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Cardiovascular Research 2004 63(2):331-337; doi:10.1016/j.cardiores.2004.04.008
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Copyright © 2004, European Society of Cardiology

Endothelium-dependent hyperpolarization in small gastric arteries

B Vanheel* and J Breyne

Department of Physiology and Physiopathology, Gent University, U.Z.-Blok B, De Pintelaan 185, Ghent B-9000, Belgium

* Corresponding author. Tel.: +32-9-240-3341; fax: +32-9-240-3059. Email address: bert.vanheel{at}ugent.be

Received 7 August 2003; revised 31 March 2004; accepted 14 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: In many blood vessels, stimulation of the endothelium with various vasoactive substances induces, besides the nitric oxide (NO) and prostacyclin pathways, a third mechanism evoking dilatation. It is based on hyperpolarization of the vascular smooth muscle cell membrane. In the present study, we investigated the existence of endothelium-dependent hyperpolarization in small gastric arteries of the rat and explored its underlying mechanism. Methods: Membrane potentials were recorded by conventional microelectrode techniques in isolated segments of small gastric arteries, the normalized diameter of which was determined from the passive wall tension–internal circumference characteristics as measured with a myograph. Results: After blocking NO and prostaglandin synthesis, application of acetylcholine (3 x 10–7 M) resulted in a membrane hyperpolarization in endothelium intact but not in endothelium-denuded arteries. This membrane potential change was increased by pre-exposure to a low concentration (30 µM) of Ba2+, which selectively inhibits inward rectifying potassium channels. Moreover, the acetylcholine-induced hyperpolarization was unaffected by additional pre-exposure to high concentrations (0.5 mM) of the Na/K-ATPase inhibitor ouabain, which by itself caused a secondary slow endothelium-independent hyperpolarization after an initial peak depolarization. Conclusions: We conclude that acetylcholine produces endothelium-dependent hyperpolarization in gastric small arteries, which does not rely on activation of smooth muscle cell inward rectifying K+ channels or Na/K pumps, and might prove to be another important regulator of gastric mucosal blood flow.

KEYWORDS Blood flow; Vascular smooth muscle membrane potential; Vasoactive agents; Endothelial factors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Gastric mucosal blood flow is of primary importance in maintaining the integrity of the mucosa. The blood provides oxygen, nutrients and gastrointestinal hormones to maintain mucosal function and turnover. The production and secretion of mucus, and the secretion of bicarbonate ions which protect the mucosa, are fully dependent on this blood flow. Moreover, the circulating blood removes waste materials and back-diffusing hydrogen ions. Therefore, disturbances in the mucosal microcirculation can result in mucosal injury and influence the development of peptic ulcers [1].

An increase in gastric mucosal blood flow is brought about by dilation of submucosal arterioles. Systemic as well as local factors such as prostaglandins, leukotrienes and other endogenous chemical mediators of the mucosa influence arteriolar tone. Moreover, gastric blood flow is substantially increased by stimulation of cholinergic vasodilator nerves, within seconds [2], and the reduction in blood flow immediately after vagotomy suggests a basal vasodilatory tone exerted by the vagus nerve [3]. Often, the vasodilators act primarily on the arteriolar endothelial cells to stimulate the production of endothelium-derived relaxing substances.

During the last two decades, the important paracrine role of the vascular endothelium in regulation of blood vessel tone has become increasingly clear. Nitric oxide (NO), formed by the constitutive NO synthase (NOS) in response to stimulation by acetylcholine and other vasodilators, is a well-known endothelium-dependent relaxing factor [4,5], also involved in the regulation of the gastric mucosal blood flow [6–8]. Besides NO, prostacyclin (PGI2) is liberated by the endothelial cells upon stimulation with various agonists, similarly eliciting vasorelaxation [9]. Moreover, in the presence of inhibitors of NOS and prostaglandin synthesis, endothelium-dependent dilation persists in most arteries. This NO- and PGI2-independent relaxation is associated with a hyperpolarization of the membrane of the vascular smooth muscle cells [10,11]. Hyperpolarization of the membrane potential immediately brings about dilatation of blood vessels [12]. It decreases the open probability of voltage-dependent calcium channels, lowering calcium influx [13]. In addition, it reduces calcium release from internal stores [14] and also diminishes the impact of intracellular calcium on the contractile proteins by altering their calcium sensitivity [15].

The mechanism underlying the endothelium-dependent hyperpolarization in various vessels is still debated (for recent reviews, see Refs. [16–18]). In hepatic and small mesenteric arteries of the rat, a transient rise in K+ concentration in the restricted myoendothelial extracellular space, resulting from K+ efflux through agonist-induced activation of calcium-dependent K+ channels on the endothelial cells, was reported [19,20] to act as endothelium-derived hyperpolarizing factor (EDHF). Indeed, in some vessels, small increases in extracellular K+ might influence the inward rectifier and stimulate the Na/K-ATPases of vascular smooth muscle cells, producing the expected membrane potential change. In the superior mesenteric artery of the rabbit, however, it was shown that acetylcholine-induced EDHF-mediated relaxation requires the transfer of a cytosolic factor from the endothelial cells to the smooth muscle cells via heterocellular gap junctions [21]. In smaller arteries, such as intestinal submucosal arterioles, the flow through gap junctions of hyperpolarizing current from endothelial cells might be sufficient to hyperpolarize the electrotonically coupled smooth muscle cells [22,23]. It appears, therefore, that different hyperpolarizing mechanisms may exist in different vascular beds. Moreover, the smooth muscle cells of some arteries such as the femoral artery of the rat are devoid of endothelium-dependent hyperpolarization responses [24].

In previous tension measurements, we showed endothelium-dependent, NO- and PGI2-independent vasorelaxations to occur in isolated small gastric arteries stimulated with acetylcholine [25]. In the present study, we extended these observations by directly measuring the membrane potential responses of the gastric arteriolar smooth muscle cells to acetylcholine using electrophysiological techniques. Moreover, the endothelium-dependent hyperpolarization was further characterized by investigating the involvement of inward rectifying K+ channels and Na/K-ATPases by the use of the blockers Ba2+ and ouabain, respectively. To the best of our knowledge, this study reports the first detailed measurements of endothelium-dependent membrane potential responses in small gastric arteries.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals
Young female Wistar rats (200–250 g body weight) were purchased from Iffa Credo (Brussels, Belgium). The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996).

2.2. Preparation
Experiments were approved by the ethical committee on animal research of Ghent University. Animals were killed with an intraperitoneal injection of a lethal dose (200 mg/kg) of pentobarbitone and laparotomized. The stomach was rapidly excised and transferred to a chilled medium of the following composition (mM): NaCl 135, KCl 5, NaHCO3 20, CaCl2 2.5, MgSO4·7H2O 1.3, KH2PO4 1.2, EDTA 0.026 and glucose 10, gassed with 95% O2, 5% CO2. Several segments of first- and second-order branches of the gastric artery were dissected and transferred to fresh oxygenated medium. After removal of the adherent tissue, one of these segments was reduced to 1.5 mm, taking care not to injure the endothelium, and transferred to a small recording chamber where it was continuously superfused at 35 °C with control fluid. The control fluid consisted of the isolation medium supplemented with NG-nitro-L-arginine (L-NA; 10–4 M) and indomethacin (5 x 10–5 M) to exclude interference from NO and prostanoids, respectively. In some experiments, the endothelium was removed from the arteries by rubbing the intimal surface.

For electrophysiological recordings, the preparation was pinned down to the silicone bottom of the experimental chamber using small pins. Near both attachment sides, the vessel was incised such as to facilitate later access of acetylcholine to the endothelium. After mounting, the vessel segment was allowed to equilibrate for at least 60 min before starting the microelectrode impalements. Microelectrode penetrations were performed from the adventitial side. At the end of the experiments, some representative vessels were moved to an automated wire myograph (model 500A, JP Trading, Aarhus, Denmark) in order to calculate their internal diameter. For this purpose, two stainless-steel wires were guided through the lumen, one was connected to a force transducer and the other fixed to a micrometer, and the passive wall tension–internal circumference characteristics were determined. From this relation, the mean internal diameter of these vessels at an effective transmural pressure of 100 mm Hg was calculated according to the method of Mulvany and Halpern [26].

2.3. Electrophysiological measurements
Transmembrane potentials were measured with standard microelectrode techniques, as described previously [27,28]. Briefly, conventional microelectrodes were pulled with a vertical pipette puller (David Kopf, model 750, Tujunga, CA) from 1 mm o.d. filamented glass tubings (Hilgenberg, Germany). Micropipettes were filled with 1 M KCl and connected to the input stage of a laboratory made MOS/FET operational amplifier. The electrical resistance of the microelectrodes, measured in the normal Krebs–Ringer solution, ranged from 40 to 80 M{Omega}. The measured potential was followed on an oscilloscope and traced with a pen recorder at low speed. Absolute values of membrane potential were taken as the difference of the stabilized potential after cell impalement and the zero potential upon withdrawal of the microelectrode from the cell. Changes in membrane potential produced by applications of acetylcholine in control conditions and after experimental intervention (barium, ouabain) were usually measured in the same cell during continuous recordings. Barium chloride was added to the superfusate for at least 10 min before challenging the preparations with acetylcholine. With ouabain, pre-exposure time was minimally 5 min but varied between experiments, as described in the Results. Exposures to barium and acetylcholine were made by addition of these substances from the appropriate stock solutions to the superfusion solution.

2.4. Chemicals
Indomethacin, L-NA, acetylcholine chloride, ouabain and barium chloride were obtained from Sigma (St. Louis, MO). All concentrations are expressed as final molar concentrations in the superfusion chamber. Stock solutions of L-NA and BaCl2 were made in pure water, indomethacin was dissolved in anhydrous ethanol. Acetylcholine was dissolved in 50 mM potassium hydrogen phthalate buffer, pH 4.0, as a 10–2 M solution. Further dilutions (1:10 or 1:100) were made in the control fluid immediately before addition of aliquots to the superfusate. Ouabain was dissolved directly in the warmed superfusion solution.

2.5. Statistics
Results are expressed as means±S.E.M. Statistical significance was evaluated using Student's t test for paired or unpaired observations, as appropriate, a p value <0.05 indicating a significant difference; n indicates the number of preparations, each obtained from a different animal.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
The small gastric arteries used in this study had an average normalized diameter (at a transmural pressure of 100 mm Hg) of 214±26 µm, as determined in four preparations at the end of the experiments (see Materials and Methods). Smooth muscle cells of such arteries, superfused with the normal L-NA (10–4 M) and indomethacin (5 x 10–5 M) containing Krebs–Ringer solution, had a stable resting membrane potential with a mean value of –64.5±1.3 mV (n=15).

Exposure of intact vessels to a submaximal concentration of acetylcholine (3 x 10–7 M), a concentration previously shown to relax norepinephrine preconstricted gastric arteries by about 50% of the developed active tension, induced a transient peak hyperpolarization of 5.1±0.6 mV (n=14). In the continuous presence of the vasodilator, the membrane potential slowly recovered towards its control level. Recovery was accelerated by washout of the agonist (Fig. 1A).


Figure 1
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  		Fig. 1 Endothelium-dependent hyperpolarization induced by acetylcholine (ACh). Typical tracings of the membrane potential (Em) recorded in smooth muscle cells from small gastric arteries during application of 0.3 µM ACh in the presence (A, C) and the absence (B) of a functional endothelium (E) and after pre-exposure to 30 µM of Ba2+, an inhibitor of inward rectifying K+ channels (C). Notice the larger endothelium-dependent membrane potential response in the presence of Ba2+.

 
In endothelium-denuded arteries (cf. Fig. 1B), the smooth muscle cells had a mean resting membrane potential not significantly different from the control vessels (–65.0±3.1 mV, n=4). Hyperpolarization of the membrane potential did not occur after exposing endothelium-denuded arteries to acetylcholine.

In a next series of experiments (n=8), the influence on the membrane potential and its response to acetylcholine of pre-exposure to low concentrations of Ba2+, known to inhibit the inward rectifying K+ channels, was tested. Fig. 1C shows an original trace from a representative experiment. Superfusion with 30 µM Ba2+ containing fluid caused a depolarization of the resting membrane potential by 5.6±0.5 mV. Pre-exposure to this inhibitor, however, did not decrease the magnitude of the endothelium-dependent hyperpolarization elicited by acetylcholine. Conversely, in this series of experiments, an increase of the endothelium-dependent hyperpolarization was observed. The peak hyperpolarization induced by acetylcholine in the presence of Ba2+ averaged 6.2±0.8 mV, vs. 4.7±0.6 mV in control conditions in this subset of arteries. This difference is statistically significant (p<0.05).

Application of a large concentration (0.5 mM) of ouabain in the continuous presence of Ba2+ produced a transient peak depolarization followed by a slow return of the membrane potential towards baseline. Traces from two succesfull long-term impalements in which the microelectrode was kept in a cell for several hours are shown in Fig. 2A and B. Due to the more compressed time scale used to construct these figures, acetylcholine-induced hyperpolarizations merely appear as inverted peaks. Ouabain depolarized the smooth muscle cells by 6.4±0.7 mV (n=8). When acetylcholine was applied shortly after exposure to ouabain, at a time when the membrane potential was still more depolarized with respect to its level in barium alone (cf. Fig. 2A and B), the endothelium-dependent hyperpolarization averaged 7.0±1.7 mV (n=4), a value not significantly different from that observed in the absence of ouabain. After prolonged pre-exposure to ouabain, acetylcholine still evoked membrane hyperpolarization, averaging 4.3±0.8 mV (n=4), a value slightly but not significantly smaller than that observed in the absence of ouabain. Effective inhibition of the Na/K pump by ouabain was additionally demonstrated by the transient hyperpolarizations of the smooth muscle cells after washout of the drug, being, evidently, more important with increasing exposure time to the inhibitor (Fig. 2A and B). From all ouabain experiments, the mean values for the resting membrane potential before application of acetylcholine and for the magnitude of the peak vasodilator-induced hyperpolarization are summarized in Fig. 3. In the combined presence of Ba2+ and ouabain, the endothelium-dependent hyperpolarization evoked by acetylcholine was not significantly different from that in Ba2+ alone, or from that in control conditions.


Figure 2
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  		Fig. 2 Influence of additional pre-exposure to the Na/K-ATPase inhibitor ouabain. Original recordings of the membrane potential (Em) recorded in smooth muscle cells of small gastric arteries, showing its responses to superfusion with acetylcholine (0.3 µM) containing fluid (as indicated by the short horizontal lines) in control conditions, in the presence of Ba2+ (30 µM), and during and after additional exposure to ouabain (0.5 mM).

 

Figure 3
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  		Fig. 3 Acetylcholine-induced hyperpolarizations of gastric artery smooth muscle cells in control conditions (Co), in the presence of barium (Ba2+, 30 µM) and after additional application of ouabain (Ba2+/ouabain). Data are means±S.E.M. (n=8) and represent the membrane potential (Em) values before and after application of acetylcholine. *Significantly different from the control value (p<0.05).

 
It might be argued that the rather unexpected secondary hyperpolarization of the membrane potential in the continuous presence of 0.5 mM ouabain is due to incomplete inhibition of all membrane Na/K-ATPases, some residual pump activity being stimulated by the rise in intracellular sodium concentration caused by the inhibited pumps. To test this possibility, the ouabain concentration was doubled during the slow hyperpolarization phase in some experiments. Increasing the concentration of the glycoside from 0.5 to 1 mM did not further depolarize the membrane or otherwise modify the course of the slow hyperpolarization (Fig. 4A). Moreover, the slow membrane potential change in the presence of ouabain was independent of an intact endothelium, as depicted in Fig. 4B (n=4).


Figure 4
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  		Fig. 4 Typical tracings showing the effect of doubling the ouabain concentration (A) and of endothelium (E) removal (B) on the membrane potential (Em) of gastric artery smooth muscle cells. Vessels were exposed to Ba2+ (30 µM) throughout. Short horizontal lines in A show times of application of acetylcholine (0.3 µM).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The membrane potential of the smooth muscle cells is an important determinant of arterial tone [12]. In this study, the resting membrane potential of smooth muscle cells of small gastric arteries was measured, and the membrane response to cholinergic stimulation of the intact vessel was quantified and characterized.

A physiological concentration (3 x 10–7 M) of acetylcholine, previously shown to relax norepinephrine-stimulated arteries in an NO- and prostacyclin-independent way to about 50% of their preconstriction level, caused a 5-mV hyperpolarizing shift of the smooth muscle cell membrane potential from a resting level of about –65 mV. The experiments with the endothelium-denuded arteries showed that the acetylcholine-induced hyperpolarization was completely endothelium-dependent, and that the agonist did not exert a direct depolarizing action on the smooth muscle cells as has been reported for some arteries. These direct measurements, therefore, confirm the existence of endothelium dependent hyperpolarization in gastric arteries, as has been suggested from earlier tension measurements [25,29].

The magnitude of the membrane potential response to the submaximal acetylcholine concentration might seem relatively small. However, it should be noted that a change in membrane potential of just a few millivolts can result in a substantial change in vessel diameter [30]. In rat mesenteric arteries in the presence of NG-nitro-L-arginine, an acetylcholine-induced hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone [31]. This implies, therefore, that the endothelium-dependent hyperpolarization as measured in the present study makes an important contribution to the regulation of the diameter of small gastric arteries in situ. Moreover, alterations of the membrane potential generally evoke faster changes in arteriolar diameter than those mediated by second messengers, which more slowly influence gastric blood flow.

Low concentrations of Ba2+ are known to specifically block the inward rectifier K+ current in vascular tissue. In the present conditions, we found 30 µM of Ba2+ to significantly depolarize the smooth muscle cells of small gastric arteries, indicating that the inward rectifier contributes to the setting of the resting membrane potential in these vessels. After inhibition of the inward rectifier, however, endothelium-dependent smooth muscle cell responses to submaximal concentrations of acetylcholine were not inhibited but significantly increased. This excludes a role for inward rectifying K+ channels in the acetylcholine-induced endothelium-dependent hyperpolarization of small gastric arteries. It also suggests that in tonically contracted vessels, in which the membrane potential is similarly depolarized with respect to resting conditions, endothelium-dependent hyperpolarization might be larger than observed in the resting arteries as used in the present study. The increased response might be due to the enhanced driving force on intracellular K+ after Ba2+-induced depolarization, or to the diminished total membrane conductance after blocking the open K+ channels with Ba2+, enlarging the impact of any hyperpolarizing current on the smooth muscle cell membrane potential. With submaximal concentrations of acetylcholine, this current is not expected to drive the membrane potential to values as negative as EK, explaining the less negative absolute level of membrane potential after the Ba2+-induced depolarization. Similarly, in smooth muscle cells from guinea-pig coronary arteries, 100 µM BaCl2 increased EDHF-attributed hyperpolarization [32], while in cells of ilial submucosal arterioles and of mesenteric arterioles of the same species 500 µM Ba2+ was used to depolarize the membrane potential away from the K+ equilibrium potential in order to observe significant endothelium-dependent hyperpolarizations after application of acetylcholine [22,33].

In gastric arteries, the additional inhibition of the Na/K pump with 0.5 mM ouabain further depolarized the smooth muscle cells, as expected from the sudden loss of electrogenic pump activity. Since the Kd value for ouabain of the ubiquitous low affinity {alpha}1-isoform containing Na/K-ATPase in rat tissue is about 15 µM [34], and the other isoenzymes are much more sensitive to the cardiotonic steroid [35], it can reasonably be assumed that in the present conditions all pump isoenzymes are effectively inhibited by 0.5 mM of the cardiotonic steroid. In mesenteric arteries of the rat, the vasodilation occurring on readmission of K+ ions after K+-free perfusion was completely inhibited by 100 µM of ouabain [36]. Moreover, in the present study, the complete inhibition of Na/K-ATPase activity by 0.5 mM ouabain was directly verified by the lack of additional depolarization on doubling the ouabain concentration.

In the combined presence of barium and ouabain, the magnitude of the endothelium-dependent hyperpolarization induced by acetylcholine was not significantly changed. These observations strongly suggest that small gastric artery smooth muscle cells do not rely on their inward rectifiers and Na/K pumps to produce this hyperpolarization. In a previous study, it was found that small increases in extracellular K+ concentration were unable to consistently relax preconstricted gastric arteries [25]. Taken together, these findings argue against a role for extracellular K+ to act as an EDHF in this preparation.

In the prolonged presence of ouabain, the membrane potential slowly became more hyperpolarized than before exposure to the cardiotonic glycoside, in an endothelium-independent way. A plausible explanation for this observed shift is that the ouabain-induced rise in intracellular Na+ concentration leads, via an influence on the Na/Ca exchanger, to a secondary rise in intracellular Ca2+ concentration, as has been documented in several cell types. This raised Ca2+ is expected to subsequently open the Ca2+-dependent K+ channels of the smooth muscle cell. In the pancreatic B-cell, e.g., ouabain was shown to increase 86Rb (used as a tracer for K+) outflow [37]. Thus, a gradually increasing membrane K+ conductance might explain both the slow hyperpolarization as well as the observed tendency for the endothelium-dependent hyperpolarization to decrease in magnitude after prolonged ouabain exposure.

In a previous study, shorter exposures to ouabain were found to inhibit the EDHF-mediated relaxation of gastric arteries [25]. The present measurements showing that the endothelium-dependent hyperpolarization of the vascular smooth muscle cells is not affected by the pump inhibitor suggest, therefore, a defective coupling between the change in membrane potential and the mechanical response, presumably involving a hampered calcium extrusion. Moreover, they stress the importance of electrophysiological measurements when studying the EDHF phenomenon [38].

In summary, we have shown that acetylcholine hyperpolarizes the membrane potential of the vascular smooth muscle cells of small gastric arteries. This electrical change, expected to exert a substantial influence on vessel tone and resistance, is entirely dependent on an intact endothelium, but independent from the endothelial NO/prostacyclin pathways. The mechanism underlying the endothelium-dependent hyperpolarization does not seem to rely on the stimulation of inward rectifying K+ channels or Na/K pumps, as was shown for rat hepatic arteries. Endothelium-dependent hyperpolarization might prove to be another important and rapid regulator of gastric mucosal blood flow, of utmost importance in the maintenance of mucosal function and integrity.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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