-like immunoreactivity in antral myocytes. These data suggest that the A-type current in murine antral smooth muscle cells is likely to be due to Kv4 channels. This current contributes to the maintenance of negative resting membrane potentials.