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(Hypertension. 1997;30:112-119.)
© 1997 American Heart Association, Inc.

Articles

Mechanosensitive Cation Channels in Aortic Endothelium of Normotensive and Hypertensive Rats

Joachim Hoyer; Ralf Köhler; ; Armin Distler

From the Department of Internal Medicine and Nephrology, University Hospital Benjamin Franklin, Free University, Berlin, Germany.

	Abstract

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Abstract In response to humoral and hemodynamic stimuli, vascular endothelium regulates vascular tone by releasing endothelium-derived vasoactive factors. Stretch-activated cation channels have been postulated to act as endothelial mechanosensors that respond to changes in hemodynamic forces. We report the presence of a nonselective (n=98) and K⁺-selective (n=53) stretch-activated channel in rat intact aortic endothelium and isolated aortic endothelial cells. The nonselective channel showed a permeability ratio for Na⁺, K⁺, and Ca²⁺ of 1:0.95:0.23 and was completely blocked by 50 µmol/L gadolinium, a blocker of stretch-activated channels. The K⁺-selective channel was selectively permeable for K⁺, with a K⁺-Na⁺ permeability ratio of 10.9:1. In whole-cell current recordings, hyposmotic cell swelling induced an increase in cell conductance. The swelling-induced current was completely blocked by 50 µmol/L gadolinium, showing that stretch-activated channels were activated by cell swelling and carry macroscopic cell currents. In a comparative study with normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), the K⁺-selective stretch-activated channel was observed in a 4.4-fold higher density in adult SHR compared with WKY. Also, in adult SHR, the stretch sensitivity of the nonselective channel was nearly twice as high as in WKY. In contrast, channel properties were unchanged in young SHR (5 to 6 weeks old) compared with age-matched WKY. These data suggest that stretch-activated channels are regulated in their sensitivity and density when subjected to increased hemodynamic forces such as in hypertension. Since the channels are capable of acting as endothelial mechanosensors, the altered channel properties might contribute to an altered mechanoreception in hypertension.

Key Words: ion channels • mechanoreceptors • aorta • endothelium

	Introduction

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Mechanical forces associated with blood flow play a critical role in the regulation of vascular tone¹ as well as in growth² and remodeling^{3 4} of the arterial vessel wall. Endothelial cells lining all blood vessels appear to be the primary transducers in these complex events.^{4 5} A flow-induced release of vasoactive factors by the endothelium is a major factor in the regulation of vascular tone.^{6 7} For instance, in response to acute hemodynamic stimulation of the endothelium by flow-induced shear stress, the intracellular Ca²⁺ concentration rises and Ca²⁺-dependent synthesis of vasodilator nitric oxide is stimulated.^{6 8 9} Although recent studies have brought insight into the mechanisms of endothelial mechanotransduction,¹⁰ the nature of the endothelial sensing mechanisms for hemodynamic changes is still incompletely understood. SACs have been identified in vascular endothelial cells and may act as microsensors for changes in the hemodynamic environment.^{10 11 12} An endothelial SAC that is nonselective for cations with an appreciable Ca²⁺ permeability presumably is involved in flow-induced Ca²⁺ influx into endothelial cells.^{11 12} Also, a flow-induced K⁺ current has been implicated in flow-dependent cell hyperpolarization, which provides the driving force for Ca²⁺ influx.¹³ Endothelial mechanosensitive ion channels have been studied almost exclusively in cultured or isolated vascular endothelial cells of porcine or bovine origin. Recently, we have identified SACs in the intact porcine endocardial endothelium.¹⁴ In the present study, we identified and characterized mechanosensitive channels in morphologically intact rat aortic endothelium and isolated aortic endothelial cells by use of the patch-clamp technique. In arterial hypertension, the cardinal hemodynamic disturbance is an increased total peripheral resistance.¹⁵ Endothelial dysfunction may at least partially be responsible for the increased vascular resistance.^{16 17} We tested whether channel functions of the endothelial SACs are altered in hypertensive rats compared with normotensive controls.

	Methods

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Rats were killed by cervical dislocation and exsanguinated. Segments of thoracic aorta about 2 cm in length were dissected and kept in ice-cold Ringer's solution until use in patch-clamp experiments or for further cell isolation procedures. Ringer's solution contained (mmol/L) NaCl 140, KCl 4.3, CaCl₂ 1.3, MgCl₂ 1, and HEPES 10 at pH 7.4. Isolated endothelial cells were prepared as described previously.¹² Briefly, aortic segments were incubated for 20 minutes at 36°C in phosphate-buffered NaCl solution (PBS) containing 0.25% trypsin. Incubation was stopped by washing with ice-cold PBS, and endothelial cells were mechanically isolated from thoracic aorta by gentle scraping of the luminal surface.

Morphological tests with transmission electron microscopy were performed essentially as described before.¹⁴ The aortic segments were fixed by immersion in 2.5% glutaraldehyde in 0.1 mol sodium cacodylate buffer, pH 7.2, for 7 hours. After a short wash in Ringer's solution, the tissue was refixed in buffered osmium tetroxide for 1 hour, incubated in 2% uranyl acetate for 2 hours, dehydrated with ethanol, and embedded in Spurr's epoxy resin. After polymerization, ultrathin sections were cut and examined with an electron microscope (CM 12, Philips) operated at 60 kV.

Isolated endothelial cells were identified by von Willebrand factor (factor VIII) immunofluorescent staining or uptake of acetylated low-density lipoprotein labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanineperchlorate (DiI-Ac-LDL). For immunofluorescent staining of von Willebrand factor, freshly isolated cells were fixed on a coverslip with 100% methanol for 10 minutes at -20°C. After saturation of nonspecific binding sites by preincubation with fetal calf serum for 10 minutes, cells were incubated with von Willebrand factor antibodies (Sigma Chemical Co) diluted in PBS (1:100) for 1 hour at room temperature.

Patch-clamp experiments were carried out as described.^{14 18} Single-channel membrane currents were recorded with a patch clamp amplifier (EPC-9, HEKA) low-pass filtered (-3 dB, 800 Hz) at a sample time of 0.5 millisecond and stored on removable cartridges until data analysis. Patch pipettes were pulled from borosilicate glass capillaries with 0.3 mm wall thickness and had a tip resistance of 5 to 7 M in symmetrical KCl solution. The seal resistance in cell-attached patches ranged from 5 to 20 G. Experiments were performed at 35°C. In the displayed current traces, single-channel currents carried by cations moving from the extracellular to the intracellular side are depicted as downward (negative) currents. The given potential value resembles the patch-clamp potential, and the sign of the potential refers to the cytosolic side. P_o was calculated as described previously.¹⁹ Slow whole-cell experiments were carried out as described.²⁰ Whole-cell currents were recorded at a sample time of 0.5 millisecond and low-pass filtered at 200 Hz. Patch pipettes had a tip resistance of 5 M. Computer-generated voltage ramps and pulse sequences were performed with EPC-9 software (HEKA). Experiments were conducted at 35°C.

If not otherwise stated, in single-channel recordings, the patch pipette solution contained (mmol/L) KCl 140, CaCl₂ 1, MgCl₂ 1, and HEPES 10 (pH 7.2). The bath solution contained normal saline solution ([mmol/L] NaCl 140, KCl 4.3, CaCl₂ 1.3, MgCl₂ 1, and HEPES 10 at pH 7.4). In experiments using ion substitution protocols, the composition of the NaCl bath solution was (mmol/L) NaCl 140, CaCl₂ 1.3, MgCl₂ 1, and HEPES 10 at pH 7.4. The composition of the CaCl₂ solution was (mmol/L) CaCl₂ 90, MgCl 1, and HEPES 10 at pH 7.4. In the case of chloride substitution by cyclamate in the pipette solution, the patch pipette was backfilled with a KCl pipette solution. Cyclamate solution contained (mmol/L) potassium-cyclamate 140, calcium-cyclamate 1, and HEPES 10 at pH 7.2. For whole-cell recordings, the patch pipette was filled with a KCl solution containing (mmol/L) KCl 140, CaCl₂ 0.1, MgCl₂ 1, and HEPES 10 as well as 150 to 200 µg/mL nystatin at pH 7.2. The normal saline bath solution was used as the isosmotic bath solution. Hyposmotic cell swelling experiments were performed as described²¹ with a hyposmotic solution containing (mmol/L) NaCl 90, KCl 4.3, CaCl₂ 1, MgCl₂ 1, and HEPES 10 at pH 7.4. Whole-cell recordings were performed in the presence of 1 mmol/L 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to avoid disturbance of the recording by large conductance Cl^- channels. Gadolinium was applied by bath solution exchange at a concentration of 50 µmol/L Gd³⁺ in normal saline bath solution. All chemicals were of analytical grade.

For a comparative study, 14 WKY and 14 SHR were obtained from Møllegard breeding center (Skensved, Denmark) and kept on a regular rat chow. Before experiments, body weight and blood pressure were monitored. Systolic pressure was measured by tail-cuff sphygmomanometry (Harvard Apparatus). Dry heart weight was determined after hearts were dried for at least 24 hours. Young rats were used for experiments between 5 and 6 weeks of age; adult rats were 15 to 20 weeks old. Channel density and stretch sensitivity were compared by Friedman's two-way ANOVA test. Values of P<.05 were considered significant. If not otherwise stated, data are given as mean±SD.

	Results

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Patch-clamp recordings were performed in tissue slices from rat aorta with an intact endothelium as well as in freshly isolated aortic endothelial cells.

Transmission electron microscopy of rat aortic tissue slices was performed to ascertain that the luminal surface of the tissue slices was covered by endothelial cells. A typical electron micrograph showed a monolayer of endothelial cells at the luminal surface. The presence of tight junctions in the intercellular cleft between two endothelial cells indicated that the cell monolayer remained morphologically intact during the preparation procedure.

Isolated aortic endothelial cells were immunostained with von Willebrand factor (factor VIII) or oxidized LDL. The endothelial cells showed a homogenous fluorescence signal for either substrate compared to controls of non-endothelial cells with no staining signals (data not shown).

Cell-attached patch-clamp recordings from intact aortic endothelium usually were without spontaneous channel activity. Application of negative pressure to the rear of the patch pipette and thus stretching the patched cell membrane induced an activation of an SAC (Fig 1A) with a mean channel conductance of 23.1±3.6 pS (n=31) at negative membrane potentials. In excised inside-out patches, the current-voltage relation was ohmic, with a channel conductance of 23.5±4.1 pS (n=10) at membrane potentials between -90 and +85 mV. Up to four channels could be simultaneously activated in a single patch. Channel activity measured as P_o depended on the strength of membrane stretch, as shown in Fig 1B. In this typical patch-clamp experiment, no channel activity was observed in the absence of membrane stretch. Pipette pressure of -30 mm Hg induced a channel activity with a P_o of 0.19. Channel activity further increased to a P_o of 0.37 when the cell membrane was stretched by -50 mm Hg. Channel activity immediately stopped when the pipette pressure was terminated and could repeatedly be stimulated afterward. In a series of nine experiments, the relationship between pipette pressure and channel activity was investigated over a range of -10 to -50 mm Hg pipette pressure in each experiment. As shown in Fig 1C, P_o gradually increased from 0.06±0.03 at -20 mm Hg to 0.16±0.3 at -30 mm Hg and 0.36±0.05 at -50 mm Hg. Rarely, SAC activity decreased slowly over a short time of 2 to 3 seconds after cessation of pipette pressure. Also, channel activity showed a spontaneous rundown when negative pipette pressure was maintained for longer than 1 minute. The channel activity was not responsive to positive pipette pressure. The channel was also permeable to divalent cations, as shown in Fig 2A. This experiment was performed with a CaCl₂ solution in the patch pipette in the cell-attached recording mode. At negative clamp potentials, the channel was active, with channel kinetics similar to those in experiments performed with a KCl pipette solution reflecting Ca²⁺ influx into the cell.

View larger version (27K): [in this window] [in a new window]   Figure 1. A, Activation of nonselective SAC by negative pipette pressure of -50 mm Hg in cell-attached patch of rat intact aortic endothelium. Activation was immediately reversed after cessation of negative pipette pressure. C denotes closed state of channels. Pipette holding potential was 80 mV. Three channels were simultaneously active in the patch. B, Gradual activation of SAC with rise in Po to 0.19 and 0.37 in response to pipette pressures of -30 and -50 mm Hg applied to the rear of the patch pipette, respectively. Three channels were active simultaneously in this cell-attached patch. C denotes closed state of channels. Pipette holding potential was 80 mV. C, Relationship between pipette pressure and channel activity. Cell-attached experiments (n=9) were performed at a pipette holding potential of +80 mV with KCl pipette solution and NaCl bath solution. Data are mean±SEM.

View larger version (20K): [in this window] [in a new window]   Figure 2. A, Ca2+ current through the SAC after activation by membrane stretch of a cell-attached patch at pipette holding potential of +80 mV in an isolated aortic endothelial cell. The patch pipette contained (mmol/L) CaCl2 90, MgCl2 1, and HEPES 10, pH 7.4 adjusted with Ca(OH)2. Up to four channels were active simultaneously. C denotes closed state of channels. B, Determination of cation selectivity with cation substitution protocols. Current-voltage relation of the SAC in inside-out patches was determined in the presence of the following solutions: CaCl2 pipette solution and NaCl bath solution (, n=5); CaCl2 bath and pipette solution (, n=3); and KCl pipette solution and NaCl bath solution (, n=10). Curves were fitted by linear regression ( and ) and Goldman-Hodgkin-Katz equation ().

After the patch was excised, SAC activity showed a rapid rundown within 2 to 3 seconds; in 121 of 136 experiments and in 15 of 136 experiments, a stable channel activity was preserved for more than 30 seconds. In the experiments with preserved channel activity in excised inside-out patches, ionic substitution protocols with bath solutions containing Ca²⁺ or Na⁺ and pipette solutions containing Ca²⁺ or K⁺ as main cations were performed to determine the cation selectivity and permeability ratio of the channel. Mean channel conductance was 13.0±4.2 pS (n=3) with CaCl₂ solution in both the pipette and bath and 24.1±4.3 pS with a KCl pipette solution and NaCl bath solution. When experiments were performed with an NaCl bath solution, reversal potentials were -1.7±0.9 mV (n=6) with a KCl pipette solution and -35.1±5.4 mV (n=4) with a CaCl₂ pipette solution (Fig 2B). Using the Goldman-Hodgkin-Katz equation,²² a permeability ratio for Na⁺, K⁺, and Ca⁺ of 1:0.98:0.23 was calculated. When cyclamate was substituted for chloride in the pipette solution (n=3), the channel current at negative membrane potentials was not altered and the reversal potential was not changed, thus excluding appreciable chloride permeability of the channel. In excised inside-out patches (n=3), channel activity was completely blocked (P_o <0.002) by 50 µmol/L gadolinium (Gd³⁺), an SAC blocker.²³ Channel properties with respect to channel conductance, ion selectivity, and pressure sensitivity were not different in freshly isolated endothelial cells compared with measurements in intact aortic endothelium.

In isolated endothelial cells, another type of SAC was identified, with mean channel conductances of 70.2±15.7 pS (n=21) in cell-attached patches and 89.8±18.1 pS (n=5) in inside-out patches in symmetrical K⁺ solutions. Channel activity depended on the degree of the negative pipette pressure applied. In a typical experiment, as shown in Fig 2A, the channel activated in response to increasing membrane stretch, with a rise of P_o from 0.15 to 0.35 in the presence of negative pressures of -40 and -60 mm Hg, respectively (Fig 3A). Also, this SAC was not active in the absence of membrane stretch and did not respond to positive pipette pressure. Similar to the nonselective SAC, this SAC rapidly inactivated when patches were excised. In a few experiments with preserved channel activity, the ion selectivity and permeability ratio was estimated by ion substitution protocols in excised inside-out patches (Fig 3B). In experiments with KCl pipette solution and NaCl bath solution, the reversal potential was 52.1±3.9 mV (n=10), corresponding to a K⁺-Na⁺ permeability ratio of 10.9:1. In some of these experiments, no outwardly directed current could be evoked even by applying high positive clamp potentials between 70 and 120 mV to the patch. However, a distinct inward rectification characteristic was not observed. The channel showed no appreciable Cl^- permeability or permeability for divalent cations such as Ba²⁺ or Ca²⁺. In a series of seven experiments, the channel was completely blocked by 50 µmol/L gadolinium in five experiments and partially blocked in two, with a remaining channel activity P_o of about 0.05. The K⁺-selective channel was almost exclusively re-corded in isolated aortic endothelial cells and only twice observed in intact tissue slices.

View larger version (28K): [in this window] [in a new window]   Figure 3. A, Activation of K+-selective SAC by negative pipette pressure in cell-attached patch of isolated aortic endothelial cell. Single-channel Po was 0.15 at -40 mm Hg and 0.35 at -60 mm Hg, whereas no activity was present in the absence of negative pipette pressure. C denotes closed state of channels. Pipette holding potential was 80 mV. B, Ion selectivity determined by ion substitution in excised inside-out patches (n=11). Pipette was filled with a KCl solution; bath contained NaCl solution () or KCl solution (). Values are given as mean±SEM. Data were fitted to Goldman-Hodgkin-Katz equation. Reversal potential was 55.4 mV. Channel conductance under symmetrical KCl solutions was g=87.5 pS. C, Coactivation of nonselective and K+-selective SACs in a single cell-attached patch in isolated aortic endothelial cell. Pipette holding potential was +50 mV; pipette was filled with KCl solution. C denotes closed state; O1, open state of nonselective SAC; O2, open state of K+-selective SAC; and O1+O2, current level of both channels being in the open state.

In a few experiments (n=7) in cell-attached patches, a coactivation of both SACs was observed, as shown in Fig 3C.

In whole-cell current recordings, isolated aortic endothelial cells showed low cell conductance under control conditions (Fig 4), with a mean cell conductance of 0.7±0.1 nS (±SEM) (n=28). Osmotic swelling was performed as described by Oike and coworkers²¹ by reducing the osmolarity of the bath solution from 300 to 200 mOsm. Hyposmotic swelling of the endothelial cells was observed within 20 seconds after replacement of isosmotic bath solution by hyposmotic bath solution. Cell swelling induced an increase in outwardly directed current and smaller increase in inwardly directed current. Accordingly, mean cell conductance was increased to 1.4±0.2 nS (±SEM) (n=23). After the bath solution was exchanged to isosmotic control solution, the whole-cell current returned to the control level as well. The swelling-induced whole-cell current was completely blocked by 50 µmol/L gadolinium (n=3, Fig 4).

View larger version (8K): [in this window] [in a new window]   Figure 4. Swelling-induced increase in whole-cell current of single rat aortic endothelial cell. Pipette was filled with KCl solution. Isosmotic bath solution consisted of a 300 mOsm NaCl solution (ISO); a 200 mOsm NaCl solution was used as hyposmotic bath solution (HYPO). Gadolinium (50 µmol/L) was added to hyposmotic bath solution to block SACs. Voltage ramp ranged from -120 to +60 mV. Cell capacity was 16.4 pF.

In a comparative study, we examined the channel properties of six young (5 to 6 weeks old) and eight adult (15 to 20 weeks old) WKY and SHR each. Systolic pressure was significantly higher in adult SHR (203±24 mm Hg) than in adult WKY (141±33 mm Hg, P<.01). Also, dry heart weight was increased in SHR (272±18 mg) compared with WKY (323±24 mg, P<.01). As expected, young SHR and young WKY did not differ significantly with respect to systolic pressure (131.0±5.5 versus 123.0±5.5 mm Hg) and dry heart weight (85.0±12.1 versus 85.5±7.9 mg). Rats were investigated in a randomized order, and the origin of the tissue slices with respect to hypertensive or normotensive rats was blinded to the investigator performing the patch-clamp experiments.

We examined the channel density and stretch sensitivity of both SACs. In isolated aortic endothelial cells from each rat, 40 experiments with cell-attached tight seal patches were performed. Therefore, in each strain, 320 tight seal experiments were performed. Channel density in endothelial cells from a rat was estimated as the fraction of all the experiments with cell-attached patches in which SAC activity was detected.²⁴ The density of the K⁺-selective SAC was low in WKY (channel activity in 4.7±3.9% [n=8] of patches) and significantly higher (P<.005) in SHR (channel activity in 20.6±10.1% [n=8] of patches; Fig 5A). The channel density of the K⁺-selective SAC was unchanged in young SHR (2.3±0.9%) compared with young WKY (1.6±0.8%). The density of the nonselective SAC was not different between the adult (14.3±9.2% versus 13.8±8.2%, WKY versus SHR) and young (15.4±7.9% versus 12.6±8.3%, WKY versus SHR) rats of both strains.

View larger version (23K): [in this window] [in a new window]   Figure 5. A, Channel density of K+-selective SAC measured as percentage of patches with channel activity was increased in adult SHR compared adult WKY (P<.005) and was not different between young SHR and young WKY. Channel density of the nonselective SAC did not differ between SHR and WKY of either age. *P<.005. B, Stretch sensitivity of nonselective SAC was higher in adult SHR compared with adult WKY (P<.05). Stretch sensitivity was measured as the increase in Po after pipette pressure was changed from -30 to -50 mm Hg. *P<.005.

Stretch sensitivity of an SAC was measured as the increase in P_o induced by an increase of negative pipette pressure from -30 to -50 mm Hg. Only in some experiments was the channel activity stable enough at different stretch maneuvers to give recording traces with stable channel activity for more than 30 seconds needed for the determination of the P_o and stretch sensitivity. Thus, from each rat strain, data from 11 experiments could be used for comparison of stretch sensitivity. In aortic endothelial cells from SHR, the P_o of the nonselective SACs increased by a factor of 2.6, from a P_o of 0.17±0.09 to one of 0.45±0.18 (n=11) in endothelial cells in adult SHR compared with a 1.4-fold increase from a P_o of 0.23±0.1 to one of 0.32±0.12 (n=11) in adult WKY (P<.05, Fig 5B). Stretch sensitivity was not different between young SHR (2.6-fold increase in P_o) and young WKY (2.1-fold increase in P_o). Because of the low channel density of the K⁺-selective SAC in WKY and therefore small number of patches with stable channel activity, the stretch sensitivity could not be compared between the two rat strains. Channel conductance and ion selectivity of both SACs were not different between SHR and WKY.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we have identified and characterized two types of SACs in endothelium from rat aorta, ie, a Ca²⁺-permeable, nonselective cation channel and a K⁺-selective channel. The channel density or stretch sensitivity of the SACs was increased in SHR compared with WKY. This is the first report on SACs in rat arterial endothelium and on changes of SACs in hypertension.

Activation of the K⁺-selective SAC will hyperpolarize the endothelial cells because of its K⁺ selectivity. Flow-induced hyperpolarization has been reported in endothelial cells.^{13 25} Endothelial hyperpolarization presumably is transmitted through gap junctions to the underlying smooth muscle cells.^{13 26} Subsequent smooth muscle cell hyperpolarization would lead to vasodilation. However, a K⁺-selective SAC has not been identified so far in single-channel recordings from endothelial cells. In whole-cell current recordings performed in isolated bovine aortic endothelial cells, a shear stress–induced K⁺ current has been reported,¹³ without identification of a corresponding SAC in single-channel recordings. The K⁺ current showed a strong inward rectification and was blocked by Ba²⁺. Recently, a single-channel recording of a shear stress–activated K⁺ channel has been reported whose basal channel activity increased during flow in a microcapillary tube.²⁷ The K⁺-selective SAC we demonstrate in rat aortic endothelial cells appears to be distinct from this shear stress–induced K⁺ current because it was not blocked by Ba²⁺, showed no inward rectification, and had a higher single-channel conductance. Involvement of endothelial Ca²⁺-dependent K⁺ channels (K_Ca) in flow-mediated nitric oxide release has been suggested by studies using tetraethylammonium, charybdotoxin, or apamin as blocker substances of K_Ca.²⁸ Endothelial Ca²⁺-dependent K⁺ channels in general are not mechanosensitive, and involvement of K_Ca in flow-dependent endothelial responses might be due to secondary activation of these channels by flow-induced increases in intracellular Ca²⁺ or cGMP concentrations.^{8 29} The K⁺-selective SAC was not Ca²⁺ dependent and not blocked by typical K⁺ channel blockers and thus does not represent the K⁺ channel implicated in flow-dependent vasodilation by pharmacological proof. A K⁺-selective SAC in chick heart cells had a comparable channel conductance but higher K⁺ selectivity.³⁰ The K⁺-selective SAC was almost exclusively present in freshly isolated endothelial cells and was rarely detected in patches of the luminal membrane from intact endothelium. This could be explained by a possible localization of the channel in the abluminal cell membrane.

The ion selectivity, stretch sensitivity, and gadolinium block of the nonselective SAC resemble those reported for SACs in cultured endothelial cells from neonatal pig aorta¹¹ and in intact endothelium from atrial endocardium¹⁴ or brain capillaries.¹⁹ However, the latter SACs have a slightly higher channel conductance (32 to 39 pS) than the rat aortic nonselective SAC. SACs comparable in channel conductance and ion selectivity have been described in porcine vascular smooth muscle cells,³¹ proximal tubular cells,³² mice skeletal muscle,²⁴ and embryo chick heart cells.³⁰ In contrast to the SAC reported in mice skeletal muscle,²⁴ the nonselective SAC in rat aortic endothelium was not activated by positive pipette pressure. The SAC was nonselective for monovalent and divalent cations, with a relatively high permeability for Ca²⁺ ions. Thus, activation of the nonselective SAC under physiological saline concentrations leads to Na⁺ and Ca²⁺ influx into the endothelial cell. Because of the large inwardly directed electrochemical gradient for Ca²⁺ ions, a large proportion of this current will be carried by Ca²⁺ ions, inducing a rise in intracellular Ca²⁺ concentration of the endothelial cells.¹⁴ This cation influx depolarizes the endothelial cell, thus limiting the Ca²⁺ influx. However, this depolarizing effect will be counteracted by a concomitant stretch activation of the K⁺-selective SAC that hyperpolarizes the endothelial cell via K⁺ efflux and therefore provides the driving force for Ca²⁺ influx. This is reflected by the whole-cell current recordings under hyposmotic cell swelling. Cell swelling is a useful tool to increase membrane tension and thus activate SACs.³³ The increase in outward current could be due to an activation of the K⁺-selective SAC and the inward current. The involvement of the SACs in the activation of whole-cell current by hyposmotic cell swelling is shown by the gadolinium block of the swelling-induced currents. Additionally, activation of Ca²⁺-dependent K⁺ channels by Ca²⁺ influx through the nonselective SAC, as has been shown in endocardial endothelium,¹⁴ might contribute to the swelling-induced outward current.

Both signals, Ca²⁺ influx and cell hyperpolarization, are important stimuli of endothelial nitric oxide production.^{34 35} Thus, activation of both SACs might induce a vasodilator response of the endothelium via activation of nitric oxide production in response to mechanical or hemodynamic stimulation. Recently, it was reported that in perfused rabbit iliac artery segments, the initial Ca²⁺-dependent nitric oxide production was abolished when the artery segments were stretched to their assumed in vivo length.³⁶ However, it remains to be determined whether these observations apply to other animal species.

In human and experimental hypertension, endothelial dysfunction has been reported that results in increased vascular tone or resistance caused by impaired secretion of vasodilating factors and increased secretion of vasoconstricting agents by the endothelium in response to endothelial stimulation by various humoral factors.^{16 17 37 38} Furthermore, an impaired flow-induced endothelium-dependent vasodilation has been observed in SHR compared with WKY.³⁹ However, normal endothelial function has been reported in human and experimental hypertension.^{40 41} Also, the flow-dependent vasodilation of the brachial artery has been found not to be impaired in essential hypertension.⁴² In salt-resistant genetic hypertension, endothelial production of nitric oxide has been assumed to be normal.⁴³ Therefore, changes of endothelial function in hypertension still need to be defined. Properties of endothelial ion channels have not been investigated in hypertension so far. Therefore, we tested whether the properties of the SACs are regulated under pathological hemodynamic conditions present in hypertension¹⁵ and whether this mechanosensitive mechanism is altered in hypertension. The SACs proved to be expressed in an increased density or stretch sensitivity in adult SHR with established hypertension, whereas both channel characteristics were not different in young WKY and SHR. As changes in channel properties were observed only in rats with established hypertension and not in prehypertensive SHR, it is likely that the changes are a consequence rather than a cause of hypertension. The altered channel functions presumably do not lead to endothelial dysfunction. Rather, the increase in channel density of the K⁺-selective SAC and stretch sensitivity of the nonselective SAC may represent an adaptive mechanism in hypertension because activation of the SACs probably induces a vasodilator rather than a vasoconstrictive response of the endothelium.

	Selected Abbreviations and Acronyms

 

Po = single-channel open probability SAC = stretch-activated cation channel SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (Ho 1103/2-2) and European Community BM H4-CT96-0602.

	Footnotes

 
Reprint requests to Dr J. Hoyer, Medizinische Klinik und Poliklinik, Universitätsklinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, FRG.

Received July 24, 1996; first decision September 5, 1996; accepted December 6, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension. 1986;8:37-44.[Abstract/Free Full Text]

2. Mitsumata M, Fishel RS, Nerem RM, Alexander RW, Berk BC. Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells. Am J Physiol. 1993;265:H3-H8.[Abstract/Free Full Text]

3. Langille BL, O'Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science. 1986;231:405-407.[Abstract/Free Full Text]

4. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med. 1994;330:1431-1438.[Free Full Text]

5. Davies PF, Tripathi SC. Mechanical stress mechanisms and the cell: an endothelial paradigm. Circ Res. 1993;72:239-245.[Abstract/Free Full Text]

6. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol. 1986;250:H1145-H1149.[Abstract/Free Full Text]

7. Busse R, Fleming I. The endothelial organ. Curr Opin Cardiol. 1993;8:719-727.

8. Falcone JC, Kuo L, Meininger GA. Endothelial cell calcium increases during flow-induced dilation in isolated arterioles. Am J Physiol. 1993;264:H653-H659.[Abstract/Free Full Text]

9. Kuchan MJ, Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol. 1994;266:C628-C636.[Abstract/Free Full Text]

10. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519-560.[Abstract/Free Full Text]

11. Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers? Nature. 1987;235:811-813.

12. Naruse K, Sokabe M. Involvement of stretch-activated ion channels in Ca²⁺ mobilization to mechanical stretch in endothelial cells. Am J Physiol. 1993;264:C1037-C1044.[Abstract/Free Full Text]

13. Oleson SP, Clapham DE, Davies PF. Haemodynamic shear stress activates a K⁺ current in vascular endothelial cells. Nature. 1998;331:168-170.

14. Hoyer J, Distler A, Haase W, Gögelein H. Ca²⁺ influx through stretch-activated cation channels activates maxi K⁺ channels in porcine endocardial endothelium. Proc Natl Acad Sci U S A. 1994;91:2367-2371.[Abstract/Free Full Text]

15. Lund-Johanson P. Newer thinking on the hemodynamics of hypertension. Curr Opin Cardiol. 1994;9:505-511.[Medline] [Order article via Infotrieve]

16. Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med. 1990;323:22-27.[Abstract]

17. Taddei S, Virdis A, Mattei P, Salvetti A. Vasodilation to acetylcholine in primary and secondary forms of human hypertension. Hypertension. 1993;21:929-933.[Abstract/Free Full Text]

18. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]

19. Popp R, Hoyer J, Meyer J, Galla HJ, Gögelein H. Stretch-activated cation channels in the antiluminal membrane of porcine cerebral capillaries. J Physiol. 1992;454:435-449.[Abstract/Free Full Text]

20. Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988;92:145-159.[Abstract/Free Full Text]

21. Oike M, Droogmans G, Nilius B. Mechanosensitive Ca++ transient in endothelial cells from human umbilical vein. Proc Natl Acad Sci U S A. 1994;91:2940-2944.[Abstract/Free Full Text]

22. Hille B. Ionic Channels in Excitable Membranes. Sunderland, Mass: Sinauer Association Inc; 1984:1-19.

23. Yang X, Sachs F. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science. 1989;243:1068-1071.[Abstract/Free Full Text]

24. Franco-Oregón A, Lansman JB. Mechanosensitive ion channels in skeletal muscle from normal and dystrophic mice. J Physiol. 1994;481:299-309.[Abstract/Free Full Text]

25. Nakache M, Gaub HE. Hydrodynamic hyperpolarization of endothelial cells. Proc Natl Acad Sci U S A. 1988;85:1841-1843.[Abstract/Free Full Text]

26. Cooke JP, Rossitch E, Andon N, Loscalzo J, Dzau VJ. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest. 1991;88:1663-1671.

27. Jacobs ER, Cheliakine C, Gebremedhin D, Birks EK, Davies PF, Harder DR. Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells. Pflugers Arch. 1995;431:129-131.[Medline] [Order article via Infotrieve]

28. Latorre R, Oberhauser A, Lbarca P, Alvarez O. Varieties of calcium-activated potassium channels. Annu Rev Physiol. 1989;51:385-399.[Medline] [Order article via Infotrieve]

29. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP. Circulation. 1993;88:193-197.[Abstract/Free Full Text]

30. Ruknudin A, Sachs F, Bustamante JO. Stretch-activated ion channels in tissue-cultured chick heart. Am J Physiol. 1993;264:H960-H972.[Abstract/Free Full Text]

31. Davis MJ, Donovitz JA, Hood JD. Stretch-activated single-channel and whole cell recordings in vascular smooth muscle cells. Am J Physiol. 1992;262:C1083-C1088.[Abstract/Free Full Text]

32. Hunter M. Stretch-activated channels in the basolateral membrane of single proximal tubule cells of frog kidney. Pflugers Arch. 1990;416:448-453.[Medline] [Order article via Infotrieve]

33. Morris CE. Mechanosensitive ion channels. J Membr Biol. 1990;113:93-107.[Medline] [Order article via Infotrieve]

34. Lückhoff A, Busse R. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pflugers Arch. 1990;416:305-311.[Medline] [Order article via Infotrieve]

35. Groschner K, Graier WF, Kukovetz WR. Histamine induces K⁺, Ca²⁺, Cl^- currents in human vascular endothelial cells. Circ Res. 1994;75:304-314.[Abstract/Free Full Text]

36. Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear-stress-induced nitric oxide production in native endothelial cells. Circ Res. 1996;78:750-758.[Abstract/Free Full Text]

37. Lüscher TF. The endothelium: target and promoter of hypertension. Hypertension. 1990;15:482-485.[Free Full Text]

38. Dominiczak AF, Bohr DF. Nitric oxide and its putative role in hypertension. Hypertension. 1995;25:1202-1211.[Free Full Text]

39. Koller A, Huang A. Impaired flow-induced vasodilation in hypertension. Circ Res. 1994;74:416-421.[Abstract/Free Full Text]

40. Cockcroft JR, Chowienczyck PJ, Benjamin N, Ritter JM. Preserved endothelium-dependent vasodilation in patients with essential hypertension. N Engl J Med. 1994;330:1036-1040.[Abstract/Free Full Text]

41. Angus JA, Dyke AC, Jennings GL, Korner PI, Sudhir K, Ward JE, Wright CE. Release of endothelium derived relaxing factor from resistance arteries in hypertension. Kidney Int. 1992;41(suppl 37):S73-S78.

42. Laurent S, Lacolley P, Brunel P, Laloux B, Pannier B, Safar M. Flow-dependent vasodilation of brachial artery in essential hypertension. Am J Physiol. 1990;258:H1004-H1011.[Abstract/Free Full Text]

43. Arnal J-F, Michel J-B, Harrison DG. Nitric oxide in the pathogenesis of hypertension. Curr Opin Nephrol Hypertens. 1995;4:182-188.[Medline] [Order article via Infotrieve]

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