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

Abstract

Heptanol, 18α‐glycyrrhetinic acid (18αGA) and 18β‐glycyrrhetinic acid (18βGA) 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. Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca2+‐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. Heptanol (150 μM) 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 μM did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 18βGA (30 μM) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18αGA (100 μM) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. We conclude that in mesenteric small arteries, heptanol and 18βGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18βGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18αGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded. British Journal of Pharmacology (2004) 142, 961–972. doi:

Sustained conduction of vasomotor responses in rat mesenteric arteries in a two‐compartment in vitro set‐up

“…It should be noted that although 18βGA has been widely used as gap junction inhibitor,27 this compound, as many of the gap junction blockers, may have non‐specific effects28 and clearly, future work should include more robust testing of their role, also by including membrane potential recordings.…”

Effect of heptanol on noradrenaline-induced contractions in rat vas deferens

“…Although some studies have been conducted previously on the contribution of cell-to-cell coupling to agonist-induced contractions in a variety of smooth muscle organs (Christ, 1995;Christ et al, 1996;Matchkov et al, 2004), in rat vas deferens the dynamics of recruitment of cells via different pathways, e.g. the Representative traces of the effect of 2 μM nifedipine and 2 μM nifedipine with 2 mM heptanol on contractions elicited by addition of 20 μM NA to the organ bath for 2 min.…”

“…Furthermore, the reduction observed with heptanol alone was not significantly different than that induced by heptanol in tissues preincubated with nifedipine (P>0.01), indicating involvement of similar mechanisms in the presence or absence of nifedipine. Heptanol has also been suggested to exert other non-junctional actions in various tissues (activation of KCa channels, reduction of [Ca 2+ ]i (Matchkov et al, 2004); voltage-dependent Na + current suppression (Nelson and Makielski, 1991); decreased voltage-dependent K + and Ca 2+ currents (Perez-Armendariz et al, 1991)). However, in a recent detailed characterization of its effects on vascular smooth muscle cells (Matchkov et al, 2004), it was shown that heptanol, over the range of concentrations used in our study (0.5-2.0 mM), potently increased the input resistance of the cells and desynchronized their [Ca 2+ ]i oscillations, indicating that it powerfully uncoupled the cells.…”

“…Heptanol has also been suggested to exert other non-junctional actions in various tissues (activation of KCa channels, reduction of [Ca 2+ ]i (Matchkov et al, 2004); voltage-dependent Na + current suppression (Nelson and Makielski, 1991); decreased voltage-dependent K + and Ca 2+ currents (Perez-Armendariz et al, 1991)). However, in a recent detailed characterization of its effects on vascular smooth muscle cells (Matchkov et al, 2004), it was shown that heptanol, over the range of concentrations used in our study (0.5-2.0 mM), potently increased the input resistance of the cells and desynchronized their [Ca 2+ ]i oscillations, indicating that it powerfully uncoupled the cells. Indeed, in several recent studies carried out on a range of cell / tissue preparations, including mouse heart (Knapp et al, 2005), rabbit heart (Przyklenk et al, 2005), bovine oocytes (Atef et al, 2005), ciliary body (Do et al, 2004), and human HeLa cells (Bader and Weingart, 2004), heptanol has been either shown (Knapp et al, 2005;Atef et al, 2005) or assumed (Do et al, 2004;Bader and Weingart, 2004) to act as a specific gap junction blocker over a range of concentrations (≤ 10 mM), substantially larger than those used in our study (≤ 2 mM).…”

“…The mechanisms resulting in the generation of these three components, in particular the role of gap junctions remain to be elucidated. In the present study, we have used 1-heptanol, a relatively selective gap junction blocker in smooth muscle systems (Christ et al, 1991;Hennig et al, 2002;Huizinga et al, 1988;Manchanda and Venkateswarlu, 1997;Matchkov et al, 2004;Saez et al, 1989), to elucidate the role of gap junctions in the NA-induced contractions.…”