Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.     

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ "window" current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.     

Low Ba2+ and Ca2+ induce a sustained high probability of repolarization openings of L-type Ca2+ channels in hippocampal neurons: physiological implications.

Openings of single L-type Ca2+ channels following repolarization to negative membrane potentials from a depolarizing step (repolarization openings, ROs) have been described previously in brain cell preparations. However, these ROs have been reported to occur only infrequently. Here we report that the frequency of ROs in cell-attached patches of cultured rat hippocampal neurons can be increased dramatically by lowering the pipette Ba2+ concentration to 20 mM from the usual 90-110 mM. This increased opening probability can last for hundreds to thousands of milliseconds following repolarization. Current-voltage analyses of open probability show that the depolarization pulse threshold for inducing ROs in 20 mM Ba2+ is -10 to 0 mV but that the probability of ROs reaches maximal levels following depolarizing pulses that approach the apparent null (equilibrium) potential for Ba2+. Comparable current-voltage curves in 110 mM Ba2+ from a more positive holding potential (-50 mV) indicate that membrane surface charge screening accounts for some, but not all, of the effect of lowering the Ba2+ concentration. Consequently, current-dependent inactivation or some other ion-dependent mechanism (e.g., ion binding inside the pore) also appears to regulate this potentially major pathway of Ca2+ entry. A high probability of ROs also can be induced under relatively physiological conditions (5-ms depolarizing steps, 2-5 mM Ca2+ in the pipette). Thus, the high open probability state at negative potentials may underlie the long Ca2+ tail currents in hippocampus that were described previously and appears to have major implications for physiological functions (e.g., the slow Ca(2+)-dependent afterhyperpolarization), particularly in brain neurons.     

Molecular coupling of a Ca2+-activated K+ channel to L-type Ca2+ channels via alpha-actinin2

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca(2+)-activated K(+) channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca(2+) concentration [Ca(2+)(i)] with changes in K(+) conductance and membrane potential, associate with L-type Ca(2+) channels; Ca(v)1.3 and Ca(v)1.2 through a physical bridge, alpha-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca(2+) channels, instead, the 2 channels colocalize via their interaction with alpha-actinin2 cytoskeletal protein. The association of SK2 channel with alpha-actinin2 localizes the channel to the entry of external Ca(2+) source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Ca(v)1.3 Ca(2+) channels. Null deletion of Ca(v)1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca(2+) channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.     

Ca2+ influx through stretch-activated cation channels activates maxi K+ channels in porcine endocardial endothelium.

The endocardial endothelium is an important modulator of myocardial function. The present study demonstrates the existence of a stretch-activated Ca(2+)-permeable cation channel and of a Ca(2+)-activated K+ channel in the endocardial endothelium of the porcine right atrium. The stretch-activated channel is permeable for K+, Na+, Ca2+, and Ba2+, with mean conductances of approximately 32 pS for the monovalent cations and approximately 13 pS for divalent cations. The Ca(2+)-activated K+ channel has a mean conductance of 192 pS in symmetrical KCl. solution. Channel activity is strongly dependent on membrane potential and the cytosolic Ca2+ concentration. Half-maximal activation occurs at a cytosolic Ca2+ concentration of approximately 5 microM. The influx of Ca2+ through the stretch-activated channel is sufficient to activate the Ca(2+)-activated K+ channel in cell-attached patches. Upon activation of the stretch-activated channel, the cytosolic Ca2+ concentration increases, at least locally, to values of approximately 0.5 microM, as deduced from the open probability of the Ca(2+)-dependent K+ channel that was activated simultaneously. The stretch-activated channels are capable of inducing an intracellular Ca2+ signal and may have a role as mechanosensors in the atrial endothelium, possibly activated by atrial overload.     

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats

OBJECTIVE : Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS : Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS : ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS : We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.     

Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels

It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+. With 2 mmol/L Ca2+ in the external solution, we observed sparks at the beginning of every evoked AP at numerous locations. Each spark was observed repeatedly at a fixed location and began during a limited interval after the AP peak. These sparks occurred with a probability of approximately unity. However, the chance that an LCC does not open during the interval when a spark is triggered is quite high ( approximately 0.13). Therefore, because single channels open with a probability significantly lower than 1, more than one LCC must be available to ensure that sparks are triggered with a probability of approximately unity. We conclude that it is likely that a cluster of LCCs is involved in gating a cluster of ryanodine receptors at the beginning of an AP.     

AKAP150 is required for stuttering persistent Ca2+ sparklets and angiotensin II-induced hypertension

Hypertension is a perplexing multiorgan disease involving renal primary pathology and enhanced angiotensin II vascular reactivity. Here, we report that a novel form of a local Ca2+ signaling in arterial smooth muscle is linked to the development of angiotensin II-induced hypertension. Long openings and reopenings of L-type Ca2+ channels in arterial myocytes produce stuttering persistent Ca2+ sparklets that increase Ca2+ influx and vascular tone. These stuttering persistent Ca2+ sparklets arise from the molecular interactions between the L-type Ca2+ channel and protein kinase Calpha at only a few subsarcolemmal regions in resistance arteries. We have identified AKAP150 as the key protein, which targets protein kinase Calpha to the L-type Ca2+ channels and thereby enables its regulatory function. Accordingly, AKAP150 knockout mice (AKAP150-/-) were found to lack persistent Ca2+ sparklets and have lower arterial wall intracellular calcium ([Ca2+]i) and decreased myogenic tone. Furthermore, AKAP150-/- mice were hypotensive and did not develop angiotensin II-induced hypertension. We conclude that local control of L-type Ca2+ channel function is regulated by AKAP150-targeted protein kinase C signaling, which controls stuttering persistent Ca2+ influx, vascular tone, and blood pressure under physiological conditions and underlies angiotensin II-dependent hypertension.     

Inflammatory modulation of calcium-activated potassium channels in canine colonic circular smooth muscle cells

BACKGROUND & AIMS: The characteristics of colonic circular smooth muscle slow waves are altered during inflammation. The aim of this study was to examine whether inflammation modulates the open-state probability of Ca2+-activated K+ (KCa) channels in these cells to contribute to these alterations. METHODS: The experiments were performed on freshly dissociated single smooth muscle cells from the canine colon using standard patch clamp methods. Inflammation was induced by mucosal exposure to ethanol and acetic acid. RESULTS: Inflammation decreased the open-state probability of large-conductance KCa (BK) channels in the cell-attached and excised inside-out configurations. The voltage sensitivity of the channels was also reduced during inflammation. Inflammation had no significant effect on the large, medium, and small conductances or the unitary current levels of channel openings. However, it decreased the maximum number of simultaneous channel openings. The channels were Ca2+-dependent and were blocked by tetraethylammonium and charybdotoxin in normal and inflamed cells. CONCLUSIONS: Inflammation decreases the open-state probability of BK channels. This may partially reverse the decrease in duration and amplitude of slow waves and depolarization of membrane potential seen in inflammation.     

Ca2+-activated synexin forms highly selective, voltage-gated Ca2+ channels in phosphatidylserine bilayer membranes.

Synexin, a cytosolic protein that mediates Ca2+-dependent membrane fusion, was incorporated into acidic phospholipid bilayers, formed at the tip of a patch pipet. The pipet was filled with a high-Ca2+ solution (50 mM) and immersed in a chamber containing a low-Ca2+ solution (1 mM). Brief exposures of the bilayer to synexin increased the capacitance of the bilayer by a factor of 10 and decreased the membrane resistance by a factor of 20. Reduction of Ca2+ in the chamber to 1 microM caused an abrupt increase in the current required to hold the pipet potential at 0 mV. Under certain conditions channel events could be detected, often occurring in bursts. Consistently, open-time histograms were found to be voltage-dependent and to exhibit one time constant in the time range examined here. The slope conductance for the synexin channel was estimated as 10.2 +/- 2.1 pS for the large Ca2+ gradient with low chamber Ca2+. However, for symmetrical, low-Cl- solutions containing 25 mM Ca2+ the conductance was 26.5 +/- 5.2 pS. Ion-replacement studies showed the synexin channel to much prefer Ca2+ over Ba2+ or Mg2+. Cd2+, a potent blocker of other voltage-gated Ca2+ channels at 100 microM, blocked synexin channels only at very high concentrations (greater than or equal to 10 mM). Similarly, nifedipine, an inhibitor of the nonactivating Ca2+ channel, was effective only at extremely high concentrations (greater than 300 microM). The high selectivity for Ca2+ and the lack of response of the channel to various drugs known to block Ca2+ channels thus distinguish the synexin channel from other types of Ca2+ channels hitherto reported.     

Beta-adrenergic stimulation of L-type Ca2+ channels in cardiac myocytes requires the distal carboxyl terminus of alpha1C but not serine 1928

Beta-adrenoceptor stimulation robustly increases cardiac L-type Ca2+ current (ICaL); yet the molecular mechanism of this effect is still not well understood. Previous reports have shown in vitro phosphorylation of a consensus protein kinase A site at serine 1928 on the carboxyl terminus of the alpha1C subunit; however, the functional role of this site has not been investigated in cardiac myocytes. Here, we examine the effects of truncating the distal carboxyl terminus of the alpha1C subunit at amino acid residue 1905 or mutating the putative protein kinase A site at serine 1928 to alanine in adult guinea pig myocytes, using novel dihydropyridine-insensitive alpha1C adenoviruses, coexpressed with beta2 subunits. Expression of alpha1C truncated at 1905 dramatically attenuated the increase of peak ICaL induced by isoproterenol. However, the point mutation S1928A did not significantly attenuate the beta-adrenergic response. The findings indicate that the distal carboxyl-terminus of alpha1C plays an important role in beta-adrenergic upregulation of cardiac L-type Ca2+ channels, but that phosphorylation of serine 1928 is not required for this effect.     

Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.     

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.     

Behavior of nonselective cation channels and large-conductance Ca2+-activated K+ channels induced by dynamic changes in membrane stretch in cultured smooth muscle cells of human coronary artery

INTRODUCTION: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery. METHODS AND RESULTS: In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+-activated K+ (BK(Ca)) channel. The single-channel conductances of SA cation and BK(Ca) channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BK(Ca) channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BK(Ca) channel activity was greatly enhanced, but the activity of SA cation channels disappeared. CONCLUSION: This study is the first to demonstrate that the behavior of SA cation and BK(Ca) channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.     

Modulation of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in iron overload.

Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca2+ channel modifiers to alter Fe2+ uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L 59Fe2+ and 5 mmol/L ascorbate resulted in specific 59Fe2+ uptake of 20.4+/-1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific 59Fe2+ uptake by 60+/-7% (P<0.01), which suggested that a component of myocardial Fe2+ uptake depends on membrane voltage. Accordingly, 59Fe2+ uptake was inhibited by 10 micromol/L nifedipine (45+/-12%, P<0.02) and 100 micromol/L Cd2+ (86+/-3%; P<0. 001) while being augmented by 100 nmol/L Bay K 8644 (61+/-18%, P<0. 01) or 100 nmol/L isoproterenol (40+/-12%, P<0.05). By contrast, uptake of 100 nmol/L ferric iron (59Fe3+) was significantly lower (1. 4+/-0.3 ng Fe per gram dry wt; P<0.001) compared with divalent iron. These data suggest that a component of Fe2+ uptake into heart occurs via the L-type Ca2+ channel in myocytes. To investigate this further, the effects of Fe2+ on cardiac myocyte L-type Ca2+ currents were measured. In the absence of Ca2+, noninactivating nitrendipine-sensitive Fe2+ currents were recorded with 15 mmol/L [Fe2+]o. Low concentrations of Fe2+ enhanced Ca2+ current amplitude and slowed inactivation rates, which was consistent with Fe2+ entry into the cell, whereas higher Fe2+ levels caused dose-dependent decreases in peak current. Fe3+ had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe2+ uptake occurs via L-type Ca2+ channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.     

Ca2+ overload evokes a transient outward current in guinea-pig ventricular myocytes.

There are 2 types of transient outward currents (Ito) in the hearts of various mammals: a 4-aminopyridine (4-AP) sensitive K+ current and a 4-AP resistant Ca2+ activated current, carried by Cl-, (referred to as I(to1) and I(to2), respectively). However, the I(to) has been considered to be absent in guinea-pig ventricular myocytes and so this study tested the hypothesis that I(to1) is generally absent in guinea-pig ventricular myocytes, but I(to2) appears under the condition of Ca2+ overload. Membrane currents were recorded by the whole-cell patch-clamp technique and Ca2+ overload was achieved by adding internal, and eliminating external, Na+ with subsequent enhancement of Ca2+ influx via the Na+-Ca2+ exchange. Under physiological conditions, I(to) could not be elicited by 300 ms-test pulse from -70 mV to 0 mV (n=32). However, under Ca2+ overload, a biphasic current resulting from the overlap of the L-type Ca2+ channel current and Ito was elicited (n=38). This I(to) was resistant to 4-AP (3 mmol/L, n=30) but sensitive to both anthrancene-9-carboxylic acid (9-AC, 3 mmol/L, n=8) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (100 micromol/L, n=3). Replacing K+ with Cs+ on both sides of the membrane failed to abolish I(to) (n=38). I(to) disappeared by lowering the external Cl- (n=3). The amplitude of I(to) was dependent on that of the L-type Ca2+ channel current (n=4). Because Ca2+ release from the sarcoplasmic reticulum was prevented by caffeine (5 mmol/L), I(to) was negligible (n=6). These results suggest that I(to1) is absent, but Ca2+ overload evokes I(to2) in guinea-pig ventricular myocytes.     

Transient receptor potential channel 6-mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger-mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 micromol/L) and 2-amino-4-phosphonobutyrate (75 micromol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 micromol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 micromol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6-containing channels.