This study had two major findings. First, in porcine pulmonary arterial vascular smooth muscle, Dex increased contraction tension that had been induced by depolarization stimulation with high KCl and reduced the increases in contraction tension induced by adrenaline receptor stimulation. These effects were concentration-dependent in both cases. Second, Dex suppressed receptor-activated Ca2+ channels (RACCs), which allow extracellular Ca2+ into the cells, and phosphatidylinositol–1,4,5-triphosphate (IP3)-induced Ca2+ release (IICR), which releases intracellular Ca2+ within the cells. Dex did not suppress CICR.
In porcine pulmonary arterial vascular smooth muscle, Dex enhanced the contraction induced by high KCl stimulation. Conversely, the Dex-induced enhancement of contraction induced by high KCl was completely suppressed by yohimbine and rauwolscine, which are α2-receptor antagonists, but not by prazosin, also an α1-receptor antagonist (Fig. 2. 3). These results suggest that Dex’s enhancing effect on vascular smooth muscle contraction induced by high KCl depolarization is mediated by an α2-receptor mechanism.
A study of endothelium-denuded human gastroepiploic arteries showed that the enhancement of high KCl-induced vascular smooth muscle contraction induced by adding Dex was completely antagonized by the α2-receptor antagonists yohimbine and rauwolscine, leading the authors to conclude that the enhancing effect of Dex is mediated by α2 receptors . Another study, on the human forearm, showed that the vasoconstriction effect of Dex after administration of a β- or α2-receptor antagonist was completely antagonized by the α2-receptor antagonist yohimbine .
In general, high KCl-induced contraction of vascular smooth muscle is mediated by increased Ca2+ brought about by an influx of extracellular Ca2+ via voltage-dependent Ca2+ channels (VDCCs). These channels open in response to cell membrane depolarization, resulting in intracellular CICR via ryanodine receptors on endoplasmic reticulum (ER) . Dex-induced increases in high KCl-induced contraction tension (Fig. 2) may promote VDCC-mediated influx of extracellular Ca2+ and/or CICR. Stimulation with caffeine activates ryanodine receptors on the ER and promotes CICR to increase Ca2+, resulting in contraction. In the present experiment, Dex had no effect on caffeine-induced increases during contraction tension in the Ca2+-free HBBS solution (Fig. 6). Therefore, the mechanism by which Dex increases depolarization-induced contraction of the pulmonary arterial vascular smooth muscle is not facilitation of CICR from the Ca2+ reservoir. Rather, the increase is likely to result from facilitated influx of extracellular Ca2+ via VDCCs. α2-Receptor-induced contraction of human subcutaneous resistance arteries depends, at least in part, on Ca2+ influx via L-type VDCCs . α2-Receptor stimulants directly promote VDCC by a mechanism that depends on a G protein associated with protein kinase C activation . It has also been reported that α2-receptor stimulation in rat saphenous vein vascular smooth muscle results from depolarization of the cell membrane, which indirectly enhances Ca2+-dependent contraction and Ca2+ sensitivity through VDCC activation . Because Dex has an imidazole group, it is believed to act not only on the α2-receptor but also on imidazoline receptors . We therefore administered imidazoline, which is an imidazoline-receptor stimulant, and compared its effects with those of Dex. Imidazoline did not increase the contraction resulting from depolarization with high KCl (Fig. 2).
The α2-receptor stimulant Dex, imidazoline-receptor stimulant imidazoline, and α2-receptor antagonists yohimbine and rauwolscine produced concentration-dependent decreases in contraction induced by the α1α2-receptor stimulant adrenaline. Dex and imidazoline suppressed contraction resulting from adrenaline, suggesting that receptor stimulants containing an imidazoline group inhibit receptor stimulation involving both α1 and α2. In the present study, adrenaline-induced contraction was suppressed by the α2-receptor stimulant Dex, the imidazoline-receptor stimulant imidazoline, and the α2-receptor antagonists yohimbine and rauwolscine. These findings suggest that the effect of Dex on adrenaline-induced contraction is attributable to its α2-receptor-blocking action.
Dex inhibited RACC and IICR but not CICR. Cell membrane Ca2+ channels regulated by receptor stimulation include RACCs, which are receptors that have a channel function coupled with receptor stimulants and that mediate the influx of extracellular Ca2+. Receptor stimulants activate phospholipase C by activating G protein-coupled receptors on the cell membrane, resulting in the production of IP3 from phosphatidylinositol, a lipid component of the cell membrane. IP3 production leads to activation of IICR from the intracellular reservoir . Influx of extracellular Ca2+ and IP3 activate ryanodine receptors on the ER, causing CICR from the Ca2+ reservoir. Both IP3 and ryanodine receptors, which are present on the ER, play an important role in the regulation of Ca2+ release . Vascular smooth muscle contraction is regulated by changes in the Ca2+ sensitivity of contraction proteins through phospholipase C activation by receptor stimulation .
The present study showed that Dex reduced the increases in contraction tension induced by the receptor stimulant adrenaline, suggesting that it suppressed RACC-mediated influx of extracellular Ca2+, IICR, and/or CICR (Fig. 4). Dex’s suppression of adrenaline-induced increases in contraction tension after depletion of Ca2+ suggest that Dex reduces the RACC-mediated influx of extracellular Ca2+ (Fig. 5). Dex’s suppression of adrenaline-induced increases in contraction tension in the absence of extracellular Ca2+ suggest that Dex suppresses IICR and/or CICR (Fig. 6). In the absence of extracellular Ca2+, Dex did not affect caffeine-induced increases in contraction tension (Fig. 6). Caffeine stimulation activates ryanodine receptors on the ER and promotes CICR to induce contraction . This mechanism suggests that Dex suppresses IICR because it did not suppress CICR.
We also conducted experiments with histamine to confirm that Dex suppresses IICR. Receptor stimulation by histamine is coupled with phospholipase C via Gq, a G protein-mediated, seven-transmembrane receptor. Ca2+ is recruited via IP3 as a second messenger. Contraction then occurs via diacylglycerol-mediated activation of protein kinase C . Thus, histamine is believed to act specifically on IICR .
Our previous study showed that receptor stimulation in the absence of Ca2+ in the extracellular fluid, and following depletion of the Ca2+ reservoir with caffeine and ryanodine, did not cause any changes in contraction tension . This finding indicates that IP3 receptor stimulation results in no Ca2+ release from the Ca2+ reservoir when ryanodine receptors are fixed in the open state. The experiment showed that the histamine-induced increase in contraction tension was reduced in the absence of extracellular Ca2+, suggesting that Dex suppresses IICR (Fig. 6).
When the 6-month-old pig whose tissues were used in the present experiments was euthanized, the major stress of the animal could have produced depletion of the Ca2+ reservoir. All pigs at that location are euthanized routinely with an electrical method. Hence, the euthanasia protocol did not introduce bias in the results.
The selection of Dex at a concentration of 10–6 M was based on the following data. We considered that the inhibitory concentration of 50% of Dex was 2.083 µM according to the dose-dependent curve (Fig. 4). We therefore needed to have 60–70% maximum inhibition. Figure 2 shows that Dex increased the 60 mM KCl-induced contraction tension, with the observed increases reaching significance at a Dex concentration of ≥5×10–6 M. Hence, we decided to use 5 µM. Dex at high doses activates the α2β-receptors distributed in vascular smooth muscle, causing hypertension resulting from contraction of vascular smooth muscle. At low doses, Dex causes hypotension resulting from vasodilation and bradycardia due to parasympathetic dominance . The blood concentrations of Dex required to maintain a sedative effect in humans are reported to be similar, at 1.0 ×10–9 g/mL (i.e., 4.0 × 10–9 mol/L) [23, 24]. The present results show that Dex had no effect on vasoconstrictor responses in porcine pulmonary arteries when applied in clinically effective concentrations. The previous reported systemic effects of Dex observed in the clinical setting, including decreased blood pressure, may not be the result of direct actions on vascular smooth muscle but possibly due to decreases in central and peripheral sympathetic nervous system activity . One clinical report also suggested that, at large doses (>10–8 mol/L), Dex increases peripheral vascular resistance, leading to increased blood pressure . Although the mechanism of blood pressure increase is unclear, it cannot be ruled out that the vasoconstrictor effects of Dex shown in this study (i.e., those mediated by VDCC activation or in case of accidental intravenous administration) may be relevant in such cases.