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Am J Physiol Heart Circ Physiol 282: H335-H341, 2002;
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Vol. 282, Issue 1, H335-H341, January 2002

Glycyrrhetinic derivatives inhibit hyperpolarization in endothelial cells of guinea pig and rat arteries

Marianne Tare, H. A. Coleman, and Helena C. Parkington

Department of Physiology, Monash University, Melbourne, Victoria 3800, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glycyrrhetinic acid (GA) derivatives have been used to implicate gap junctions in vasorelaxation attributed to endothelium-derived hyperpolarizing factor (EDHF). The aim of this study was to assess whether GA compounds affect endothelial cell hyperpolarization. Membrane potentials were recorded from dye-identified endothelial and smooth muscle cells of guinea pig coronary and rat mesenteric arteries. GA derivatives had varied effects on the resting membrane potential: depolarization, hyperpolarization, or no effect, depending on the artery. 18alpha -GA (50 µM) had a small variable effect on ACh-induced hyperpolarizations in endothelial cells. 18beta -GA (30 µM) and carbenoxolone (100 µM) significantly reduced ACh-induced hyperpolarizations in both endothelial and smooth muscle cells. Smooth muscle action potentials in rat tail arteries were smaller and slower in the presence of 18beta -GA. Nerve-induced excitatory junction potentials were inhibited by 18beta -GA and carbenoxolone, whereas the time course of their decay initially increased and then decreased. In conclusion, the GA compounds had a range of effects. Their inhibition of the EDHF hyperpolarization and relaxation in the smooth muscle may stem from the inhibition of endothelial cell hyperpolarization.

gap junctions; glycyrrhetinic acid; action potentials; carbenoxolone; endothelium-derived hyperpolarizing factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AN INCREASING APPRECIATION of the importance of gap junction intercellular communication (GJIC) in diverse tissues is resulting in greater interest in agents that can alter the activity of gap junctions. In some blood vessels, the endothelial and smooth muscle cells are electrically (2, 5, 6, 9, 16, 26, 27) or dye coupled (15), and this has raised the possibility that, at least in some blood vessels, endothelium-dependent hyperpolarization in vascular smooth muscle may represent electrotonic spread, via myoendothelial gap junctions, of hyperpolarization generated in the endothelial cells (3). Inhibitors of GJIC would be invaluable tools with which to assess the involvement of GJIC in the prominent smooth muscle hyperpolarization that is independent of nitric oxide and prostanoid and attributed to endothelium-derived hyperpolarizing factor (EDHF). Although there are a number of putative inhibitors of gap junctions, nonspecific actions limit their usefulness.

Glycyrrhetinic acid (GA) and its derivatives are triterpenoid saponins derived from licorice. These agents inhibit intercellular transfer of metabolites, and this has been attributed to the inhibition of gap junctions (7). GA also inhibits the spread of the fluorescent dye Lucifer yellow between alveolar epithelial cells (11). On the basis of these effects of GA on GJIC, some studies (8, 28, 29) using GA derivatives have concluded that EDHF is due to electrotonic spread. However, even though smooth muscle cells are coupled electrically, Lucifer yellow does not move from smooth muscle cells either to neighboring smooth muscle or into endothelial cells (5, 6, 9, 15, 23). Therefore, inhibition of dye coupling may not necessarily mean that electrical activity is uncoupled. Furthermore, GA derivatives have been reported to have a range of effects, and this is not surprising in view of the detergent-like nature of these compounds. A reputed inhibition of arachidonic acid metabolism (14) is particularly pertinent to EDHF because there have been suggestions that EDHF may be a product of the cytochrome P-450 pathway of arachidonic acid metabolism in some vessels (4, 10, 18). The aim of the present study was to evaluate the effects of GA compounds on the ACh-induced hyperpolarization recorded from identified endothelial cells. The study focused on the guinea pig coronary artery because stimulation of its endothelium in the presence of nitric oxide and prostaglandin synthesis inhibitors results in a very pronounced hyperpolarization of the smooth muscle that is attributed to EDHF (13, 17, 24). The results of the present study indicate that GA derivatives have a range of effects that limit their usefulness in evaluating the involvement of electrical coupling in cellular processes, especially in the actions of EDHF.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Guinea pigs and rats were killed by cervical dislocation with ethics committee approval. For membrane potential recordings from endothelial cells, guinea pig coronary and rat mesenteric arteries were cut open and secured to the Silicone rubber floor of the recording chamber with the endothelium uppermost. In other experiments, ring segments of guinea pig coronary and rat tail arteries, 1-2 mm in length, were mounted on a wire myograph for simultaneous measurement of smooth muscle membrane potential and isometric tension (17, 24). Silver-silver chloride electrodes mounted on the jaws of the myograph, one on either side of the tissue, were used to stimulate the perivascular sympathetic nerves of the rat tail artery. Pulse strengths of 20-40 V (stimulator dial settings) and 0.1 ms in duration were used. Responses evoked by electrical stimulation were sensitive to tetrodotoxin. Tissues were superfused at 3.6 ml/min and 35°C with physiological saline solution (PSS) consisting of (in mM) 120 NaCl, 5 KCl, 25 NaHCO3, 1 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11 glucose and bubbled with 95% O2-5% CO2. Except where indicated, Nomega -nitro-L-arginine methyl ester (L-NAME; 100 µM) and indomethacin (1 µM) were included in the superfusate to inhibit nitric oxide and prostaglandin production. The endothelium was stimulated with ACh using two methods of application: for short exposures, ACh was injected directly into the superfusion line for 10 s; for long exposures, ACh was added to the superfusate for 1 min.

Membrane potentials were recorded with intracellular glass microelectrodes. The tips of the electrodes were initially filled with 2% Lucifer yellow CH as the dilithium salt dissolved in 1 M LiCl, and the electrodes were then backfilled with 1 M KCl, resulting in electrode resistances of ~100 MOmega . Diffusion of the Lucifer yellow into the cells from which the recordings were made enabled the unequivocal identification of the cells when viewed with epifluorescence optics. Every endothelial cell studied was identified in this manner.

The following drugs were used: ACh, L-NAME, indomethacin, dilithium Lucifer yellow CH, phenylephrine, 18alpha -GA, 18beta -GA, carbenoxolone, tetraethylammonium (all from Sigma), and U46619 (Cayman Chemicals). Stock solutions of 18alpha -GA and 18beta -GA were prepared immediately before use to minimize possible cytotoxicity of these compounds (7). 18alpha -GA and 18beta -GA were dissolved in dimethyl sulfoxide (BDH Chemicals), which was without effect at 100 µM. Carbenoxolone was dissolved directly in the PSS.

Data were compared using Student's t-test, paired or unpaired as appropriate, using the software packages InStat3 or Prism3 (both from GraphPad). P values <0.05 were considered statistically significant. Means ± SE and the number of animals (n) are quoted throughout. Concentration-response data for each tissue were fitted to a sigmoidal curve using the least-squares method (Prism3). The time constant of decay of excitatory junction potentials (EJPs) was determined by fitting a single exponential to the decay of the EJPs using the software CLAMPfit 8 (Axon Instruments).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of cell types. An important aspect to this study was the unequivocal identification of the type of cell from which the recordings were made. Figure 1, left, shows an endothelial cell that was identified under epifluorescence optics after its loading with Lucifer yellow. These cells were orientated along the longitudinal axis of the arteries. Smooth muscle cells loaded with Lucifer yellow were identified by their transverse orientation to the vessel axis (Fig. 1, right).


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  		Fig. 1.   Left: example of a dye-identified endothelial cell in the guinea pig coronary artery from which recordings of membrane potential were made. The cell, loaded with Lucifer yellow, was aligned along the longitudinal axis of the artery. This image was obtained under epifluorescence illumination alone, resulting in the dark background. Calibration bar: 33 µm. Right: a smooth muscle cell in the guinea pig coronary artery that was identified after loading with Lucifer yellow. The cell was orientated transversely to the axis of the artery. This image was obtained under epifluorescence plus bright-field illumination, enabling greater detail of the tissue to be seen. Calibration bar: 66 µm.

Guinea pig coronary artery smooth muscle. The effects of 18alpha -GA, 18beta -GA, and carbenoxolone on the EDHF hyperpolarization were examined in recordings of membrane potential made from the smooth muscle of coronary arteries mostly mounted on a wire myograph and superfused with PSS containing L-NAME and indomethacin. The resting membrane potential of coronary artery smooth muscle cells in arteries mounted on the myograph was -52 ± 3 mV (n = 8). In tissues at rest, 18beta -GA (30 µM) evoked an initial hyperpolarization of 8 ± 3 mV (n = 4), but this tended to decrease over time. 18beta -GA decreased the amplitude of the hyperpolarization evoked by 1-min exposure to 0.1 µM ACh from 14 ± 1 to 5 ± 1 mV (n = 4, P = 0.0017). An example of a response recorded from a smooth muscle cell in a tissue pinned out flat is shown in Fig. 2A,a. From concentration-response curves for 10-s applications of ACh, 18beta -GA reduced the pD2 from 7.23 ± 0.07 to 6.87 ± 0.16 (n = 4), but these were not significant (P = 0.085) (Fig. 2C,c).


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  		Fig. 2.   Effects of 18beta -glycyrrhetinic acid (GA) and carbenoxolone (Carb) on endothelium-derived hyperpolarizing factor (EDHF)-mediated responses in guinea pig coronary and rat mesenteric arteries. A,a: membrane potential recordings in an endothelial cell (top) and smooth muscle cell (bottom) from the same segment of guinea pig coronary artery. ACh (0.1 µM, 1 min) was applied before and during exposure to 30 µM 18beta -GA. A,b: membrane potential recording in a coronary artery endothelial cell from a different animal. Shown are responses to 0.1 µM ACh (1 min) before and during exposure to Carb (100 µM). A,c: membrane potential recording in a rat mesenteric artery endothelial cell. Responses evoked by 1-min application of 0.1 µM ACh are shown before, during, and after washout of Carb (100 µM). This was a continuous recording in the same cell. B: inhibitory effect of Carb (100 µM) and of 18beta -GA (30 µM) on endothelial cell hyperpolarization in response to 1-min application of 0.1 µM ACh (guinea pig coronary artery, n = 5; rat mesenteric artery, n = 3). C: concentration-response curves for the smooth muscle response to 10-s applications of ACh in rings of guinea pig coronary artery mounted on a wire myograph. The effects of Carb (100 µM) on the amplitude of hyperpolarization (C,a) and corresponding relaxation (C,b; expressed as a percentage of contraction evoked by U46619 or Carb, n = 4) are shown. C,c: effect of 18beta -GA (30 µM) on amplitude of hyperpolarization (n = 4). *Significantly different from control (P < 0.05).

Carbenoxolone (100 µM) alone depolarized the smooth muscle by 24 ± 4 mV (n = 4); hence, control responses were obtained in arteries depolarized and constricted to a similar degree using the thromboxane analog U46619 (80-100 nM). Carbenoxolone decreased the amplitude of the EDHF-attributed hyperpolarization evoked by 1-min exposure to 0.1 µM ACh from 19 ± 3 to 4 ± 1 mV (n = 4, P = 0.0043) and reduced the accompanying relaxation from 101 ± 5% to 31 ± 10% (n = 4, P = 0.0007) (data not shown). Carbenoxolone reduced the sensitivity of the artery to ACh (10-s application) by decreasing the pD2 for the hyperpolarization from 7.28 ± 0.09 to 6.76 ± 0.09 (n = 4, P = 0.0065) (Fig. 2C,a) and for the accompanying relaxation from 7.63 ± 0.08 to 7.02 ± 0.06 (n = 4, P = 0.001) (Fig. 2C,b).

18alpha -GA (50 µM) had no significant effect on either the resting membrane potential or on the hyperpolarization evoked by ACh (Fig. 3B, bottom).


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  		Fig. 3.   The effects of 18alpha -GA (50 µM) on the membrane potential recorded from the guinea pig coronary artery. A: in a dye-identified endothelial cell, 18alpha -GA had little effect on the resting membrane potential and produced only a small inhibition of the ACh-induced hyperpolarizations (1-min application). B, top: histogram summarizing the results from 6 tissues showing that the inhibition of the ACh-induced hyperpolarizations recorded from endothelial cells was maximal and statistically significant after 10-min exposure to 18alpha -GA but that the effect then declined with longer exposures. B, bottom: histogram showing that 18alpha -GA had no effect on the EDHF-attributed hyperpolarizations recorded from smooth muscle cells in 6 arteries mounted on a wire myograph. *Significantly different from control (P < 0.05).

Guinea pig coronary artery endothelial cells. Because the EDHF-attributed hyperpolarization may well be due to electrotonic spread of hyperpolarization from the endothelium, the effects of the GA compounds were assessed on dye-identified (Lucifer yellow) endothelial cells in coronary arteries. L-NAME and indomethacin were present in the PSS throughout the experiments. 18beta -GA hyperpolarized the membrane from -41 ± 1 to -44 ± 2 mV (n = 5) and decreased the amplitude of the hyperpolarization evoked by ACh (1 min) from 16 ± 1 mV to 6 ± 1 mV (n = 5, P = 0.0001) within 6 min (Fig. 2, A,a, and B).

Carbenoxolone depolarized the membrane of the endothelial cells by 14 mV from -48 ± 4 to -34 ± 2 mV (n = 5). Carbenoxolone also reduced the ACh (1 min)-induced hyperpolarization, from 17 ± 2 to 4 ± 1 mV (n = 5, P = 0.0004) within 6 min (Fig. 2, A,b, and B).

18alpha -GA (50 µM) had no significant effect on the resting membrane potential. It had a small and variable effect, which declined after 30 min, on the hyperpolarization evoked by ACh (Fig. 3, A and B, top).

Rat mesenteric artery endothelial cells. The EDHF hyperpolarization of the rat mesenteric artery has been the subject of a number of studies, including one study (8) in which carbenoxolone reduced the smooth muscle hyperpolarization. In the present study, we recorded the effects of carbenoxolone on the ACh-induced hyperpolarization of dye-identified endothelial cells in this artery. Carbenoxolone (100 µM) had a small, variable, and insignificant effect on the resting potential (-59 ± 1 mV in control and -55 ± 6 mV in carbenoxolone, n = 3). However, it reduced the amplitude of the hyperpolarization induced by 1-min application of 0.1 µM ACh from 18 ± 2 to 7 ± 2 mV (n = 3, P = 0.0177) (Fig. 2, A,c, and B).

It was observed in both coronary and mesenteric arteries that Lucifer yellow readily diffused from the impaled endothelial cells to neighboring endothelial cells in control conditions. It was predominantly visible only in the impaled cell due to the continued diffusion of dye from the microelectrode. In the presence of GA compounds, the Lucifer yellow appeared to become localized within the cell from which the recordings were made, resulting in more intense fluorescence, consistent with inhibition of dye transfer due to inhibition of gap junctions. After washout of the GA compounds, it was observed that inhibition of dye coupling persisted, whereas the electrical responses in endothelial and smooth muscle cells returned to normal.

Rat tail artery junction and action potentials. EJPs and action potentials can be readily and consistently evoked in the rat tail artery. The resting membrane potential of smooth muscle cells of the rat tail artery was -67 ± 1 mV (n = 10). In this vessel, 18beta -GA (30 or 50 µM) evoked 7.0 ± 1.2 mV (n = 6) depolarization of the membrane and decreased the amplitude of subthreshold EJPs from 4.1 ± 0.3 mV (77 EJPs, n = 5) to 2.5 ± 0.2 mV (75 EJPs, n = 5, P < 0.001) (Fig. 4A). 18beta -GA had a biphasic effect on the time constant of decay of the EJPs, with an initial increase and then a decrease (Fig. 4B).


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  		Fig. 4.   The effects of GA compounds on nerve-evoked junction and action potentials in the rat tail artery. A: 18beta -GA and Carb produced similar monotonic decreases in the amplitudes of the excitatory junction potentials (EJPs). B: 18beta -GA initially increased and then decreased the decay time constant of the EJPs. Carb also transiently increased the decay time constant. A subsequent trend to a decreased time constant was not statistically significant. C: action potentials evoked in the presence of tetraethylammonium (2 mM) by nerve stimulation () were not affected by Carb (100 µM) but were inhibited by 18beta -GA (50 µM) even when greater stimulus strengths were used.

Carbenoxolone (100 µM) initially hyperpolarized the membrane by 1.8 ± 0.5 mV and then depolarized it by 4.5 ± 0.7 mV (n = 6). It decreased the amplitude of EJPs, from 4.0 ± 0.2 mV (94 EJPs, n = 5) to 2.3 ± 0.1 mV (77 EJPs, n = 5, P < 0.001) (Fig. 4A), and increased the decay time constant (Fig. 4B). Subsequently, the time constant decreased to a value that was not significantly different from control (Fig. 4B).

When the sympathetic nerves were stimulated with a greater stimulus strength, the EJPs were larger and initiated active responses. In the presence of tetraethylammonium (2 mM), the active responses became pronounced action potentials. 18beta -GA (50 µM) decreased the amplitude of the action potentials from 46.7 ± 1.0 mV (n = 9) to 29.3 ± 0.5 mV (n = 7, P < 0.0001), often with a shoulder appearing on the repolarization phase (Fig. 4C) (L-NAME and indomethacin were present on 4 of 9 or 7 occasions and had no additional effects). In contrast, carbenoxolone (100 µM) had no effect on the amplitude of the action potentials (47.4 ± 0.8 mV, n = 5) (Fig. 4C).

In another set of experiments, brief (30 s) applications of phenylephrine (1 µM) were used to depolarize and evoke action potentials and contract the rat tail artery (in the presence of 2 mM tetraethylammonium). 18beta -GA (30 µM) reduced the phenylephrine-induced depolarization, the action potentials, and contraction (Fig. 5). In contrast, carbenoxolone (100 µM) had no obvious effect on any of these responses (Fig. 5) (4 tissues studied; 2 included L-NAME and indomethacin).


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  		Fig. 5.   The effect of GA compounds on phenylephrine (Phen)-induced responses recorded from the rat tail artery. A: membrane potentials recorded simultaneously with isometric tension (B) from an artery mounted on a wire myograph. In control solution, Phen (1 µM) induced depolarization, action potentials, and contraction of the artery. Carb (100 µM) had no appreciable effects on these responses, whereas 18beta -GA (50 µM) inhibited the depolarization, the action potentials, and the contraction.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A striking feature of the results presented in this study is the similarity in recordings made from identified endothelial cells with recordings from identified smooth muscle cells. The membrane potentials before, as well as during, the hyperpolarizations induced by ACh were very similar in both cell layers in the guinea pig coronary artery in both the absence and presence of GA compounds (e.g., Fig. 2A). Such observations are consistent with strong electrical coupling between the two layers of cells, as indicated in a number of other blood vessels (2, 5, 6, 9, 16, 26). The persistent similarity in the responses in the presence of GA compounds indicate that the effects of the three GA compounds on electrical coupling between the two layers of cells in the guinea pig coronary artery are likely to be weak. These results support the notion that the inhibitory actions of GA compounds on the EDHF-attributed hyperpolarization and relaxation of vascular smooth muscle cells (Ref. 8 and the present study) are due to their inhibition of the responses of endothelial cells to agonist stimulation.

In the rat mesenteric artery the reduction by carbenoxolone of the endothelial hyperpolarization to 39% (present study) is reasonably similar to the inhibition of smooth muscle hyperpolarization (to 56%) reported by Edwards et al. (8). Thus these observations on the rat mesenteric artery are also consistent with strong electrical coupling, the electron microscopic evidence of myoendothelial gap junctions in this artery (20), and with the GA compounds not appreciably affecting the electrical coupling.

In inhibiting the initiation of the EDHF-attributed hyperpolarization, there are a number of sites at which the GA compounds could act, of which some may involve the smooth muscle cells. Inhibition of muscarinic receptors may be unlikely because binding studies have ruled out an effect of carbenoxolone on a wide range of receptors including muscarinic receptors (19). Phospholipase activity has been implicated in the production and/or actions of EDHF (1), and phospholipase activity has been reported to be inhibited by GA compounds (14). Furthermore, inhibition of the activation or activity of intermediate and small conductance Ca2+-activated K+ channels, which underlie the EDHF-attributed hyperpolarization (6), could explain its reduced amplitude. This is a real possibility because action potentials initiated by nerve stimulation or phenylephrine were of smaller amplitude and slower time course in the presence of 18beta -GA (although not carbenoxolone), indicating an inhibitory effect of this GA compound on voltage-operated Ca2+ channels. This observation is consistent with, and extends the conclusions of, Santicioli and Maggi (21), who showed that 18beta -GA (30 µM) inhibited electrical and contractile activity of the guinea pig renal pelvis and ureter. The sucrose gap technique used in that study could not distinguish between an effect on electrical coupling or an effect on the action potentials. Our results strongly support the latter and not the former possibility. At 100 µM, 18beta -GA was found to have nonspecific effects that limited its usefulness in determining the involvement of gap junctions (21).

The GA compounds (18beta -GA in particular) also decreased the amplitude of the EJPs and, after a transient increase, resulted in a marked decline in the decay time constant of the EJPs, the latter being indicative of a decrease in membrane resistance. The transient increase in the decay of the EJPs may have resulted from an inhibition of ion transport processes that contribute to the resting conductance, and this would be consistent with the changes in the resting membrane potential. The subsequent decrease in membrane resistance may well be an increase in membrane leakiness due to the detergent-like nature of these triterpenoid saponins. Although the decrease in membrane resistance may explain some of the decrease in the amplitude of the EJPs, the decline in the amplitudes preceded the decline in the decay time constant of the EJPs. There are myriad steps between the initiation of the presynaptic action potential and the EJP at which the GA compounds could exert their effects.

The GA compounds that were used in the present study were also capable of inducing changes in the resting membrane potential of the smooth muscle and endothelial cells. The effects on the resting potential depended on the particular compound and on the blood vessel, but in any individual vessel the effects on the endothelial and smooth muscle cells were similar. Thus 18beta -GA tended to hyperpolarize the smooth muscle and endothelial cells of the guinea pig coronary artery, whereas carbenoxolone depolarized both cell layers. In contrast, in the rat tail artery, 18beta -GA depolarized the membrane, and carbenoxolone had a small biphasic effect. In guinea pig submucosal arterioles, 18beta -GA and carbenoxolone both produced large depolarizations of the membrane (6, 12). GA-induced depolarization may be due, at least in part, to inhibition of Na+-K+-ATPase (25). The reasons for the differences in the effects of the GA compounds between blood vessels are not clear but may reflect different complements of ion transport processes.

Carbenoxolone (18beta -GA, 3beta -O-hemisuccinate) is a more water-soluble form of 18beta -GA and has been presumed to have similar effects to 18beta -GA. Both agents inhibited the ACh-induced hyperpolarization in endothelial and smooth muscle cells and also reduced the amplitude of the EJPs. However, in the present study, there were some differences between the two. 18beta -GA had an inhibitory effect on the smooth muscle action potential, whereas carbenoxolone did not. In the guinea pig coronary smooth muscle cells, 18beta -GA evoked hyperpolarization, whereas carbenoxolone depolarized the cells. The reasons for the different effects are not clear but may reflect differences in their lipophilic nature.

That carbenoxolone inhibited the ACh-induced hyperpolarization in guinea pig coronary artery endothelial cells that were unequivocally identified with Lucifer yellow is at variance with previous reports of carbenoxolone having no effect on the ACh-induced hyperpolarization of endothelial cells in the guinea pig carotid artery (8). The reason for this discrepancy is not clear but may reflect variability between tissues.

Some evidence indicates that the alpha -form of GA (18alpha -GA) is a more specific and less toxic inhibitor of gap junctions than other GA compounds (7). In the present study, 18alpha -GA was less effective than either 18beta -GA or carbenoxolone, consistent with an ineffectiveness of 18alpha -GA on myoendothelial coupling in the rat mesenteric artery (22). It had only a small and variable effect on the ACh-induced hyperpolarization of endothelial cells, which did not persist beyond 30 min, and no appreciable effect on the hyperpolarization recorded from smooth muscle cells. A small difference in effect between the two cell types also occurred with the other two GA compounds when the smooth muscle recordings were made from tissues mounted on the wire myograph. This difference in effect may reflect differences in accessibility of the GA compounds to the endothelial cell layer, and this depended on the experimental setup that was used. During recordings from the endothelial cells, the superfusate flowed directly over the exposed endothelial cells, whereas during recordings from the smooth muscle cells in arteries mounted on the wire myograph, the endothelial cells were less directly exposed to the superfusate, remaining in the very limited "lumen" of the wire myograph preparation. If the hyperpolarizations attributed to EDHF were generated in the endothelial cells, then reduced accessibility to the endothelial cells could result in a lesser effect of the GA compounds. Nevertheless, that the hyperpolarizations recorded from the smooth muscle and endothelial cells were of similar amplitude suggests that 18alpha -GA, similar to 18beta -GA or carbenoxolone, had no appreciable effects on electrical coupling between the two cell layers.

The effectiveness of various putative gap junction inhibitors, including GA compounds, has been assessed in dye transfer studies, usually with the fluorescent dye Lucifer yellow. In the present study, Lucifer yellow rapidly spread from the impaled endothelial cell to neighboring cells. Dye localization in the impaled cell persisted after removal of the GA compounds even though endothelial and smooth muscle hyperpolarization returned to normal. This is consistent with the notion that inhibition of dye transfer may not be a good indicator of the state of electrical coupling. Good electrical coupling of smooth muscle cells has been shown to occur in the absence of dye coupling (9, 23). It is possible that good electrical coupling between the two cell layers may require the patency of only a small proportion of the total number of connexons present.

In conclusion, these results show that GA compounds can exert a variety of effects, some of which are likely to alter the activity of ion transport processes, including ion channels. The various effects of the GA compounds raise concerns regarding the interpretation of results that are based on the assumption that the only effect of these agents is to inhibit GJIC. In particular, the inhibition by these compounds of hyperpolarization induced in endothelial cells by ACh indicates that these agents are limited in their usefulness in the evaluation of the involvement of electrical coupling in the EDHF-attributed hyperpolarization and relaxation of smooth muscle in blood vessels.


    ACKNOWLEDGEMENTS

This work was supported by the National Health and Medical Research Council of Australia. This work was carried out during the tenure of a grant from the National Heart Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: H. A. Coleman, Dept. of Physiology, PO Box 13F, Monash Univ., Melbourne, Victoria 3800, Australia (E-mail: h.coleman{at}med.monash.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 June 2001; accepted in final form 17 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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6.   Coleman, HA, Tare M, and Parkington HC. K+ currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels. J Physiol (Lond) 531: 359-373, 2001[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 282(1):H335-H341
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