Rem-GTPase regulates cardiac myocyte L-type calcium current

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca²⁺-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK–LTCC interactions is untested.

Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca²⁺ current (I_Ca,L) via LTCC in murine cardiomyocytes.

Methods and Results: Rem knockout mice (Rem^?/?) were engineered, and I_Ca,L and Ca²⁺-handling properties were assessed. Rem^?/? ventricular cardiomyocytes displayed increased I_Ca,L density. I_Ca,L activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type I_Ca,L. Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem^?/?. Cell shortening was not significantly different. Increased I_Ca,L density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I_Ca,L density, Rem^?/? cardiomyocyte Ca²⁺ twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem^?/? LTCC can account for the paradoxical decrease of Ca²⁺ transients.

Conclusions: This is the first demonstration that loss of an RGK protein influences I_Ca,L in vivo in cardiac myocytes.     

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I_Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca²⁺ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I_Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca²⁺ influx via LTCCs on AC activity, leading to the inability to inhibit AC.     

A Xanthine-Derivative K+-Channel Opener Protects against Serotonin-Induced Cardiomyocyte Hypertrophy via the Modulation of Protein Kinases

This study investigated whether KMUP-1, a xanthine-derivative K⁺ channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca²⁺ channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 μM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 μM), and attenuated by the LTCC blocker verapamil (1 μM) and the 5-HT_2A antagonist ketanserin (0.1 μM), but unaffected by the 5-HT_2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca²⁺ currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (I_Ca,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 μM). Serotonin-increased I_Ca,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 μM and chelerythrine, 1 μM) while the current was enhanced by the PKC activator PMA, (1 μM) but not the PKA activator 8-Br-cAMP (100 μM), and was abolished by KMUP-1. In contrast, serotonin-increased I_Ca,L was blunted by the PKG activator 8-Br-cGMP (100 μM), but unaffected by the PKG inhibitor KT5823 (1 μM). Notably, KMUP-1 blocked serotonin-increased I_Ca,L but this was partially reversed by KT5823. In conclusion, serotonin-increased I_Ca,L could be due to activated 5-HT_2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Regulation of the slow Ca++ channels of myocardial cells

Contraction of the heart is regulated by a number of mechanisms, such as neurotransmitters, hormones, autacoids, pH, intracellular ATP, and Ca⁺⁺ ions. These actions are mediated, at least in part, by actions on the sarcolemmal slow (L-type) Ca⁺⁺ channels, exerted directly or indirectly. The major mechanisms for the regulation of the slow Ca⁺⁺ channels of myocardial cells includes the following. cAMP/PK-A phosphorylation stimulates the slow Ca` channel activity, whereas cGMP/PK-G phosphorylation inhibits. DAG/PK-C phosphorylation and tyrosine kinase phosphorylation are suggested to stimulate the slow Ca⁺⁺ channel activity. Intracellular application of G_s protein increases the slow Ca⁺⁺ currents (ICa(L)). Lowering of intracellular ATP inhibits I_Ca(L). Acidosis and increase in [Ca]_i inhibits I_Ca(L). A number of changes in the Ca⁺⁺ channels also occur during development and aging. Thus, it appears that the slow Ca⁺⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors, and thereby control can be exercised over the force of contraction of the heart.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Ginseng Gintonin Activates the Human Cardiac Delayed Rectifier K+ Channel: Involvement of Ca2+/Calmodulin Binding Sites

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca²⁺]_i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K⁺ (I_Ks) channel is a cardiac K⁺ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I_Ks channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I_Ks channel activity by expressing human I_Ks channels in Xenopus oocytes. We found that gintonin enhances I_Ks channel currents in concentration- and voltage-dependent manners. The EC₅₀ for the I_Ks channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I_Ks channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP₃ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I_Ks channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca²⁺]_i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I_Ks channel. However, gintonin had no effect on hERG K⁺ channel activity. These results show that gintonin-mediated enhancement of I_Ks channel currents is achieved through binding of the [Ca²⁺]_i/CaM complex to the C terminus of KCNQ1 subunit.     

Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca²⁺ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of I_Ca,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late I_Ca,L (Ca²⁺ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak I_Ca,L, which is essential for proper excitation–contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late I_Ca,L with minimal effect on peak current. Scaling our investigation from a human Ca_V1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces I_Ca,L noninactivating component in a human Ca_V1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late I_Ca,L suppresses EADs and EATs (early after Ca²⁺ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca²⁺ transient, and (3) late I_Ca,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late I_Ca,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of Ca_V1.2 channels rather than blocking their pore, largely preserving contractility.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Properties of the apamin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigtaenia coli

With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca²⁺-dependent K⁺ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I _SK(Ca)). This component did not show the temporal inactivation;I _SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI _SK(Ca) is very sensitive to changes in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ): a decrease in [Ca²⁺] _i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca²⁺ channels of L-type was not an obligatory condition for activation ofI _SK(Ca). The current-voltage relationship forI _SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Regulation of calcium current through L-type calcium channels (I _Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI _Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI _Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI _Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI _Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI _Ca,L. 8-Bromo-cAMP (1 μM) increasedI _Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI _Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI _Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI _Ca,L (probably through activation of PKA). PKG apparently mediatesI _Ca,L drops evoked by cAMP at high concentrations and by cGMP.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).