Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid derivates in rat mesenteric small arteries

Abstract

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

Figure 1

Effect of heptanol on membrane potential, cell input resistance and tension. (a) Original recordings of simultaneous measurements of membrane potential and isometric tension in isolated mesenteric small arteries activated with noradrenaline (NA). (b) Bars illustrate the reversibility of heptanol responses on membrane potential and tension: arteries (n=4) were stimulated with NA, superfused with 150 and 500 μM heptanol, rinsed for 20 min and stimulated with NA again. No significant difference between first and second NA stimulation was found. (c) Original recordings of the effects of heptanol on membrane potential and cell input resistance (rapid deflections) in isolated mesenteric small arteries under resting conditions. Heptanol was present as indicated. Note the change in time scale.

Figure 2

Concentration–response relations for effects of heptanol (a, b) and 18β-glycyrrhetinic acid (c, d) on membrane potential (a, c) (differences in membrane potential after and before addition of chemicals) and on cell input resistance (b, d) (the increase in amplitude of voltage responses to 1 nA current injection). Experiments were performed under resting conditions (open circles) and during activation with noradrenaline (filled circles).

Figure 3

Effects of 18β-glycyrrhetinic acid on membrane potential, cell input resistance (rapid deflections) and tension. (a) Original recordings of simultaneous measurements of membrane potential and isometric tension of isolated mesenteric small arteries activated with noradrenaline (NA). (b) Bars illustrate the reversibility of 18β-glycyrrhetinic acid responses on membrane potential and tension: arteries (n=7) were stimulated with NA, superfused with 18β-glycyrrhetinic acid, rinsed for 15 min and stimulated with NA again. No significant difference between first and second NA stimulation was found. (c) Effects of 18β-glycyrrhetinic acid on membrane potential and cell input resistance in isolated mesenteric small arteries under resting conditions. 18β-glycyrrhetinic acid was present as indicated.

Figure 4

Current–voltage relations obtained in isolated smooth muscle from rat mesenteric small arteries. (a) I/V relation under control conditions, in the presence of 150 μM heptanol and in the presence of 150 μM heptanol and 100 nM iberiotoxin. (b) I/V relation under control conditions, in the presence of 150 μM heptanol and in the presence of 500 μM heptanol. (c) I/V relation under control conditions, in the presence of 30 μM 18α-glycyrrhetinic acid and in the presence of 100 μM 18α-glycyrrhetinic acid. (d) I/V relation under control conditions, in the presence of 10 μM 18β-glycyrrhetinic acid and in the presence of 30 μM 18β-glycyrrhetinic acid.

Figure 5

Concentration–tension relation for heptanol in arteries activated with 10 μM noradrenaline (triangles) and with 10 μM noradrenaline in the presence of 100 nM iberiotoxin (squares), n=4. Vertical bars indicate s.e.m.

Figure 6

Original recordings of the effects of heptanol (a), 18αGA (b) and 18βGA (c) on tension and [Ca²⁺]_i in arteries stimulated with a submaximal concentration of noradrenaline (NA). (d) Addition of 150 μM heptanol relaxes the artery and inhibits NA-induced oscillations, but force can be recovered by increasing the NA concentration; oscillation reappeared, but with reduced amplitude. (e) In the presence of 30 μM 18βGA NA evokes only tonic contraction.

Figure 7

Concentration-dependent effects of heptanol (a–c), 18αGA (d) and 18βGA (e–f) on [Ca²⁺]_i (open bars) and tension (filled bars). Arteries were activated with 10 μM noradrenaline, n=5 (a), 2 μM noradrenaline, n=4 (b), 125 mM potassium, n=5 (c), 2 μM noradrenaline, n=7 (d), 2 μM noradrenaline, n=10 (e) or 125 mM potassium, n=9 (f). Vertical bars indicate s.e.m.

Figure 8

Whole-cell calcium currents from smooth muscle cells isolated from rat mesenteric small arteries. Traces in (a) show (I) control current, with (II) 150 μM and (III) 500 μM heptanol and with (IV) 5 μM nifedipine; (V) shows the voltage protocol applied. n=5. Traces in (b) show (I) control current, with (II) 10 μM and (III) 30 μM 18βGA and (IV) 5 μM nifedipine; (V) shows the voltage protocol applied; n=4. Vertical bars indicate s.e.m.

Figure 9

Confocal recordings of [Ca²⁺]_i in regions of interest (ROI, see Methods). Each trace represents one ROI corresponding to a single smooth muscle cell in the arterial wall. (a) Effects of 150 and 500 μM heptanol on an artery activated with noradrenaline (NA). (b) Effects of 10 and 30 μM 18βGA.