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To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase [D.sub.2] (PL[D.sub.2]), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-[delta] regulates the VEGF-induced activation of PLD/ERK. Thus PL[D.sub.2] could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-[delta] pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-[beta]-cyclodextrin (M[beta]CD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to M[beta]CD-treated ceils restored caveolar structures. Pretreatment with M[beta]CD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade. caveolin-1; protein kinase C-[delta]; signaling; vascular endothelial growth factor; phospholipase D

Jo, Su-Hyun, Chung-Hyun Cho, Soo Wan Chae, and Chin O. Lee. Role of protein kinase C in [[Alpha].sub.1]-adrenergic regulation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in guinea pig ventricular myocytes. Am J Physiol Heart Circ Physiol 279: H1661-H1668, 2000.--We investigated the role of protein kinase C (PKC) in [Alpha]-adrenergic regulation of intracellular [Na.sup.+] activity [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in single guinea pig ventricular myocytes. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and membrane potentials were measured with the [Na.sup.+]-sensitive indicator sodium-binding benzofuran isophthalate and conventional microelectrodes, respectively, at room temperature (24-26 [degrees] C) while myocytes were stimulated at a rate of 0.25-0.3 Hz. The PKC activator 4[Beta]-phorbol 12-myristate 13-acetate (PMA) decreased [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in a concentration-dependent manner. PMA (100 nM) produced a maximal decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] of 1.5 mM from 6.5 [+ or -] 0.4 to 5.0 [+ or -] 0.4 mM (means [+ or -] SE, n = 12, P [is less than] 0.01). The PMA concentration required for a half-maximal decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was 0.46 [+ or -] 0.13 nM (n = 3, P [is less than] 0.01). An inactive phorbol, [Alpha]-phorbol 12-myristate 13-acetate, did not decrease [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. The decrease caused by PMA could be blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I (GF-109203X). Stimulation of the [[Alpha].sub.1]-adrenoceptor with 50 [micro]M phenylephrine decreased [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from 6.1 [+ or -] 0.3 to 4.6 [+ or -] 0.3 mM (n = 11, P [is less than] 0.01). The decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] produced by phenylephrine was blocked by pretreatment with staurosporine, GF-109203X, or PMA. The decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] produced by PMA was not prevented by pretreatment with tetrodotoxin but was blocked by pretreatment with strophanthidin or high extracellular [K.sub.+] concentration. The results suggest that [[Alpha].sub.1]-adrenergic receptor activation results in a decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] via PKC-induced stimulation of the [Na.sup.+]-[K.sup.+] pump in cardiac myocytes. [[Alpha].sub.1]-adrenergic receptor; phenylephrine; phorbol 12-myristate 13-acetate; sodium-potassium ion pump; intracellular sodium ion activity

Double-barreled Na+-selective microelectrodes and the fluorescent Na+ indicator sodium-binding benzofuran isophthalate were used in measuring the intracellular Na+ activity in sinoatrial node (SA) cells of rabbit hearts. The roles of isoprenaline, carbachol and Cs+ on intracellular Na+ activity and pacemaker potential in SA cells have also been studied. Results indicated that isoprenaline, carbachol and Cs+ can modify intracellular Na+ activity and pacemaker depolarization.

To clarify the role of caveoine in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes, Interestingly, VEGFR-2, phospholipase D2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas die negative control for l-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-δ regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-δ pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-β-cyclodextrin (MβCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MβCD-treated cells restored caveolar structures. Pretreatment with MβCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for die VEGF/VEGFR-2-mediated MEK/ERK signaling cascade. [ABSTRACT FROM AUTHOR]

We have investigated the effect of external H+ concentration ([H+]o)on the human-ether-a-go-go-related gene (HERG) current ( IHERG), the molecular equivalent of the cardiac delayed rectifier potassium current ( IKr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the IHERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance ( gmax) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in gmax caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V1/2) and the decrease in gmax. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing gmax, their interaction is non-competitive. [ABSTRACT FROM AUTHOR]

SBFI in Cardiac Myocytes. Introduction: Although some properties of the fluorescent sodium indicator "SBFI" are known, there is no accepted method by which the SHFI signal might he calibrated for intracellular Na (Nai) in cardiac cells. The first aim of this study was to characterize the loading and compartmentalization of this indicator in single cardiac myocytes. The second aim was, from experimental observation, to develop a rational calibration method for SBFI. The third aim was to use this Na indicator to study the dependence of tonic contraction on Nai in voltage-clamped ventricular myocytes. Methods and Results: SBFI was incorporated into myocytes by incubating with the acetoxymethyl ester (10 µM) for 2 hours. This led to an intracellular concentration of SBFI free acid of 122 ± 17 µM. We considered a number of issues with respect to compartmentalization of indicator and, under our conditions, we found the majority (71%) of indicator was situated in the cytoplasm. Therefore, SBFI indicates mainly changes of cytoplasmic Na. Calibration of the indicator for Nai was performed by equilibrating internal and external Na. We investigated the conditions required for optimum transmembrane Na equilibration and found it necessary to use a calibration solution free of both Ca and magnesium (Mg). The Na ionophores gramicidin D and monensin were both required, and it was also necessary to inhibit the Na/K pump for optimum Na equilibration. Using these conditions, the Nai concentration in quiescent rat ventricular myocytes was 10.9 ± 0.74 mM (mean ± SEM, n = 40; equivalent to an Na activity of 8.3 mM). The concentration of Nai was significantly lower in quiescent rabbit mycoytes: 3.8 ± 0.23 mM (n = 24; equivalent to an Na activity of 2.9 mM). In voltage-clamped rabbit myocytes, inhibition of the Na/K pump caused a rise of Nai; there were also marked effects on the tonic shortening elicited by ramp depolarizations. As Nai rose, the magnitude of tonic shortening increased and its voltage dependence also changed. Conclusion: These results confirm the suitability of SBFI for measuring Na, in cardiac cells. Provided that steps are taken to optimize transmembrane Na equilibration, the indicator can be calibrated for Nai. The different Nai of rat and rabbit myocytes has implications for the function of sarcolemmal Na/Ca exchange in each cell type. An Nai of 10.9 mM in rat myocytes gives a calculated reversal potential for the exchange of-35 mV. In comparison, an Nai of 3.8 mM in rabbit myocytes leads to a reversal potential for the exchange +45 mV. Therefore, relatively small changes of Nai can shift the reversal potential of (he exchange to values that are substantially more positive or negative than zero. The behavior of voltage-dependent tonic contraction in rabbit mycoytes was consistent with the Na/Ca exchange reversal potential being more positive than zero in these cells. [ABSTRACT FROM AUTHOR]

Glibenclamide, a potent cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel blocker, is frequently used to study function and regulation of CFTR Cl(-) channels. In this study, the effects of glibenclamide on intracellular Na(+) concentration ([Na(+)](i)), contraction, Ca(2+) transient, and membrane potential were investigated in isolated guinea pig ventricular myocytes. Glibenclamide increased [Na(+)](i) and decreased contraction and Ca(2+) transient. However, glibenclamide did not change membrane potential. To determine whether inhibition of Na(+)-K(+) pumps and L-type Ca(2+) channels is responsible for the increase of [Na(+)](i) and the decrease of contraction, we tested the effects of glibenclamide on Na(+)-K(+) pump current and L-type Ca(2+) current (I(Ca,L)). Glibenclamide decreased Na(+)-K(+) pump current and I(Ca,L) in a concentration-dependent manner. In the presence of Cl(-) channel inhibitors, glibenclamide depolarized diastolic membrane potential and reduced action potential duration. This result suggests that the reason for lack of effect of glibenclamide on membrane potential might be due to its combined inhibitory effects on the Na(+)-K(+) pump, the L-type Ca(2+) channel, and Cl(-) channels, which may have opposing effects on membrane potential. These results indicate that glibenclamide increases [Na(+)(i)] by inhibiting the Na(+)-K(+) pump and decreases contraction and Ca(2+) transient, in addition, by blocking the L-type Ca(2+) channel.

Chlorpromazine, a commonly used antipsychotic drug, has been known to induce QT prolongation and torsades de pointes, which can cause sudden death. We studied the effects of chlorpromazine on the human ether-a-go-go-related gene (HERG) channel expressed in Xenopus oocytes and on delayed rectifier K current of guinea pig ventricular myocytes. Application of chlorpromazine showed a dose-dependent decrease in the amplitudes of steady-state currents and tail currents of HERG. The decrease became more pronounced at increasingly positive potential, suggesting that the blockade of HERG by chlorpromazine is voltage dependent. IC50 for chlorpromazine block of HERG current was progressively decreased according to depolarization: IC50 values at -30, 0, and +30 mV were 10.5, 8.8, and 4.9 microM, respectively. The block of HERG current during the voltage step increased with time starting from a level 89% of the control current. In guinea pig ventricular myocytes, bath application of 2 and 5 microM chlorpromazine at 36 degree C blocked rapidly activating delayed rectifier K current (IKr) by 31 and 83%, respectively. How-ever, the same concentrations of chlorpromazine failed to significantly block slowly activating delayed rectifier K current (IKs). Our findings suggest that the arrhythmogenic side effect of chlorpromazine is caused by blockade of HERG and rapid component of delayed rectifier K current rather than by blockade of the slow component.

The null mutation of cardiac Na(+)-Ca(2+) exchanger (NCX1) gene in mice caused death of embryo in utero at embryonic day (ED) 9.0-9.5 and this embryonic lethality appears resulted from abnormal heart development. In the present study, we investigated whether transgenic re-expression of NCX1 in mutant cardiac myocytes could rescue these lethal defects. Transgenic mice expressing the canine NCX1 in a cardiac specific manner were bred into the NCX1 knock-out background but did not prevent the fetal lethality associated with the NCX1 null allele. However, the NCX1 knock-out embryos with an NCX1 transgene survived with heart beatings until ED 10.5 which was one day longer than the survival of the NCX1 knock-out embryos (ED 9.5). At ED 10.5, however, the partially rescued NCX1 embryos might have succumbed to the lack of an organized vasculature in the yolk sacs. The placental labyrinth layer was reduced in size and largely avascular. The transgenic re-expression of NCX1 rescued heart beatings and survived longer, but was still insufficient for the mice to be completely rescued. Importantly, NCX1 was observed to express in the yolk sac and the placenta of wild type mice. The results suggest that defects in extra-embryonic compartments are causal to the lethality, and that NCX1 may play an important role in establishing vascularization in extra-embryonic tissues.

Betacellulin (BTC) is a member of the epidermal growth factor (EGF) family, and it acts through EGF receptors. We asked whether BTC could be an angiogenic factor. Using human umbilical vein endothelial cells (HUVECs), we examined the effect of BTC on kinases and angiogenic processes. BTC induced ERK1/2 and Akt phosphorylation in a dose- and time-dependent manner. BTC induced phosphorylation of all three EGF receptors present on HUVECs: ErbB2, ErbB3, and ErbB4. Pretreatment with effective concentrations of ErbB1 inhibitor did not suppress BTC-induced kinase phosphorylation. BTC, EGF, VEGF (all at 10 ng/ml) produced similar increases in DNA synthesis. BTC, EGF, and VEGF all significantly increased endothelial cell migration. In addition, BTC promoted survival in a dose-dependent manner, and its effect was inhibited by pretreatment with PtdIns 3'-kinase inhibitor wortmannin or MEK1/2 inhibitor PD98059. Both BTC and EGF produced similar increases in tube formation in collagen gels. BTC-induced tube formation was suppressed by PD98059, wortmannin, and LY294002. In the mouse Matrigel plug assay, BTC (100 ng/ml) promoted neovessel formation, and its effect was suppressed by a combination of wortmannin and PD98059. Taken together, these data show that BTC exerts potent angiogenic activity through activation of EGF receptors, mitogen-activated protein kinase, and PtdIns 3'-kinase/Akt in endothelial cells.