Lack of tyrosine protein kinase regulation of L-type Ca2+ channel current in transfected cells stably expressing alpha1C-b Subunit.

Tyrosine protein kinase (Tyr-PK) regulation of L-type Ca2+ channel (CaL) current was studied in COS-7 cells expressing vascular smooth muscle-type alpha1C-b with no auxiliary subunit by using a whole-cell voltage clamp. The averaged peak amplitude of CaL currents was -0.33 +/- 0.03 at holding potential of -60 mV. Na(3)VO(4), genistein and phosphorylated p60(c-src) peptide had no effect on the current. Thus the alpha1C-b subunit may not be involved in Tyr-PK regulation of CaL current.

Inhibition of Ca2+-activated K+ channels by tyrosine phosphatase inhibitors in rat mesenteric artery.

To investigate the possible regulation of large-conductance Ca2+-activated K+ channels (BKCa) by tyrosine phosphatases (Tyr-PPs), single-channel currents of myocytes from rat mesenteric artery were recorded in open cell-attached patches. Two structurally different Tyr-PP inhibitors, sodium orthovanadate (Na3VO4) and dephostatin, were used. The channels (236 pS) evoked at +40 mV and pCa 6, were significantly inhibited by 1 mM Na3VO4 (-81+/-3%, n = 10; P < 0.005). Similarly, 100 microM dephostatin strongly inhibited the BKCa channels (-80+/-7%, n = 7 ; P < 0.05). Therefore, BKCa channels in vascular smooth muscle cells may be regulated by tyrosine phosphatase-dependent signal transduction pathways, whose inhibition could attenuate the channel activity.

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells.

To clarify interactions between the cytoskeleton and activity of L-type Ca(2+) (Ca(L)) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca(2+) current [I(Ba(L))] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-alpha-actin and anti-alpha-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I(Ba(L)) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I(Ba(L)) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I(Ba(L)) due to another unknown mechanism, e.g., a direct block of Ca(L) channels. In summary, actin filament disruption of VSM cells inhibits Ca(L) channel activity, whereas disrupting the microtubules does not.

Role of voltage-dependent and Ca(2+)-activated K(+) channels on the regulation of isometric force in porcine coronary artery.

We investigated the role of K(+) channels in the regulation of vascular tone in de-endothelialized porcine coronary artery. Isometric force and intracellular Ca(2+) ([Ca(2+)](i)) under resting conditions were increased by treatment with 4-aminopyridine (4-AP, 1 mM), an inhibitor of voltage-dependent K(+) (K(v)) channels, but not by tetraethylammonium chloride (TEA, 1 mM) or charybdotoxin (100 nM), both inhibitors of Ca(2+)-activated K(+) (K(Ca)) channels, or glibenclamide (10 microM), an inhibitor of ATP-sensitive K(+) channels. Under stimulated conditions with 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619), 4-AP as well as TEA or charybdotoxin increased isometric force and [Ca(2+)](i), but not glibenclamide. 4-AP was the most potent in terms of depolarization of membrane potential compared with TEA or glibenclamide in the presence or absence of EGTA. In the presence of U46619, a high concentration of 4-AP (10 mM) caused a further contraction with oscillations. The force oscillations induced by 4-AP were inhibited by diltiazem (10 microM), an inhibitor of voltage-dependent Ca(2+) channels, or TEA (1 mM), but not by glibenclamide (10 microM). These force oscillations may be associated with the periodic activation of K(Ca) channels. These findings suggested that 4-AP-sensitive K(v) channels play an important role in the control of vascular tone in both resting and stimulated conditions. Moreover, under stimulated conditions, K(Ca) channels also have an important role in the regulation of vascular tone. Dysfunction of these channels induces abnormal vasoconstriction and may be implicated in vascular diseases such as hypertension and vasospasm.

Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.

We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 degrees C). The peak IBa (Ca2+ channel current with 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca]i) to pCa 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 microM CMZ reduced the peak amplitude of IBa to 36.7+/-4.9% (n = 8); maximal effect occurred within 7-8 min. Peak IBa continued to decrease even after washing out the CMZ. Recovery of IBa was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 microM) in the pipette solution, CMZ inhibited IBa to 27.8 +/-5.3% (n = 7). To exclude the possibility that other Ca2+/ CaM-dependent kinases and phosphatases may regulate Ca2+ channel activity, we examined the effect of CMZ on IBa when [Ca]i was reduced by use of Ca2+/EGTA-buffered pipette solutions. At pCa approximately equal to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inhibited IBa to 33.4+/-5.9% (n = 14) with a median inhibitory concentration (IC50) value of 0.29 microM. The activation curve (pCa approximately equal to 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased IBa progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca2+ channels.

Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002.

Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca2+. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60(c-src), and PKC have been reported to stimulate the Ca2+ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca2+ channels. The Ca2+-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application of 50 nM AngII significantly increased the amplitude of Ba2+ currents through the voltage-gated Ca2+ channels (IBa) by 34. 5+/-9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 microM), a selective inhibitor of Tyr-PK, AngII failed to stimulate IBa (n=5). AngII stimulation of IBa was also prevented by (5 microM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 microM), an inhibitor of PKC, did not prevent the effect of AngII on IBa (n=6). These results suggest that AngII may stimulate the Ca2+ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C.

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells.

To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5'-triphosphate (ATP)-sensitive K+ (KATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal KATP currents (at 0 mV) induced by metabolic inhibition were 48.9+/-2.8 pA/pF in control (n=48), 34.3+/-3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5+/-3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na+ current and inward-rectifying K+ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of KATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional KATP channels. Furthermore, in four out of seven control cells tested, outward K+ currents were stimulated by diazoxide, which is a potent K+ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional KATP channels. The larger population of KATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2.

Carbachol inhibition of Ca2+ currents in ventricular cells obtained from neonatal and adult rats.

We investigated the postnatal developmental changes produced by the muscarinic receptor agonist, carbachol, on the L-type Ca2+ current (ICa(L)) in neonatal (aged 5 to 7 days) and adult (aged 2 to 5 months) rat ventricular cells by using the whole-cell voltage clamp technique. Carbachol inhibited the isoproterenol-stimulated ICa(L). The maximal inhibition was 89.3 +/- 4.8% (n = 5) in neonatal cells and 17.7 +/- 7.7% (n = 9) in adult cells. Carbachol inhibited the forskolin-stimulated ICa(L) to almost same extent as the isoproterenol-stimulated ICa(L). In the cells pretreated with pertussis toxin, carbachol failed to inhibit the isoproterenol-stimulated ICa(L), indicating that carbachol produced its effect via a pertussis toxin-sensitive G-protein pathway. The effects of carbachol in adult cells became more pronounced, increasing from 17.7% to 54.8% (n = 11), with the addition of the synthetic inhibitory G-protein alpha subunit (Gi alpha) (1 microM) to the reaction. Conversely, the alpha subunit of another pertussis toxin-sensitive synthetic G-protein (G(o) alpha, 1 microM) failed to mimic the effect of Gi alpha. These results suggest that, in rat ventricular cells, (1) the action of carbachol on ICa(L) showed a marked decrease during development; (2) the decrease in the effect of carbachol in adult cells is in part due to a decrease in the activity of pertussis toxin-sensitive G protein, especially Gi alpha.

Intracellular application of calmidazolium increases Ca2+ current through activation of protein kinase A in cultured vascular smooth muscle cells.

In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak IBa (Ca2+ channel using 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To test the effect of CaM on IBa, 1 microM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]i). The amplitude of maximally activated IBa was -4.3 +/- 0.5 pA/pF (n = 13) for control and -8.1 +/- 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of IBa was not abolished when 5 microM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (-9.5 +/- 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal IBa. On the other hand, the CMZ stimulation of IBa was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca2+/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated IBa: control, -4.6 +/- 0.4 pA/pF (n = 10); MIBMX, -9.6 +/- 1.2 (n = 8), and CMZ, -7.9 +/- 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated IBa by +42%: before, -3.7 +/- 0. 7 pA/pF; after, -5.2 +/- 1.0 (n = 6). In conclusion, Ca2+ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of IBa might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.

Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes.

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 microM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 microM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

Inhibition of Ca2+ current in neonatal and adult rat ventricular myocytes by the tyrosine kinase inhibitor, genistein.

Yokoshiki et al. (Yokoshiki, H., Sumii, K., Sperelakis, N., 1996. Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor, genistein and its inactive analog, daidzein. J. Mol. Cell. Cardiol. 28, 807-814) reported that genistein and daidzein inhibited L-type Ca2+ current (I(Ca)(L)) in young rat ventricular cells. Therefore, we investigated the developmental differences in the effect of genistein, an inhibitor of tyrosine kinases, on I(Ca)(L) in freshly-isolated neonatal (3-7 days) and adult (2-5 months) rat ventricular myocytes using whole-cell voltage clamp and single-channel recordings (cell-attached configuration). For whole-cell voltage clamp, I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV by applying a 300 ms pulse from a holding potential of -40 mV. To isolate I(Ca(L), the pipette solution was Cs+-rich and the bath solution was Na+-, K+-free. Ca2+ (1.8 mM) was used as charge carrier. Bath application of 100 microM genistein (sufficient for maximal effect) decreased the basal I(Ca)(L) by 43.3% (n = 27) in neonatal cells and by 30.6% (n = 14) in adult cells (P < 0.05). In the current/voltage relationships, the potential of peak I(Ca)(L) was shifted to the right by genistein by 8.6 mV in neonatal and by 9.3 mV in adult cells. Genistein produced a shift of the steady-state inactivation curve (to the left) in neonatal cells (from -16.0 +/- 3.9 mV to -26.1 +/- 4.2 mV; P < 0.05) and in adult cells (-15.9 +/- 3.2 mV to -22.9 +/- 3.3 mV; P < 0.05); the slope factor was not affected. For single-channel recordings in cell-attached patches, Ca2+ currents were evoked by applying a 150 ms pulse from a holding potential of -40 mV to a test potential of 0 mV. The pipette solution contained 110 mM Ba2+ (as charge carrier), and the bath solution contained 150 mM K+ (to bring resting potential to near zero). Genistein (50 microM) decreased the open probability of the channels from 2.8% to 0.75% (P < 0.05) in absence of Bay K 8644, and from 24% to 7.9% (P < 0.05) in presence of Bay K 8644; the mean open time and the slope conductance of the currents were not affected. In conclusion, (1) genistein inhibits the basal I(Ca)(L) in rat ventricular cells and (2) the inhibition of I(Ca)(L) by genistein is greater in immature cells than in adult cells.

Review of some actions of taurine on ion channels of cardiac muscle cells and others.

1. Taurine has recently been known to protect against ischemia and heart failure. Taurine possesses plenty of actions on the ion channels and transports, but is very non-specific. 2. Taurine may directly and indirectly help to regulate the [Ca]i level by modulating the activity of the voltage-dependent Ca2+ channels (also dependent on [Ca]i/[Ca]o), by regulation of Na+ channels, and secondly via Na-Ca exchange and Na(+)-taurine cotransport. 3. Taurine can prevent the Ca2+ ([Ca]o or [Ca]i)-induced cardiac functions. 4. Therefore, it seems possible that taurine could exert the potent cardioprotective actions even under the condition of low [Ca]i levels as well as under the Ca2+ overload condition. 5. The electrophysiological actions of taurine on cardiomyocytes, smooth muscle cells, and neurons from recent studies are summarized.

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

beta-adrenergic receptor (beta-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 (IBa) instead of Ca2+ (ICa). The EC50 and maximum increase of peak currents were 43.7 +/- 7.9 nM and 1.8 +/- 0.1-fold for ICa and 23.3 +/- 4.7 nM and 2.4 +/- 0.1-fold for IBa. When cells were dialyzed with the faster Ca2+ chelator, BAPTA, both sensitivity and maximum response of ICa 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 ICa 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 ICa 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 beta-AR stimulation.

ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells.

ATP-sensitive K+ (KATP) channels are therapeutic targets for several diseases, including angina, hypertension, and diabetes. This is because stimulation of KATP channels is thought to produce vasorelaxation and myocardial protection against ischemia, whereas inhibition facilitates insulin secretion. It is well known that native KATP channels are inhibited by ATP and sulfonylurea (SU) compounds and stimulated by nucleotide diphosphates and K+ channel-opening drugs (KCOs). Although these characteristics can be shared with KATP channels in different tissues, differences in properties among pancreatic, cardiac, and vascular smooth muscle (VSM) cells do exist in terms of the actions produced by such regulators. Recent molecular biology and electrophysiological studies have provided useful information toward the better understanding of KATP channels. For example, native KATP channels appear to be a complex of a regulatory protein containing the SU-binding site [sulfonylurea receptor (SUR)] and an inward-rectifying K+ channel (Kir) serving as a pore-forming subunit. Three isoforms of SUR (SUR1, SUR2A, and SUR2B) have been cloned and found to have two nucleotide-binding folds (NBFs). It seems that these NBFs play an essential role in conferring the MgADP and KCO sensitivity to the channel, whereas the Kir channel subunit itself possesses the ATP-sensing mechanism as an intrinsic property. The molecular structure of KATP channels is thought to be a heteromultimeric (tetrameric) assembly of these complexes: Kir6.2 with SUR1 (SUR1/Kir6.2, pancreatic type), Kir6.2 with SUR2A (SUR2A/ Kir6.2, cardiac type), and Kir6.1 with SUR2B (SUR2B/Kir6.1, VSM type) [i.e., (SUR/Kir6.x)4]. It remains to be determined what are the molecular connections between the SUR and Kir subunits that enable this unique complex to work as a functional KATP channel.

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats.

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.

Tyrosine kinase inhibitor, genistein, inhibits macroscopic L-type calcium current in rat portal vein smooth muscle cells.

The effect of genistein, a specific tyrosine kinase inhibitor, was tested on the slow (L-type) Ca2+ current (ICa(L)) of vascular smooth muscle cells from freshly isolated rat portal vein, using whole-cell voltage clamp. To isolate ICa(L), the pipette contained high Cs+ and the bath contained 140 mM tetraethylammonium (TEA) to block K+ currents. Bath application of genistein decreased ICa(L) in a concentration-dependent manner within 3-6 min. The concentration for half-maximal inhibition (IC50) was 54.9 microM (at a holding potential of -40 mV). At a concentration of 300 microM, genistein produced nearly complete inhibition of ICa(L). The inhibitory effect of genistein was not reversed after washout for up to 5 min. The potential for half-inhibition (V1/2) of the steady-state inactivation curve for ICa(L) was shifted to the left by genistein (10.6 mV at 50 microM), suggesting that genistein exerts a voltage-dependent block. Superfusion with daidzein, an inactive analog of genistein, had no inhibitory effect on ICa(L) at concentrations as high as 300 microM. These results may suggest that the L-type Ca2+ channels in vascular smooth muscle cells are possibly modulated by endogenous tyrosine kinase activity. That is, tonic phosphorylation by tyrosine kinases maintains the Ca2+ channels in an available state for activation by depolarization. Thus, the vascular tone may be controlled by tyrosine kinase activity.

Tyrosine kinases modulate the activity of single L-type calcium channels in vascular smooth muscle cells from rat portal vein.

In a previous study, we demonstrated that extracellular application of the tyrosine kinase inhibitor genistein produced a dose-dependent inhibition of the macroscopic slow (L-type) Ca2+ currents of vascular smooth muscle (VSM) cells, and that daidzein, an inactive analog of genistein, had no such effect. These results suggested that the L-type Ca2+ channels in VSM cells may be modulated by endogenous tyrosine kinase activity. To confirm and extend those findings, the effect of genistein on the activity of single Ca2+ channels was examined in freshly isolated single VSM cells from rat portal vein, using the cell-attached patch-clamp technique. The pipette solution contained 90 mM Ba2+ as charge carrier and 0.5 microM Bay K 8644 (to enhance basal activity of the channels), and the bath contained 140 mM KCl to "zero" the resting membrane potential. Depolarizing pulses to 0 mV, from a holding potential of -80 mV, elicited inward unitary currents that were blocked by 1 microM nifedipine (n = 6). The slope conductance of the unitary Ca2+ currents gave a value of 21.5 +/- 0.4 pS (n = 9) for the Ca2+ channels. Bath application of genistein (50 microM) did not change the unit amplitude and slope conductance: the conductance in the presence of genistein was 22.2 +/- 0.5 pS (n = 6). However, compared with controls, the activity of single Ca2+ channels was significantly inhibited by genistein in a dose-dependent fashion. The ensemble-averaged currents were decreased by 48.4 +/- 11.2% with 50 microM genistein; 100 microM genistein inhibited the Ca2+ currents by 76.8 +/- 11.8%. The open probability (NPo) was decreased by 50 microM genistein from 0.24 +/- 0.09 to 0.11 +/- 0.07. Single-channel kinetic analysis showed that genistein decreased the mean open time and prolonged the mean closed time. The inhibitory effect of genistein on the Ca2+ channel activity occurred within 3 min, and it could be reversed by washout within 3-5 min. Daidzein, in concentrations up to 300 microM, produced no change in the activity of the single Ca2+ channels. These results demonstrate that genistein inhibits the activity of the L-type Ca2+ channels in VSM cells, suggesting that the availability of the channels for voltage activation may be maintained through tonic tyrosine kinase phosphorylation.

The novel calcium sensitizer levosimendan activates the ATP-sensitive K+ channel in rat ventricular cells.

Levosimendan, a new Ca++-sensitizing and positive inotropic agent, was reported to act as a coronary vasodilator and protect ischemic myocardium. To elucidate the mechanisms of these actions, the possible electrophysiological effects of levosimendan on isolated rat ventricular cells were examined by the patch-clamp technique with whole-cell and single-channel recordings. Levosimendan (3 and 10 microM) markedly shortened action potential duration and activated an outward current at potentials positive to -70 mV. The increased current was abolished by glibenclamide, a blocker of the ATP-sensitive K+ (K[ATP]) current. Stimulation of K[ATP] current was dose dependent, with an EC50 value of 4.7 microM; a maximal effect occurred at 30 microM. The L-type Ca++ current was not affected by levosimendan (0.2-10 microM). In single-channel current recording in open cell-attached patches, K[ATP] channels, which had been inhibited by 0.3 mM ATP, were activated by levosimendan. However, levosimendan did not stimulate the K[ATP] channels that exhibited high spontaneous activity in ATP-free solution. Levosimendan also could not stimulate K[ATP] channels that had rundown in ATP-free solution. However, levosimendan could stimulate rundown K[ATP] channels that were reactivated by nucleotide diphosphates. K[ATP] channels inhibited by 0.5 mM AMP-PNP, a nonhydrolyzable ATP analog, were not stimulated by levosimendan; however, the channels were stimulated by levosimendan in the presence of 30 to 50 microM ADP. Levosimendan stimulates cardiac K[ATP] channels that are suppressed by intracellular ATP. It appears that levosimendan acts synergistically with nucleotide diphosphates. These properties of levosimendan may help protect ischemic myocardium because activation of K[ATP] channels by levosimendan would likely occur in ischemic regions in which intracellular ADP concentration is increased and intracellular ATP concentration is decreased.