INTRODUCTION

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VASCULAR ENDOTHELIAL CELLS form the primary interface between the circulating blood and the vessel wall. In addition, the endothelium regulates the structure and function of vascular tissue through release of vasoactive substances such as prostacyclin, endothelium-derived relaxation factor (nitric oxide), and endothelins (14). Endothelial cells obtained from large conduit vessels express various types of ion channels including voltage-gated, stretch-activated, and hormone-regulated conductances (1). Voltage-gated inward rectifier (8, 28, 29), transient outward (29, 33), Ca²⁺-activated (24), and ATP-sensitive (15) K⁺ channels have been identified in a variety of macrovascular endothelial cells. In contrast, inward currents through voltage-gated Ca²⁺ channels or tetrodotoxin (TTX)-sensitive Na⁺ channels have not been observed in these cells (1, 29, 33).

Although the electrophysiological properties of macrovascular endothelial cells have been studied in some detail, limited information is available concerning ion channels in cardiac microvascular endothelial cells (CMEC) (9). Both T- and L-type Ca²⁺ channels are known to exist in microvascular cells obtained from bovine adrenal capillary tissues (4, 5). If present in the CMEC, inward Ca²⁺ or Na⁺ currents could be important in promoting endothelium and cardiac muscle interactions in the beating heart (19). In addition, these channels could function in transmitting electrical signals along the vessel wall as observed in arterioles from other tissues (26, 27). In the present study, we report the existence of a voltage- and TTX-sensitive Na⁺ channel in primary cultures of CMEC isolated from rat ventricular muscle.

	MATERIALS AND METHODS

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Isolation and characterization of CMEC. CMEC were isolated according to the procedure of Nishida et al. (19). Briefly, hearts were removed from adult rats (200-250 g), mounted on a Langendorff-type column, and perfused for 5 min with Krebs solution composed of (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, and 25 NaHCO₃ and saturated with 95% O₂-5% CO₂ at pH 7.4. After a heart was perfused, the outer one-fourth of the left ventricle free wall and septum was dissected away to remove epicardial arteries and larger penetrating vessels. The remaining tissue was minced in 0.2% collagenase (type B; Boehringer Mannheim) and incubated for 30 min at 37°C in a shaking bath. Trypsin (0.02%; Sigma Chemical, St. Louis) was then added, and the tissue was sheared 10 times over a period of 30 min. Dissociated cells were filtered through a 100-µm mesh filter, washed with Ca²⁺-free solution, and centrifuged at 100 g for 5 min. CMEC were resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum and antibiotics (penicillin, streptomycin, and Fungizone). The CMEC were plated on laminin-coated coverslips at a density of ~2.5 × 10³ cells/cm². After a 2-h period, attached cells were washed with DMEM to allow differential adhesion (19). For patch-clamp studies, coverslips were transferred to a recording chamber containing the normal external solution (see Recording procedure and measurement of Na⁺ currents). Cultures were maintained in a humidified atmosphere of 5% CO₂-95% O₂ at 37°C and were normally used within 1-4 days after plating.

Recording procedure and measurement of Na⁺ currents. The patch-clamp method (13) was used to record whole cell CMEC currents using L/M EPC-7 (Adams and List) and Axopatch 200 (Axon Instruments) amplifiers. Pipettes were made from Gold Seal Accu-fill 90 Micropets (Clay Adams) and had resistances of 2-4 M when filled with internal solution. Unless stated otherwise, all experiments were conducted on isolated, noncoupled CMEC at room temperature (22-24°C). Cells were placed in an external solution consisting of (in mM) 132 NaCl, 5 KCl, 2 MgCl₂, 1 CaCl₂, 5 dextrose, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4 (with NaOH) (280 mosM). For Na⁺ current measurements, patch pipettes were filled with an internal solution consisting of (in mM) 70 CsCl, 40 Cs-aspartate, 2 MgCl₂, 1 CaCl₂, 11 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 3 ATP, and 10 HEPES, pH 7.3 (with CsOH) (280 mosM). To measure the I_Na reversal potential (E_rev) in some experiments, the internal solution contained 10 mM NaCl plus 60 mM CsCl. For resting potential and K⁺ current measurements, the internal solution consisted of (in mM) 140 KCl, 2 MgCl₂, 1 CaCl₂, 11 EGTA, 3 ATP, and 10 HEPES, pH 7.3. The ratio of EGTA to CaCl₂ in these solutions sets the free intracellular Ca²⁺ concentration to ~10 nM. A reference electrode made from a Ag-AgCl pellet was connected to the bath using an agar salt bridge saturated with external solution. Data were adjusted for liquid junction potentials that arose both between the pipette solution and the bath solution and between the reference electrode and the bath (35). Liquid junction potential values were measured at the start and end of experiments and were between 5 and 5 mV.

Materials. DMEM, TTX, saxitoxin, phorbol 12,13-dibutyrate (PDB), 4-phorbol, and the membrane-soluble adenosine 3',5'-cyclic monophosphate (cAMP) analog 8-(4-chlorophenylthio) cAMP (CPT-cAMP) were purchased from Sigma Chemical. Fluorescently labeled acetylated low-density lipoprotein (DiI-Ac-LDL) was obtained from Biomedical Technologies (Stoughton, MA).

	RESULTS

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Uptake of DiI-Ac-LDL into CMEC. Primary cultures of CMEC were analyzed with an anchored cell analysis system after overnight incubation with 10 µg/ml of DiI-Ac-LDL. Figure 1, A and B, shows fluorescent images of the CMEC obtained on day 2 (before confluency) and day 7 (at confluency) in culture. A strong fluorescent signal was also measured in bovine pulmonary artery endothelial cells (Fig. 1C). In contrast, no fluorescence was observed in NIH/3T3 fibroblasts (Fig. 1D) or rat cardiac fibroblasts (results not shown). Differential uptake of DiI-Ac-LDL, determined by fluorescence-activated cell sorting (FACS), indicated that the cultures contained >90% CMEC.

View larger version (165K): [in this window] [in a new window]   Fig. 1.   Isolation of cardiac microvascular endothelial cells (CMEC). Uptake of fluorescently labeled acetylated low-density lipoprotein (DiI-Ac-LDL) was measured in CMEC on days 2 (A) and 7 (B) of culture. Uptake of DiI-Ac-LDL was also observed in bovine pulmonary artery endothelial cells (C) but not in NIH/3T3 fibroblasts (D).

Resting potential of CMEC. Figure 2 shows a histogram of the resting membrane potential (V_m) measured in isolated CMEC using the whole cell patch-clamp technique. For the nonconfluent cells (days 1-4 in culture), the resting V_m ranged from 22 mV to 50 mV with a mean (±SE) of 39 ± 2 mV (n = 24 cells). Resting V_m was also measured when the cell culture reached confluency at days 6-8. Previous studies have shown that the resting V_m of coronary artery endothelial cells becomes more hyperpolarized at confluency (9). The resting V_m of the confluent CMEC was 42 ± 3 mV (n = 12 cells). The resting V_m values measured in the confluent and nonconfluent cells were not significantly different (P > 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Resting membrane potentials (Vm) in isolated CMEC. Vm were recorded from a total of 36 cells studied on days 2-8 of culture.

Voltage-gated Na⁺ currents in CMEC. Figure 3A displays membrane currents recorded from a CMEC during voltage steps applied from a holding potential of 80 mV to various potentials. Voltage steps to potentials more positive than 30 mV resulted in the activation of a fast, inward current. This current was defined as a voltage-gated Na⁺ current (I_Na) on the basis of the following criteria: 1) fast activation and inactivation kinetics characteristic of I_Na measured in other cells (Fig. 3A), 2) complete elimination of the current when external Na⁺ was replaced with N-methylglucamine (results not shown), and 3) block by the Na⁺-channel toxins TTX (see Fig. 5) and saxitoxin (50 nM). An I_Na based on these criteria was observed in 35 of 48 CMEC (~75%) examined on days 1-4 in culture. For cells separated using FACS, the I_Na was measured in 100% (5 of 5) of those CMEC displaying positive uptake of DiI-Ac-LDL. In contrast, inward Ca²⁺ currents were not observed under the conditions used to measure I_Na.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Measurement of voltage-gated Na+ currents (INa) in CMEC. A: currents recorded during voltage steps, given in 10-mV increments, applied from a holding potential of 80 mV to potentials ranging from 20 to 20 mV. B: average current-voltage relationship for INa measured in 20 CMEC and normalized to cell membrane capacitance.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Inactivation properties of CMEC INa. A: inactivation curves obtained using a 2-pulse protocol. Currents obtained at 0 mV were normalized and plotted as a function of prepulse potential (Vp). Data were fit with the Boltzmann equation, gNa = gNa(max)/{1 + exp[(Vp  V1/2)/k]}, where gNa is Na+ conductance, gNa(max) is maximal Na+ conductance, V1/2 is half-maximal voltage required for inactivation, and k is slope. B: recovery from inactivation determined at various times after a 40-ms prepulse to 0 mV. A single exponential curve with a time constant () of 9 ms was fit to data.

Block of CMEC Na⁺ current by TTX and saxitoxin. The results displayed in Figs. 3 and 4 provide strong evidence that a voltage-gated Na⁺ channel is expressed in the CMEC. Na⁺ channels measured in excitable tissues have been categorized as TTX-sensitive and TTX-insensitive channels (6). TTX-sensitive channels, found in nerve and adult skeletal muscle, are blocked by low nanomolar concentrations of the toxin (20, 32). In contrast, TTX-insensitive channels, found in cardiac tissues and neonatal skeletal muscle, require micromolar concentrations of TTX for block (23). Figure 5 summarizes the results of experiments determining the effect of TTX on the CMEC I_Na. A concentration of TTX as low as 50 nM produced a complete block of I_Na (Fig. 5A) that could be reversed with washout of the toxin. In Fig. 5B, the TTX concentration-I_Na inhibition relationship is plotted. The dissociation constant (K_d) for Na⁺-channel block was 5 nM. I_Na was also completely blocked by 50 nM saxitoxin (n = 4 cells; results not shown). Thus the CMEC express a TTX-sensitive subtype of the voltage-gated Na⁺ channel.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Block of CMEC INa by TTX. A: INa measured during a voltage step to 0 mV before (Con) and after addition of 50 nM TTX. B: concentration-inhibition curve of INa by TTX. Each point represents mean ± SE for 3-5 experiments. Theoretical curve for data is given by [TTX]n/(Kd + [TTX]n) where [TTX]n is TTX concentration with Hill coefficient and Kd is dissociation constant, which is 5 nM.

Modulation of CMEC Na⁺ current by protein kinase C. Both cardiac and neuronal Na⁺ channels are regulated after phosphorylation by protein kinase C (PKC) (16, 21). To determine the effect of PKC stimulation on the CMEC, cells were exposed to the phorbol ester PDB. As shown in Fig. 6, application of 50 nM PDB produced a small but consistent increase in I_Na. Significant increases (ranging from 26 ± 3 to 33 ± 5%, n= 6 cells, P < 0.05) were observed over the voltage range of 20 to 10 mV within 5 min of adding PDB. Augmentation of the I_Na during stimulation of PKC was not associated with any change in the voltage dependence of activation (Fig. 6B). In contrast to the results with PDB, treatment of the cells with 4-phorbol, a phorbol ester that does not stimulate PKC, caused no significant change in the amplitude of I_Na (2 ± 4%, n = 4 cells, P > 0.05). In addition, no change in the amplitude of I_Na was observed in the presence of bradykinin (100 nM; 2 ± 2%), thrombin (50 U/ml; 1 ± 5%), and CPT-cAMP (1 mM; 3 ± 6%).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Regulation of CMEC INa by protein kinase C (PKC). A: INa measured during a voltage step to 0 mV before (Con) and after addition of 50 nM phorbol 12,13-dibutyrate (PDB). B: current-voltage relationship for INa measured before and after PDB. Each point represents mean ± SE for 6 experiments. PDB caused a significant increase in INa at 20 mV (32 ± 2%), 10 mV (32 ± 10%), 0 mV (26 ± 3%), and +10 mV (33 ± 5%) but caused no significant change at +20 mV (9 ± 11%).

Voltage-gated K⁺ currents in CMEC. In addition to I_Na, an outward K⁺ current was also present in the CMEC. Figure 7A shows an example of this K⁺ current measured in a cell that displayed a negligible I_Na. The K⁺ current activated after a delay during voltage steps applied to potentials from 20 to 10 mV (Fig. 7A). In Fig. 7B the normalized conductance for the K⁺ current is plotted as a function of the test voltage. The line shows the best fit of the data points to a Boltzmann distribution. The V_1/2 required for activation was 14 mV, and the slope of the fitted curve was 7 mV.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Properties of outward K+ currents recorded in CMEC. A: currents recorded during voltage steps, given in 10-mV increments, applied from a holding potential of 80 mV to potentials ranging from 30 to 10 mV. B: activation curve for currents displayed in A. K+ conductance (gK) was determined by dividing peak current amplitude at each potential by driving force for K+ (Vm  EK, where EK is K+ equilibrium potential). Line represents best fit of Boltzmann equation, gK = gK(max){1 + exp[(Vm  V1/2)/k]}, where gK(max) is maximal K+ conductance, V1/2 is half-maximal voltage required for activation, and k gives steepness of voltage dependence to data points.

	DISCUSSION

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Voltage-gated Na⁺ channels in CMEC. The results of this study demonstrate that CMEC express voltage-gated Na⁺ channels. This study represents the first measurement of ion channels in rat CMEC and the first report of Na⁺ channels in microvascular endothelial cells isolated from any vascular tissue. At least five distinct mammalian voltage-gated Na⁺ channels, referred to as types I, II, III, µ1, and h1, have been identified (6). The high affinity of the CMEC I_Na for TTX (K_d = 5 nM), compared with that of the TTX-insensitive channels found in cardiac myocytes (23), indicates that different Na⁺-channel subtypes are expressed in the myocardial and endothelial cells of the heart.

Relation to previous studies. Vascular endothelial cells express voltage-gated K⁺ channels including inward rectifier (8, 28, 29) and transient outward (29, 33) channels. Of particular relevance, inward rectifier K⁺ currents, but not Na⁺ or Ca²⁺ currents, are present in guinea pig coronary endothelial cells (9). In one report, a voltage-gated Na⁺ current was measured in human umbilical vein endothelial cells (HUVEC) and in endothelial cells derived from rat interlobar arteries of the kidney (12). This Na⁺ current displayed a peak density of ~6 pA/pF, an activation V_1/2 of 37 mV, and an inactivation V_1/2 of 82 mV. The current was only partially blocked by a 1 µM concentration of TTX, suggesting that these channels might fall into the TTX-resistant category of Na⁺ channels (12). The interlobar endothelial cells used in the latter study were immortalized by transfection with a simian virus and analyzed after eight passages in cell culture. Thus it could be argued that the presence of Na⁺ channels in these cells was an artifact of either viral transfection or the length of cell culture. Another study (33) failed to measure this I_Na in freshly cultured HUVEC. In contrast to the interlobar endothelial and HUVEC I_Na, the CMEC I_Na could be measured on the first day after cell isolation and was present at least until days 6 and 7, when the cells reached confluency. These results suggest that the CMEC Na⁺ channels may be present in vivo within the endothelium of cardiac microvessels.

Physiological relevance of CMEC I_Na. Opening of voltage-gated Na⁺ channels produces the upstroke of the action potential in nerve, muscle, and other excitable tissues. However, I_Na has also been observed in nonexcitable cells such as bone (10, 34) and epithelial cells (36). Thus the physiological function of I_Na in the CMEC is unclear. With an average resting V_m of 39 mV (Fig. 2), ~30% of the CMEC Na⁺ channels would be capable of being activated during membrane depolarization (Fig. 4). A larger percentage of channels would be available for opening in those cells with resting V_m more negative than 45 mV (Fig. 2). Although previous studies (26, 27) have suggested that electrical signals can be transmitted through the microvascular endothelium, the underlying mechanism for this process has not been described. It has been proposed (27) that active electrical events, such as action potentials, may be required to account for the conduction of vasomotor responses throughout the arteriole network. If present and functional in the intact endothelium, the I_Na could play a role in vascular signaling by causing membrane depolarization. Furthermore, the CMEC I_Na could function in regulating intracellular levels of Ca²⁺ by affecting Na⁺/Ca²⁺ exchange processes (25) and thus could affect the activity of Ca²⁺-dependent enzymes such as nitric oxide synthase.