Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Cellular distribution of calcium current is unaltered during compensated hypertrophy in the spontaneously hypertensive rat

Changes in cellular calcium (Ca²⁺) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca²⁺. The present study was performed to investigate whether changes in the distribution of I _Ca between the surface and t-tubule membranes might contribute to the altered Ca²⁺ handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I _Ca in the presence and absence of t-tubules. Membrane capacitance and I _Ca were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I _Ca both decreased and were no longer significantly different in the two cell types. The density of I _Ca was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I _Ca is unchanged in SHR myocytes compared to WKY controls; I _Ca density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I _Ca, so that the density of I _Ca at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca²⁺ release in the SHR at this stage of compensated hypertrophy.     

Tetrodotoxin blocks L-type Ca2+ channels in canine ventricular cardiomyocytes

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na⁺ channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca²⁺ current (I _Ca) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I _Ca in a reversible manner without altering inactivation kinetics of I _Ca. Fitting results to the Hill equation, an IC₅₀ value of 55?±?2???M was obtained with a Hill coefficient of unity (1.0?±?s0.04). The current was fully abolished by 1???M nisoldipine, indicating that it was really I _Ca. Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I _Ca, which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I _Ca and I _Na. This is the first report demonstrating TTX to interact with L-type calcium current in the heart.     

Role of Ca2+-Dependent Cl− Current on Delayed Afterdepolarizations. A Simulation Study

Calcium-dependent chloride current (I _Cl,Ca) is the second component (I _to2) of the transient outward current (I _to) that provokes the action potential (AP) phase 1 repolarization. This current contributes to the transient inward current (I _ti) that generates delayed afterdepolarizations (DAD) in several pathological conditions. The present work uses a computer AP model of rabbit atrial myocyte and a one-dimensional (1D) tissue model of 400 cells to study the role of I _Cl,Ca on the generation of DAD and triggered activity under calcium-overload conditions. A mathematical model describing the dependence of I _Cl,Ca on intracellular Ca²⁺ is proposed. This model takes into account the experimentally recorded characteristics of I _Cl,Ca: (1) calcium dependence, (2) voltage-dependent inactivation, and (3) I–V field-diffusion relation. Our results support the hypothesis that I _Cl,Ca plays an important role in action potential repolarization, mainly at high frequencies. In the calcium-overload conditions tested in this work, I _Cl,Ca represents between 28% and 44% of the total I _ti that provokes DADs. Our simulations also show that the blockage of I _Cl,Ca reduces the calcium overload range in which DADs provoke triggered activity.     

Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

Aim

Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca²⁺ entry and intracellular Ca²⁺ release. The small‐conductance Ca²⁺‐activated K⁺ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.

Methods

Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real‐time PCR. The expression of proteins was analysed by Western blotting. Ca²⁺‐activated K⁺ current (I_K,Ca) in infected adult mouse cardiac myocytes was recorded using whole‐cell voltage‐clamp technique. The intracellular Ca²⁺ transient was measured using an IonOptix photometry system.

Results

We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N‐terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I_K,Ca and reduced the amplitude of the Ca²⁺ transient in infected cardiomyocytes.

Conclusion

The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.     

Effects of calcium release from sarcoplasmic reticulum on membrane currents in guinea pig atrial cardioballs

(1) Ca current (I _Ca) and membrane currents related to Ca-entry during activation ofI _Ca have been studied in cultured atrial myocytes from hearts of adult guinea pigs by means of patch clamp pipettes. The pipettes were filled with solutions containing citrate (65 mM) as major Ca-chelating compound and Cs ions in order to block K currents. (2) In myocytes dialysed with such solutions a monophasic time course of inactivation ofI _Ca is observed, which is 1–2 orders of magnitude slower as compared to studies on intact cardiac cells or cells dialysed with EGTA as only Ca-chelating compound. (3) During long-lasting or repetitive depolarization a second component ofI _Ca inactivation, apart from the slow decay observed in cells dialysed with such solutions, can be seen. This component of inactivation is not related to the depolarization as such but to loading of the cells with Ca²⁺. Whenever the rapid component of inactivation occurs, a transient inward current (I _ti) after repolarization to the holding potential (–40 to –50 mV) is recorded. Both,I _Ca inactivation andI _ti can be mimicked by extracellular application of caffeine (5–10 mM), suggesting both current changes to be caused by a rise in Ca_i due to Ca release from sarcoplasmic reticulum. In the presence of caffeine the rapid component ofI _Ca-inactivation andI _ti are abolished. (4) In addition toI _Ca inactivation and activation ofI _ti sarcoplasmic Ca release causes openings of a novel ion channel with large conductance (>200 pS), the function of which is unknown. (5) The results are consistent with the concept of Ca_i-dependent inactivation of Ca current, which can be caused either by Ca-entry or by Ca-release from the SR. The transient inward current is likely to reflect a process of Ca-removal from the cell, namely Na–Ca exchange.     

Aldosterone-induced changes in the cardiac L-type Ca2+ current can be prevented by antioxidants in vitro and are absent in rats on low salt diet

Mineralocorticoid receptor (MR) activation modulates cardiac L-type Ca²⁺ current (I _CaL) and transient outward K⁺ current (I _to). The exact circumstances of MR activation, however, remain elusive. Here, we investigate the influence of corticosteroids on MR-mediated changes in cellular electrophysiology. In vitro incubation of adult rat ventricular myocytes with the MR agonist aldosterone (100 nM, 24 h) increased I _CaL density by 34% (n = 16; p < 0.01). This effect was abrogated by co-incubation with the MR antagonist spironolactone (10 μM). To investigate whether an increase in serum aldosterone concentration is sufficient for an increase in I _CaL in vivo, rats were subjected to low Na⁺ diet (LSD, 0.013% Na⁺) for 28 days. This increased serum aldosterone concentration from 0.19 ± 0.04 nM (n = 6) in control animals (0.3% Na⁺, CSD) to 16.1 ± 2.1 nM (n = 6; p < 0.0001). Strikingly, I _CaL density was similar in both CSD and LSD rats (−12.9 ± 0.9 pA pF⁻¹, n = 18 and −13.7 ± 1.1 pA pF⁻¹, n = 16, respectively), as was I _to density. In vitro, the glucocorticoid corticosterone (1 μM) also increased I _CaL and this effect was blocked by spironolactone (10 μM). Co-incubation with corticosterone (1 μM, the normal serum concentration) and aldosterone (100 nM, mimicking low Na⁺ intake) did not further increase I _CaL compared to corticosterone alone. Moreover, co-incubation of myocytes with N-acetylcysteine (10 mM) prevented the aldosterone (100 nM) or corticosterone (1 μM)-induced increase in I _CaL. In conclusion, an increase in serum aldosterone concentration in response to LSD is not sufficient for an increase in I _CaL density in cardiomyocytes in vivo. This is supported in vitro by the absence of an effect of aldosterone on I _CaL in the presence of a physiological concentration of corticosterone. Moreover, the cellular redox state may modulate MR activation. Michael Wagner and Elena Rudakova contributed equally to this work.     

Effects of atrionatriuretic factor on Ca2+current and Cai-independent transient outward K+ current in human atrial cells

The effect of 10 nM atrial natriuretic peptide (ANF) on macroscopic L-type calcium current, I _Ca, and calcium-independent outward potassium current, I _lo, were studied in myocytes isolated from human atrial trabeculae using the whole-cell-recording patch-clamp technique. When cells were dialysed with pipette media containing 0.2 mM GTP, ANF reduced I _Ca by 37.81%±5.4% at +20mV and I _lo by 21.72%±3.68% at +60 mV in a reversible manner. When I _Ca was increased by -adrenoreceptor stimulation (0.1 M isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 M) ANF reduced I _Ca by 24.99±3.4% and by 39.9±6.3% respectively. In cells dialysed with GTP-free pipette media, ANF increased I _Ca markedly (39.8%±7%) and reversibly, whereas it still depressed I _lo (18.92%±2%). Addition of 0.2 mM GTP[S] to the pipette solution in the absence of GTP increased I _Ca, decreased I _lo and suppressed the effect of ANF on both I _Ca and I _lo. It is suggested that activation of the ANF receptor in human atrial cells reduces I _Ca via guanylate-cyclase-dependent cGMP production, increases I _Ca via Gs protein activation and decreases I _lo via Gi protein activation.     

Decrease in sodium–calcium exchange and calcium currents in diabetic rat ventricular myocytes

This study was designed in order to gain insight into possible changes in the inward sodium–calcium exchange current (I_Na–Ca) and the L -type calcium current (I_Ca), in ventricular myocytes isolated from streptozotocin-induced diabetic rats. Recordings were made using the nystatin-perforated patch technique which minimizes interference with the normal intracellular Ca²⁺ buffering mechanisms. The averaged I_Na–Ca current density elicited by Ca²⁺ current was smaller in diabetic than in normal myocytes at all potentials tested. I_Na–Ca activated by rapid application of caffeine was significantly reduced and the decay phase was prolonged. The density of I_Ca was also significantly reduced by diabetes in the range of test potentials between –10 and +50 mV. In addition, the fast time constant of I_Ca inactivation, which represents mainly the sarcoplasmic reticulum (SR) Ca²⁺ release-induced inactivation, was significantly higher in diabetic than in normal myocytes. The decrease in I_Ca, which is the main source of trigger Ca²⁺ for SR Ca²⁺ release, may explain the significantly lowered peak systolic [Ca²⁺]_i previously shown in diabetic myocytes. As activation of I_Ca is essential for subsequent stimulation of I_Na–Ca, reduced I_Ca may contribute to decreasing activation of the Na⁺–Ca²⁺ exchanger.     

Protective effect of oxymatrine on chronic rat heart failure

Oxymatrine is one of the alkaloids extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with anti-inflammatory, immune reaction inhibiting, antiviral, and hepatocyte and antihepatic fibrosis protective activities. However, the effect of oxymatrine on heart failure is not yet known. In this study, the effect of oxymatrine on heart failure was investigated using a Sprague-Dawley rat model of chronic heart failure. Morphological findings showed that in the group treated with 50 and 100 mg/kg of oxymatrine; intermyofibrillar lysis disappeared, myofilaments were orderly, closely and evenly arranged; and mitochondria contained tightly packed cristae compared with the heart failure group. We investigated the cytosolic Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content, and assessed the expression of ryanodine receptor (RyR2), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca²⁺ transients were markedly increased in amplitude in the medium- (ΔF/F ₀ = 26.22 ± 2.01) and high-dose groups (ΔF/F ₀ = 29.49 ± 1.17) compared to the heart failure group (ΔF/F ₀ = 12.12 ± 1.35, P < 0.01), with changes paralleled by a significant increase in the SR Ca²⁺ content (medium-dose group: ΔF/F ₀ = 32.20 ± 1.67, high-dose group: ΔF/F ₀ = 32.57 ± 1.29, HF: ΔF/F ₀ = 17.26 ± 1.05, P < 0.01). Moreover, we demonstrated that the expression of SERCA2a and cardiac DHPR was significantly increased in the medium- and high-dose group compared with the heart failure rats. These findings suggest that oxymatrine could improve heart failure by improving the cardiac function and that this amelioration is associated with upregulation of SERCA2a and DHPR.     

Nitric oxide mediates the anti-adrenergic effect of adenosine on calcium current in isolated rabbit atrioventricular nodal cells

The aim of this study was to determine if adenosine exerts an anti-adrenergic effect on rabbit isolated atrioventricular (AV) nodal cells and, if so, the dependence of this effect on nitric oxide (NO) production. Inward Ca current,I _Ca, was measured in AV nodal cells, enzymatically isolated from rabbit hearts. Isoprenaline (0.1 M) increasedI _Ca from 676 ± 59 to 1102 ± 86 pA (n = 25). This isoprenaline-induced increase inI _Ca, (178 ± 15 % of control) was abolished in the presence of 10 M adenosine (I _Ca 100 ± 2 % of control,n = 9, P < 0.05). This effect of adenosine was completely blocked by the A₁ receptor antagonist CPDPX (8-cyclopentyl 1, 3-dipropylxanthine, 0.1 M). In cells pre-treated with the NO synthase inhibitor,l-nitro-arginine methyl ester (l-NAME, 1 mM) the isoprenaline-induced increase inI _Ca(208 ± 39 % of control,n = 7) was not reduced by the addition of 10 M adenosine (195 ± 32% of control). Co-incubation of cells inl-NAME withl--arginine (1 mM, the endogenous substrate of NO synthase) restored the adenosine-induced attenuation ofI _Ca. In these cells, isoprenaline increasedI _Ca (157 ± 7% of control,n = 6), and, following addition of adenosine (10 M)I _Ca was reduced to 107 ± 8% (P < 0.05). The NO-releasing agent SIN-1 (3-morpholino-sydnonimine, 100 M) inhibitedI _Ca augmented by isoprenaline (n = 5). It is concluded that adenosine exerts an anti-adrenergic effect on the AV node via A, receptors to attenuate a catecholamine-stimulated increase inI _Ca and that this action involves the intracellular production of NO.     

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn²⁺ on whole-cell Ca²⁺-activated Cl⁻ currents (I _CLCA) in mouse inner medullary collecting duct cells (mIMCD-3). I _CLCA was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10–400 μM) to the bathing solution resulted in a dose-dependent increase in I _CLCA with little change in Cl⁻ selectivity or biophysical characteristics, whereas gadolinium chloride (30 μM) and lanthanum chloride (100 μM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn²⁺ (400 μM) stimulated an increase in intracellular Ca²⁺ to an elevated plateau. The Zn²⁺-stimulated [Ca²⁺]_i increase was inhibited by thapsigargin (200 nM), the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (10 μM) and removal of bath Ca²⁺. Pre-exposure to Zn²⁺ (400 μM) markedly attenuated the ATP (100 μM)-stimulated [Ca²⁺]_i increase. These data are consistent with the hypothesis that extracellular Zn²⁺ stimulates an increase in [Ca²⁺]_i by a release of calcium from thapsigargin/IP₃ sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca²⁺-sensing receptor, in IMCD cells is discussed.     

Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca²⁺ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N,N-tetraacetic acid (BAPTA) (a fast Ca²⁺ chelator), (2) a stimulation of peak I _Ca that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak I _Ca that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of I _Ca inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak I _Ca is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on I _Ca is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.The experimental part of this work was published during the postdoctoral stay of I. Zahradník in the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555, USA     

Regulation of L- and N-types of Ca2+ channels by intracellular ATP4– in frog dorsal root ganglion neurons

The roles of free Mg²⁺ ions, ATP^4– ions and Mg-ATP complexes in the regulation of N- and L-types of Ca²⁺ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg²⁺ ions interact with ATP^4– ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (I _BaL) and N-type (I _BaN) Ca²⁺ channels were plotted against its concentrations. The dose-response curves for ATP^4– show that I _BaL and I _BaN proportionally increased with ATP^4– concentrations up to 1 mM at three different Mg²⁺ concentrations. At a fixed concentration of ATP^4–, I _BaL and I _BaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for I _BaL and I _BaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg²⁺ ions did not exert any blocking effect on the activity of either Ca²⁺ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca²⁺ channels in frog DRG neurons are regulated by ATP^4– ions alone, and that the neuronal Ca²⁺ channels are regulated by mechanisms that are different from those regulating the cardiac Ca²⁺ channels. Received: 30 October 1998 / Received after revision: 19 February 1999 / Accepted: 8 March 1999     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l – 1 nmol/l) caused a concentration-dependent increase of Ca²⁺-sensitive K currents (I _K) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca²⁺ currents. The I _K-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca²⁺ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable I _K increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates I _K via PKA-dependent increase of Ca²⁺ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.     

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.     

On the mechanism of muscarinic inhibition of the cardiac Ca current

The mechanism of muscarinic inhibition of the Ca-current (I _Ca) was studied in ventricular myocytes of guinea pig hearts and the following results were obtained. 1. Acetylcholine (ACh) in concentrations up to 10^–4 M had little effect, if any, onI _Ca in control cells. 2. ACh reduced the isoprenaline (ISP)-induced increase ofI _Ca. The doseresponse-relation (ISP concentration vs.I _Ca density) was shifted by ACh towards higher ISP concentrations. But both, at low and high ISP concentrations ACh had nor or little effect. 3. ACh was ineffective whenI _Ca was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. 4. ACh reducedI _Ca enhanced by isobutylmethylxanthine or by forskolin. 5. ACh did not depressI _Ca when the cell was dialysed with the nonhydrolysable GTP-derivative, GMP-PNP. In this condition the -adrenergic enhancement ofI _Ca was also absent. 6. Pertussis toxin, which is known to inhibit the inhibitory transducerprotein (N_i), abolished the ACh response.We concluded from these results that ACh depressesI _Ca by inhibiting, via N_i, the cAMP production.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 (Membranforschung), Project G     

VIP and secretin augment cardiac L-type calcium channel currents in isolated adult rat ventricular myocytes

Vasoactive intestinal peptide (VIP) is colocalized in parasympathetic nerve terminals in the heart and coreleased from these nerve terminals with the “classical” neurotransmitter acetylcholine (Ach). VIP also exerts a positive inotropic effect on the intact heart and enhances adenylyl cyclase activity in isolated heart membranes. Using the whole-cell patch-clamp technique, we show here that VIP enhances Ca²⁺ and Ba²⁺ currents (I _Ba) through voltage-dependent L-type Ca²⁺ channels in adult rat ventricular myocytes. Neither the kinetics nor the voltage-dependent properties of the currents are affected. The effect of VIP on I _Ba is dose dependent with a half-maximal concentration of approximately 0.4 μM. The onset of the effect of VIP and the recovery phase are slow, suggesting the involvement of an intracellular second messenger. The effect of VIP on I _Ba is antagonized by a peptide analog of the growth hormone releasing factor ([Ac-Tyr¹, D-Phe²]-GRF) which belongs to the same peptide family as VIP. Although VIP and the β-adrenergic receptor agonist isoproterenol (ISO) enhance I _Ba peak amplitudes to approximately the same extent, the effect of VIP is not seen on all cells. Only approximately 50% of the isolated myocytes respond to 5 μM VIP, whereas 95% of the cells respond to ISO. Similar results were obtained using the amphotericin B perforated-patch whole-cell-recording technique, suggesting that the variable response to VIP does not reflect the loss of a pivotal intracellular regulator. The gastrointestinal hormone secretin, a peptide structurally related to VIP, also potentiates I _Ba in adult rat ventricular myocytes, although secretin is substantially more potent than VIP (half-maximal concentration for secretin is about 0.7 nM). Taken together, these results suggest that the VIP- (and secretin-) induced potentiation of I _Ba in adult rat ventricular myocytes is mediated through a non-VIP-preferring class of VIP receptors. Received: 7 December 1995/Received after revision and accepted: 31 May 1996     

Developmental changes in Ca2+ currents from newborn rat cardiomyocytes in primary culture

Electrophysiological characteristics of neonatal rat ventricular cardiomyocytes in primary culture were studied using the whole-cell patch-clamp recording technique. Cell size, estimated by measurement of membrane capacitance, was significantly increased throughout the culture from 22.4±5.4 pF at day 2 to 55.0±16.1 pF at day 7, reflecting the hypertrophic process which characterises postnatal cell development. The Ca²⁺ current was investigated at day 2 and 7 of the culture which constituted the early postnatal and maximally developed stages, respectively, of isolated cells in our experimental conditions. At 2 days of culture, two types of Ca²⁺ current could be distinguished, as also observed in freshly dissociated newborn ventricular cells. From their potential dependence and pharmacological characteristics, they could be attributed to the T- (I _Ca-T) and L-type (I _Ca-L) Ca²⁺ current components. After 7 days of culture, only the latterI _Ca-L was present and its density was significantly increased when compared to the density in 2-day-old cells, but lower than that obtained in freshly dissociated adult cells. As the age of the culture progressed, the steady-state inactivation curve was shifted toward negative potentials, in the direction of the inactivation curve obtained for adult cells. Compared to the serum-free control conditions, the density ofI _Ca-L was significantly increased in the presence of fetal calf serum throughout the culture. Consequently, the density ofI _Ca-L obtained in 7-day-old cells was similar to the density ofI _Ca-L obtained in freshly dissociated adult cardiac cells. These results show that in rat neonatal ventricular cardiomyocytes, the changes in Ca²⁺ current during development in primary culture can be compared to that observed in vivo during the first weeks of the postnatal period. The data suggest that the composition of the culture medium is a conditioning factor in the development of cardiac cells in culture. However, the determination and the role of specific factors contained in the serum need to be investigated. The data are also discussed in terms of a possible correlation between the expression and maturation of the Ca²⁺ current components and the capabilities of the neonatal cardiac cells to proliferate and/or to hypertrophy. For these reasons primary cultures of neonatal rat cardiac cells could constitute a valuable in vitro model for studies of postnatal development.