Endothelial Membrane Potential Research Articles

Effects of Shear Stress on [Ca2+]I and Membrane Potential of Vascular Endothelium of Intact Rat Blood Vessels

Shear stress is an important modulator of endothelial function which can affect vessel tone, endothelial morphology and gene transcription. However, the mechanism by which the endothelium detects changes in shear stress is the subject of conflicting reports. The possible contributions of changes in the cytosolic Ca2+ concentration ([Ca2+]i) and of membrane hyperpolarisation are disputed. Here we have investigated the effects of shear stress on these variables in native endothelium in situ in rat aorta and hepatic portal vein. Endothelial [Ca2+]i was recorded using the fluorescent indicator fura-2. The [Ca2+]i in unstimulated rat aortic endothelium was 86 +/- 12 nM (n = 9). Acetylcholine (ACh) evoked relaxation of the aorta and a biphasic increase in endothelial [Ca2+]i. Shear stress that evoked relaxation comparable to that evoked by ACh did not affect the endothelial [Ca2+]i. Endothelial membrane potential was recorded using the patch clamp technique. The resting membrane potentials of rat aortic and portal vein endothelium were -47 +/- 3.8 mV (n = 14) and -68 +/- 4.5 mV (n = 9), respectively. Shear stress did not affect the endothelial membrane potential of rat aorta, but evoked a small (-1.6 +/- 0.3 mV, n = 9) hyperpolarisation of the endothelium of rat portal vein in all preparations studied. These results suggest that neither changes in [Ca2+]i nor hyperpolarisation are involved in the reception of shear stress by native rat aortic endothelium in situ, but that shear stress induces small hyperpolarisations of endothelium of portal vein, a mechanism that may be present and of greater physiological significance in other parts of vascular system.

Hoechst Marion Roussel Hypertension Research Clinical Fellowship Award 1999

Hoechst Marion Roussel Hypertension Research Clinical Fellowship Award 1999

A flow-activated chloride-selective membrane current in vascular endothelial cells.

Shear stress-induced activation of endothelial ion channels, one of the earliest responses to flow, is implicated in mechano-signal transduction that results in the regulation of vascular tone. The effects of laminar flow on endothelial membrane potential were studied in vitro using both fluorescent potentiometric dye measurements and whole-cell patch-clamp recordings. The application of flow stimulated membrane hyperpolarization, which was reversed to depolarization within 35 to 160 seconds. The depolarization was caused by a Cl(-)-selective membrane current activated by flow independently of the K(+) channel-mediated hyperpolarization. Thus, flow activated both K(+) and Cl(-) currents, with the net membrane potential being determined by the balance of the responses. Membrane potential sensitivity to flow was unchanged by flow preconditioning that elongated and aligned the cells.

Endothelial membrane potential regulates production of both nitric oxide and superoxide – a fundamental determinant of vascular health

There is recent evidence that the membrane potential of vascular endothelium regulates not only nitric oxide (NO) synthesis, but also superoxide generation, such that hyperpolarization stimulates NO production while suppressing that of superoxide. Given that NO works in a variety of ways to inhibit atherothrombotic disease and hypertension, whereas superoxide not only vetoes the benefits of NO but also disrupts endothelial metabolism and promotes LDL oxidation through its oxidant activity, it is thus evident that endothelium membrane potential is a crucial determinant of cardiovascular risk. Membrane polarization can be enhanced by measures which increase the synthesis or availability of the Na+-K+-ATPase, moderately enhance serum K+and increase the conductance of membrane K+channels. Such measures may include high-K+/low-NA+natural diets, insulin sensitizing modalities, ‘euthyroid replacement therapy’ and ACE inhibitors. Epidemiological correlations of insulin resistance with hypertension and cardiovascular risk may reflect the low membrane potential of insulin-resistant vascular endothelium. Adjunctive measures for suppressing the generation or half-life of endothelial superoxide are suggested.

Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na+−H+ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH4Cl) to the superfusate, or by changing the bath solution saturated with 10% CO2+90% O2 to a solution saturated with 100% O2. Both approaches evoked hyperpolarization of EC. After intracellular Ca2+ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH4Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH4Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na+-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca2+-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca2+ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na+−H+-antiporter, inhibits influx of extracellular Ca2+ into EC under ATP stimulation.

Capillaries demonstrate changes in membrane potential in response to pharmacological stimuli.

It has been proposed that capillaries can detect changes in tissue metabolites and generate signals that are communicated upstream to resistance vessels. The mechanism for this communication may involve changes in capillary endothelial cell membrane potentials which are then conducted to upstream arterioles. We have tested the capacity of capillary endothelial cells in vivo to respond to pharmacological stimuli. In a hamster cheek pouch preparation, capillary endothelial cells were labeled with the voltage-sensitive dye di-8-ANEPPS. Fluorescence from capillary segments (75-150 microns long) was excited at 475 nm and recorded at 560 and 620 nm with a dual-wavelength photomultiplier system. KCl was applied using pressure injection, and acetylcholine (ACh) and phenylephrine (PE) were applied iontophoretically to these capillaries. Changes in the ratio of the fluorescence emission at two emission wavelengths were used to estimate changes in the capillary endothelial membrane potential. Application of KCl resulted in depolarization, whereas application of the vehicle did not. Application of ACh and PE resulted in hyperpolarization and depolarization, respectively. The capillary responses could be blocked by including a receptor antagonist (atropine or prazosin, respectively) in the superfusate. We conclude that the capillary membrane potential is capable of responding to pharmacological stimuli. We hypothesize that capillaries can respond to changes in the milieu of surrounding tissue via changes in endothelial membrane potential.

Effect of acidosis on the membrane potential of intact guinea pig aortic endothelial cells

The effects of a decrease in the extracellular pH from 7.4 to 6.9 on the membrane potential (MP) of intact non-stimulated guinea pig aortic endothelial cells and their ATP-induced electrical responses were studied using a whole-cell mode of the patch-clamp technique. Superfusion of the strip with CO2-−HCO 3 − -buffered acidic solution evoked endothelial depolarization of 6.1±1.0 mV. In Ca2+-free medium, after the MP had been stabilized at a depolarized value, there was no shift in the MP due to extracellular acidification to pH 6.9. In the case of using tris-buffered solution, the same drop in the extracellular pH in Ca2+-containing medium induced no change in the endothelial MP. Subsequent superfusion with CO2−HCO 3 − -buffered solution with pH 6.9 evoked endothelial depolarization of 7.3±1.5 mV. Changing from tris-buffered to CO2−HCO 3 − -buffered solution at a constant buffer pH 7.4 also induced endothelial depolarization, suggesting that intracellular pH is a possible factor that modulates leak Ca entry. The duration of ATP-induced endothelial hyperpolarization at pH 6.9 significantly dropped (76±5 sec, on average) compared with that at pH 7.4 (121±14 sec). It is concluded that modulatory effect of acidosis on the MP of endothelial cells and their ATP-induced electrical responses are caused by inhibition of leak and ATP-stimulated calcium entry into these cells.

Intracellular chloride activity in corneal endothelial cells

Purpose: Current models of hydration control by the corneal endothelium suggest that HCO−3 movement across the cell is driven by the energy contained in the re‐entry of NA+ into the cell down its electrochemical gradient. The nature of the coupling between HCO−3 and Na+ is not yet fully resolved. One suggestion is that the Na+/K+/2Cl− co‐transporter drives Cl− into the cell which then exits the same membrane via a HCO−3/Cl− exchanger. This would drive HCO−3 into the cell from where it could exit down its own electrochemical gradient into the aqueous. Such a system would also raise Cl− above its electrochemical equilibrium. The purpose of this study was to determine if Cl− is passively distributed across the plasma membrane or elevated above its electrochemical equilibrium.Method: To determine if this situation was present in rabbit corneal endothelial cells, we measured [Cl−]i directly using potential sensing and Cl− selective microelectrodes. To make the Cl− selective microelectrodes, filamented glass capillaries were silanized and back‐filled with Cl− ionophore (Corning or WPI) immediately before use. Electrode sensitivity was determined using the fixed interference method.Results: Endothelial membrane potential (PDm) in this series was similar to that in previous studies, 29 ± 0.9 mV (mean ± SEM, n = 22). Chloride potential (PDCl) was used to calculate [Cl−]i, which was 53 ± 2.0 mM (mean ± SEM, n = 22).Conclusions: The [Cl−]i measurement is significantly higher (P < 0.001) than that predicted from the measured PDm (39 ± 0.9 mM) and suggests that Cl− is not passively distributed in corneal endothelial cells.

Smooth muscle cells affect endothelial membrane potential in rat aorta.

The effects of vasoconstrictors on membrane potential of endothelium of intact rat aorta were investigated using the patch-clamp technique. Norepinephrine, endothelin (ET)-1, 5-hydroxytryptamine (5-HT), vasopressin, and angiotensin II evoked depolarization and oscillations in membrane potential. The alpha 1-adrenoreceptor agonist phenylephrine (PE), but not the alpha 2-agonist clonidine or the beta-agonist isoproterenol, evoked oscillations. The antagonist of 5-HT2-receptors, ketanserin, inhibited 5-HT-evoked oscillations. ET-3, unlike ET-1, did not evoke oscillations. The antagonists of voltage-operated Ca2+ channels, nifedipine and verapamil, inhibited vasoconstrictor-evoked oscillations, and the Ca2+ channel agonist BAY K 8644 enhanced oscillations. Acetylcholine and sodium nitroprusside inhibited PE-evoked oscillations. The inhibitors of NO synthase, N omega-nitro-L-arginine and NG-methyl-L-arginine, as well as methylene blue, enhanced oscillations. The intima of rat aorta with endothelium was removed from underlying smooth muscle. In this preparation, acetylcholine evoked a response similar to that in the intact vessel, but PE and ET-1 were without effect. These data suggest that vasoconstrictors acting on receptors on aortic smooth muscle evoke a response that is transferred to the endothelium and evokes depolarization and oscillations in endothelial membrane potential.

Mechanism of acetylcholine action on membrane potential of endothelium of intact rat aorta.

The membrane potential of the endothelium of isolated rat aorta was recorded using the patch-clamp technique. The inhibitor of phospholipase C, U-73122, reduced or abolished the electrical response of the endothelium evoked by acetylcholine, but its inactive analogue, U-73343, was without effect. Depletion of intracellular Ca2+ stores with thapsigargin, cyclopiazonic acid, 2,5-di-(tertbutyl)-1,4-benzohydroquinone, or ionomycin evoked an acetylcholine-like response, which in some aortas included oscillations in endothelial membrane potential and abolished electrical responses evoked by acetylcholine. All stages of acetylcholine- and ionomycin-evoked responses could be evoked in endothelium isolated from underlying smooth muscle. Buffering of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid reduced or abolished acetylcholine-evoked responses. Both buffering of intracellular Ca2+ and inhibition of phospholipase C abolished the inhibitory effect of acetylcholine on phenylephrine-evoked oscillations in endothelial membrane potential. These results suggest that acetylcholine exerts its effect on the membrane potential of intact rat aorta through activation of phospholipase C, the release of Ca2+ from intracellular stores, and so the elevation of intracellular Ca2+ by a mechanism typical for inositol(1,4,5)-trisphosphate-mediated responses.

The effects of metabolic inhibition on endothelial cellular membrane potential

The effects of metabolic inhibition on endothelial cellular membrane potential

Sustained hyperpolarization of cultured guinea pig coronary endothelial cells induced by adenosine.

Electrophysiological experiments on cultured monolayers of guinea pig coronary endothelial cells were performed to substantiate the hypothesis that variation of the endothelial membrane potential may be a functionally important mechanism that contributes to the regulation of coronary blood flow. The endothelial cells were loaded with sodium by superfusion with K(+)-free solution at 37 degrees C for 5-30 min. Readmission of external K+ produced a transient hyperpolarization of up to 85 mV, which was due to stimulation of the electrogenic pump current. In most but not all of the monolayers, superfusion with 2 microM adenosine elicited a transient or a sustained hyperpolarization. The transient hyperpolarization had an amplitude of 15 +/- 6 mV. The sustained hyperpolarization had an amplitude of 11 +/- 3 mV. Our results are in line with the hypothesis that the hyperpolarization of the endothelium induced by release of adenosine into the perivascular space of the capillaries may be conducted electronically to the terminal arterioles and may cause vasodilation via current flow through myoendothelial gap junctions.