Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4)(-3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 microM); 100 microM genistein stimulated I(Cl.vol) by 122.4 +/- 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 150 microM) and tamoxifen (20 microM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(-) current in 1T. In contrast to the stimulatory effects of genistein, 100 microM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 +/- 4.9% and 40.9 +/- 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4)(-3) (1 mM) reduced I(Cl.vol) by 53.5 +/- 4.5% (IC(50) = 249.6 microM). Pretreatment with VO(4)(-3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 microM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 microM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4)(-3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved.     

抗心律失常药RP62719对哺乳动物心室肌K+电流的抑制

使用全细胞膜片箝技术, 研究RP62719对内向整流钾电流(IK1)、瞬时外向钾电流(Ito)和延迟外向整流钾电流(IK)的作用, 并探讨其抗心律失常作用的机制.实验结果表明, 在指令电压为-100 mV时, RP62719可显著抑制豚鼠心室肌细胞IK1, 半数抑制浓度(IC50)为5.0±1.0 μmol/L.RP62719 10 μmol/L在+40 mV时对犬心室肌细胞Ito抑制率为84.0±4.4%, IC50为1.2±0.51 μmol/L.在+40 mV时, 50 μmol/L RP62719还可使豚鼠心室肌细胞IKstep 减少50.0±8.3%, IKtail减少56.0±4.9%, IC50分别为4.2±0.8 μmol/L和3.3±0.75 μmol/L.提示RP62719抗心律失常的离子机制与其对IK1、Ito及IK的抑制有关.     

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.     

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac IKs

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K(+) current (I(Ks)) via unknown mechanisms. In the present study, I(Ks) was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 microM H89; 200 microM H8; 50 microM H7; 1 microM bisindolylmaleimide I; 10 microM LY294002; 50 microM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 microM forskolin; 0.1 microM phorbol-12-myristate-13-acetate; 10 microM acetylcholine; 0.1 microM angiotensin II; 20 microM ATP), (iii) suppress G-protein activation (10 mM GDPbetaS), or (iv) disrupt the cytoskeleton (10 microM cytochalasin D), had little effect on the stimulation of I(Ks) by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 microM) strongly attenuated both the hyposmotic stimulation of I(Ks) in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Postnatal development has a marked effect on ventricular repolarization in mice

To better understand the mechanisms that underlie cardiac repolarization abnormalities in the immature heart, this study characterized and compared K(+) currents in mouse ventricular myocytes from day 1, day 7, day 20, and adult CD1 mice to determine the effects of postnatal development on ventricular repolarization. Current- and patch-clamp techniques were used to examine action potentials and the K(+) currents underlying repolarization in isolated myocytes. RT-PCR was used to quantify mRNA expression for the K(+) channels of interest. This study found that action potential duration (APD) decreased as age increased, with the shortest APDs observed in adult myocytes. This study also showed that K(+) currents and the mRNA relative abundance for the various K(+) channels were significantly greater in adult myocytes compared with day 1 myocytes. Examination of the individual components of total K(+) current revealed that the inward rectifier K(+) current (I(K1)) developed by day 7, both the Ca(2+)-independent transient outward current (I(to)) and the steady-state outward K(+) current (I(ss)) developed by day 20, and the ultrarapid delayed rectifier K(+) current (I(Kur)) did not fully develop until the mouse reached maturity. Interestingly, the increase in I(Kur) was not associated with a decrease in APD. Comparison of atrial and ventricular K(+) currents showed that I(to) and I(Kur) density were significantly greater in day 7, day 20, and adult myocytes compared with age-matched atrial cells. Overall, it appears that, in mouse ventricle, developmental changes in APD are likely attributable to increases in I(to), I(ss), and I(K1), whereas the role of I(Kur) during postnatal development appears to be less critical to APD.     

Extracellular Ba(2+) blocks the cardiac transient outward K(+) current

Ba(2+) is widely used as a tool in patch-clamp studies because of its ability to block a variety of K(+) channels and to pass Ca(2+) channels. Its potential ability to block the cardiac transient outward K(+) current (I(to)) has not been clearly documented. We performed whole cell patch-clamp studies in canine ventricular and atrial myocytes. Extracellular application of Ba(2+) produced potent inhibition of I(to) with an IC(50) of approximately 40 microM. The effects were voltage independent, and the inactivation kinetics were not altered by Ba(2+). The potency of Ba(2+) was approximately 10 times higher than that of 4-aminopyridine (a selective I(to) blocker with an IC(50) of 430 microM) under identical conditions. By comparison, Ba(2+) blockade of the inward rectifier K(+) current was voltage dependent; the IC(50) was approximately 20 times lower (2.5 microM) than that for I(to) when determined at -100 mV and was comparable to I(to) as determined at -60 mV (IC(50) = 26 microM). Ba(2+) concentrations of 相似文献   

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 muM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 muM) and the Src-family kinase inhibitor PP2 (10 muM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.     

Aging-associated changes in whole cell K(+) and L-type Ca(2+) currents in rat ventricular myocytes

The effect of aging on cardiac membrane currents remains unclear. This study examined the inward rectifier K(+) current (I(K1)), the transient outward K(+) current (I(to)), and the L-type Ca(2+) channel current (I(Ca,L)) in ventricular myocytes isolated from young adult (6 mo) and aged (>27 mo) Fischer 344 rats using whole cell patch-clamp techniques. Along with an increase in the cell size and membrane capacitance, aged myocytes had the same magnitude of peak I(K1) with a greater slope conductance but displayed smaller steady-state I(K1). Aged myocytes also had a greater I(to) with an increased rate of activation, but the I(to) inactivation kinetics, steady-state inactivation, and responsiveness to L-phenylephrine, an alpha(1)-adrenergic agonist, were unaltered. The magnitude of peak I(Ca,L) in aged myocytes was decreased and accompanied by a slower inactivation, but the I(Ca,L) steady-state inactivation was unaltered. Action potential duration in aged myocytes was prolonged only at 90% of full repolarization (APD(90)) when compared with the action potential duration of young adult myocytes. Aged myocytes from Long-Evans rats showed similar changes in I(to) and I(Ca,L) but an increased I(K1). These results demonstrate aging-associated changes in action potential, in morphology, and in I(K1), I(to), and I(Ca,L) of rat ventricular myocytes that possibly contribute to the decreased cardiac function of aged hearts.     

Antagonistic regulation of swelling-activated Cl- current in rabbit ventricle by Src and EGFR protein tyrosine kinases

Regulation of swelling-activated Cl(-) current (I(Cl,swell)) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates I(Cl,swell) and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). I(Cl,swell) evoked by hyposmotic swelling was increased 181 +/- 17% by 100 microM genistein, and the genistein-induced current was blocked by the selective I(Cl,swell) blocker tamoxifen (10 microM). Block of Src with PP2 (10 microM) stimulated tamoxifen-sensitive I(Cl,swell) by 234 +/- 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 microM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited I(Cl,swell) and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited I(Cl,swell). Several lines of evidence argue that the PP2-stimulated current was I(Cl,swell): 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl(-) gradients, and 4) the current reversed near the Cl(-) equilibrium potential. To rule out contributions of other currents, Cd(2+) (0.2 mM) and Ba(2+) (1 mM) were added to the bath. Surprisingly, Cd(2+) suppressed the decay of I(Cl,swell), and Cd(2+) plus Ba(2+) eliminated time-dependent currents between -100 and +100 mV. Nevertheless, these divalent ions did not eliminate I(Cl,swell) or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls I(Cl,swell), and regulation of I(Cl,swell) by the Src and EGFR kinase families of PTK is antagonistic.     

Tyrosine kinase and protein kinase C regulate L-type Ca(2+) current cooperatively in human atrial myocytes

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.     

Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction

Although the protein tyrosine kinase (PTK) inhibitor, genistein, has been widely used to investigate the possible involvement of PTK during reproductive functions, it is unknown whether it modulates sperm calcium channel activity. In the present study, we recorded T-type calcium currents (I(Ca,T)) in mouse spermatogenic cells using whole-cell patch clamp and found that extracellular application of genistein reversibly decreased I(Ca,T) in a concentration-dependent manner (IC(50) approximately 22.7 microM). To determine whether TK activity is required for I(Ca,T) inhibition, we found that peroxovanadate, a tyrosine phosphatase inhibitor, was ineffective in preventing the inhibitory effect of genistein. Furthermore, intracellular perfusion of the cells with ATP-gamma-S also did not alter the inhibitory effect of genistein. To further reveal the direct inhibitory mechanism of genistein on I(Ca,T), we applied into the bath lavendustin A, a PTK inhibitor structurally unrelated to genistein, and found that the current amplitude remained unchanged. Moreover, daidzein, an inactive structural analog of genistein, robustly inhibited the currents. The inhibitory effect of genistein on T-type calcium channels was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Genistein was observed to decrease sperm motility and to significantly inhibit sperm acrosome reaction (AR) evoked by zona pellucida. Using transfected HEK293 cells system, only Cav3.1 and Cav3.2, instead of Cav3.3, channels were inhibited by genistein. Since T-type calcium channels are the key components in the male reproduction, such as in AR and sperm motility, our data suggest that this PTK-independent inhibition of genistein on I(Ca,T) might be involved in its anti-reproductive effects.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

Donepezil is a strong antagonist of voltage-gated calcium and potassium channels in molluscan neurons

Donepezil is an acetylcholinesterase inhibitor used in Alzheimer's disease therapy. The neuroprotective effect of donepezil has been demonstrated in a number of different models of neurodegeneration including beta-amyloid toxicity. Since the mechanisms of neurodegeneration involve the activation of both Ca(2+)- and K(+)-channels, the study of donepezil action on voltage-gated ionic currents looked advisable. In the present study, the action of donepezil on voltage-gated Ca(2+)- and K(+)-channels was investigated on isolated neurons of the edible snail (Helix pomatia) using the two-microelectrodes voltage-clamp technique. Donepezil rapidly and reversibly inhibited voltage activated Ca(2+)-current (I(Ca)) (IC(50)=7.9 microM) and three types of high threshold K(+)-current: Ca(2+)-dependent K(+)-current (I(C)) (IC(50)=6.4 microM), delayed rectifier K(+)-current (I(DR)) (IC(50)=8.0 microM) and fast transient K(+)-current (I(Adepol)) (IC(50)=9.1 microM). The drug caused a dual effect on low-threshold fast transient K(+)-current (I(A)), potentiating it at low (5 microM) concentration, but inhibiting at higher (7 microM and above) concentration. Donepezil also caused a significant hyperpolarizing shift of the voltage-current relationship of I(Ca) (but not of any type of K(+)-current). Results suggest the possible contribution of the blocking effect of donepezil on the voltage-gated Ca(2+)- and K(+)-channels to the neuroprotective effect of the drug.     

Tyrosine kinases act directly on the alpha1 subunit to modulate Ca(v)2.2 calcium channels.

Voltage-operated calcium channels are modulated by tyrosine kinases in different cell types. In this study, I(Ba) was measured by the whole cell voltage-clamp technique in single COS-7 cells overexpressing the Ca(v)2.2 calcium channels encoding N-type currents. Bath application of genistein, a nonselective PTK inhibitor (50-300 microM), concentration-dependently inhibited calcium channel currents, whereas the inactive structural analogue, daidzein, was without effect over the same concentration range. Similarly, PP1, a src family-selective tyrosine kinase inhibitor, inhibited I(Ba) in a concentration-dependent manner (500 nM-5 microM) over a range of test potentials. Expression of the Ca(v)2.2alpha1 (alpha(1B)) subunit alone gave rise to functional channels, and genistein (100 microM) also inhibited currents elicited by the alpha(1B) subunit alone. These results indicate that tyrosine kinase inhibitors are likely to inhibit Ca(v)2.2 calcium channels via an action on the pore-forming alpha(1) subunit and suggest that an endogenous member of the Src family may play a physiological role in modulating these channels.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.     

Effects of endothelin-1 on K(+) currents from rat ventricular myocytes.

It has been suggested that the positive inotropic effect of the vasoactive peptide hormone, endothelin-1 (ET-1), involves inhibition of cardiac K(+) currents. In order to identify the K(+) currents modulated by ET-1, the outward K(+) currents of isolated rat ventricular myocytes were investigated using whole-cell patch-clamp recording techniques. Outward currents were elicited by depolarisation to +40 mV for 200 ms from the holding potential of -60 mV. Currents activated rapidly, reaching a peak (I(pk)) of 1310 +/- 115 pA and subsequently inactivating to an outward current level of 1063 +/- 122 pA at the end of the voltage-pulse (I(late)) (n = 11). ET-1 (20 nM) reduced I(pk) by 247.6 +/- 60.7 pA (n = 11, P < 0.01) and reduced I(late) by 323.2 +/- 43.9 pA (P < 0.001). The effects of ET-1 were abolished in the presence of the nonselective ET receptor antagonist, PD 142893 (10 microM, n = 5). Outward currents were considerably reduced and the effects of ET-1 were not observed when K(+) was replaced with Cs(+) in the experimental solutions; this indicates that ET-1 modulated K(+)-selective currents. A double-pulse protocol was used to investigate the inactivation of the currents. The voltage-dependent inactivation of the currents from potentials positive to -80 mV was fitted by a Boltzmann equation revealing the existence of an inactivating transient outward component (I(to)) and a noninactivating steady-state component (I(ss)). ET-1 markedly inhibited I(ss) by 43.0 +/- 3.8% (P < 0.001, n = 7) and shifted the voltage-dependent inactivation of I(to) by +3.3 +/- 1.2 mV (P < 0.05). Although ET-1 had little effect on the onset of inactivation of the currents elicited from a conditioning potential of -70 mV, the time-independent noninactivating component of the currents was markedly inhibited. In conclusion, the predominant effect of ET-1 was to inhibit a noninactivating steady-state background K(+) current (I(ss)). These results are consistent with the hypothesis that I(ss) inhibition contributes to the inotropic effects of ET-1.     

Calcium influx induced by activation of tyrosine kinase receptors in cultured bovine aortic endothelial cells

We studied the ionic currents activated by basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) in cultured bovine aortic endothelial cells (BAE-1) by using patch-clamp and single-cell fluorimetric calcium measurements. In whole-cell, voltage-clamp experiments at V(h) = -50 mV, the addition of either bFGF (20 ng/ml) or IGF-I (50 ng/ml) induced an inward current with similar amplitude, time course, and permeation properties. The response was dependent on receptor occupancy and showed a desensitisation in the continued presence of the factors. Ionic substitutions in whole-cell experiments indicated that the current barely discriminated among Na(+), Ca(+), and K(+) ions. Accordingly, stimulation with bFGF or IGF-I induced a dose-dependent [Ca(2+)](i) elevation completely due to entry from the extracellular medium, whereas no detectable release from internal stores was observed. Calcium influx was dependent on protein tyrosine kinase (PTK) activity; it was significantly inhibited by treatment with genistein or tyrphostin 47, two PTK inhibitors, and not affected by inactive analogues, daidzein, and tyrphostin 1. Moreover, addition of 200 microM Na(3)VO(4), an inhibitor of protein tyrosine phosphatase (PTP) activity, evoked the responses to the factors both in patch-clamp and in fluorimetric measurements. Cell-attached recordings using 100 mM CaCl(2) in the pipette showed that bFGF and IGF-I activate calcium-permeable channels with similar properties. These results provide evidence for a calcium influx induced by two factors that bind to tyrosine kinase receptors (RTK) in endothelial cells.