Benzodiazepines are widely used in anaesthesia, intensive care and emergency medicine for sedation and anxiolysis. The short-acting compound midazolam is preferred among the benzodiazepines for rapid recovery after administration. However, arterial hypotension is common with this compound, especially in elderly and hypovolaemic patients. The mechanisms why midazolam causes hypotension are not completely understood. Negative inotropic effects [1,2], reduced vascular resistance [3,4] or reduced sympathetic activity  have been discussed. Meanwhile there is growing evidence that midazolam directly relaxes vessel tone. This has been shown in rat aorta , rabbit mesenteric artery [7,8], porcine coronary artery  and in pulmonary artery smooth muscle cells . The objective of this investigation was to elucidate the underlying mechanisms by which midazolam relaxes vascular smooth muscle. To this end, we studied the influence of midazolam on isolated coronary arteries in the presence or absence of different inhibitors of the formation of nitric oxide, prostanoids and endothelial hyperpolarizing factors.
Materials and vessel preparation
The following compounds were used: midazolam (Dormicum®; Hoffmann-La Roche, Grenzach-Wyhlen, Germany), potassium chloride (KCl), prostaglandin F2α (PGF2α, Dinoprost, Minprostin F2α®; Pharmacia, Erlangen, Germany), a thromboxane A2 analogue and bradykinin. All other compounds were obtained from Sigma Chemicals: sodium indomethacin (inhibitor of cyclo-oxygenase), glibenclamide (inhibitor of ATP-sensitive K+ channels), Nω-nitro-L-arginine (L-NNA, inhibitor of nitric oxide synthase) and tetraethylammonium chloride (TEA, non-selective inhibitor of K+ channels, with preference for the high conductance Ca2+-sensitive K+ channel, BKCa). The Krebs-Ringer solution contained (mmol L−1): NaHCO3 25.0, glucose 11.1, titriplex III 0.026, MgSO4 × 7 H2O 1.2, KH2PO4 1.2, KCl 4.7, CaCl2 1.9 and NaCl 118.2.
This study was performed using porcine coronary artery segments. The hearts of adult pigs were obtained immediately post mortem from a nearby slaughterhouse and stored in ice-cold Krebs- Ringer solution. The left anterior descending coronary arteries were dissected, flushed with Krebs-Ringer solution, cleaned of surrounding fat and cut into 3-mm wide rings (n = 4-12 per vessel). These arterial rings were placed in organ chambers filled with Krebs-Ringer solution 10 mL at 37°C and aerated with an O2 (95%)-CO2 (5%) gas mixture. The vessel segments were suspended between two stainless steel hooks, one of which was anchored in the organ chamber and the other connected with a high fidelity force transducer (Hugo Sachs Elektronik, March, Germany), allowing continuous measurement of vessel tension and display on a printer.
The arterial rings were progressively stretched to optimal tension (20 mN) and then allowed to equilibrate for 45 min. A standard contractile response to 8 × 10−2 mol KCl was first obtained. After the segments had been flushed, a prolonged contraction was obtained with 3.5 × 10−5 mol PGF2α. Endothelial integrity was assessed by exposing the rings to 10−6 mol bradykinin. An intact endothelial function was presumed in rings with a dilation of more than 60% of the PGF2α contraction; preparations failing to meet this criterion were excluded. We studied the influence of midazolam (0.05-100 μg mL−1) on contractions obtained with KCl (8 × 10−2 mol) or PGF2α (3.5 × 10−5 mol). First, a stable contraction was established in different sets of experiments either by KCl or by PGF2α, then midazolam was added cumulatively. The resulting relaxation was measured, and concentration-response curves were constructed. After the rings had been allowed to rest for 90 min, they were again contracted in the presence of one of the following compounds: L-NNA 0.1 mmol, indomethacin 10 μmol, a combination of L-NNA 0.1 mmol and indomethacin 10 μmol, glibenclamide 100 μmol and TEA 50 and 100 mmol. Then midazolam was again added in a cumulative manner. Two rings not exposed to any of the substances mentioned above served as controls.
Relaxation after adding midazolam was expressed as relative value of precontraction induced by KCl or PGF2α. All values are given as mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA). The differences between groups were considered significant when P < 0.05.
A total of 140 rings were tested, half of them were contracted with KCl, the rest with PGF2α, 10 rings were used in each set of experiments with one inhibiting compound. Midazolam concentration dependently relaxed isolated coronary rings, beginning at 0.5 μg mL−1 in PGF2α and at 5.0 μg mL−1 in KCl precontracted segments. Full relaxation was seen at 100 μg mL−1 midazolam in both groups. The concentration-response curve for KCl precontracted rings was rightward-shifted compared with that for PGF2α-treated rings (P < 0.001) (Fig. 1). The relaxant effect of midazolam was not affected in the presence of L-NNA, indomethacin, a combination of both or glibenclamide (Fig. 1). TEA (50 and 100 mmol) attenuated the dilating effect of midazolam dose dependently at midazolam concentrations of 0.5 μg mL−1 (PGF2α) or 5 μg mL−1 (KCl) and above (TEA 50 mmol: P < 0.005; 100 mmol: P < 0.001) (Fig. 1). Full relaxation was not achieved in the presence of 100 mmol TEA.
We found that midazolam was a potent vasorelaxing compound for coronary arteries. Other workers have shown that benzodiazepines increase coronary artery blood flow under experimental conditions using the retrograde-perfused Langendorff rat-heart preparation . However, the concentration necessary for full relaxation was 100 μg mL−1 in our study, which is more than 100-fold the concentration seen in clinical practice. Peak concentrations of midazolam 575 ng mL−1 were reported during anaesthesia , which is close to 500 ng mL−1 the value predicted on the basis of theoretical calculations . As the plasma protein binding of midazolam is 98% , the free fraction of midazolam is probably even lower. However, invivo and in vitro concentrations are not easy to compare, as the in vivo situation cannot be mimicked completely in organ baths.
The mechanisms, by which midazolam reduces vascular smooth muscle tone are still poorly understood. In rabbit mesenteric artery, the influence of an intact endothelium on the vasodilating effect of midazolam was not tested [7,8]. In those studies the investigators removed the endothelium from their preparations and focussed on the modulating effect of midazolam on Ca2+ homoeostasis. They pointed out that midazolam attenuates Ca2+ influx into smooth muscle cells via inhibition of voltage and receptor operated Ca2+ channels. On the other hand, inhibition of Ca2+ release from intracellular store sites may be involved as well. In a previous study, we were able to show that midazolam dose dependently attenuates the contractile response to vasoconstrictors in coronary artery rings both by endothelium-dependent and -independent mechanisms . This hypothesis is supported by results reported by Chang and colleagues . These investigators showed that midazolam produces mixed endothelium-dependent and -independent vasodilation in rat aorta. The exact mechanisms involved remained unclear.
The endothelium is known to produce many vasoactive factors. Some of them were identified, e.g. endothelium-derived relaxing factor (EDRF) , prostacyclin (PGI2)  and endothelium-derived hyperpolarizing factor (EDHF) . While EDRF has been identified as nitric oxide , the chemical nature of EDHF is still doubtful. The experiments presented in this study were designed to identify possible mechanisms, including endothelial factors, which contribute to the vasoactive role of midazolam. Blocking nitric oxide synthase by L-NNA did not affect the vasorelaxing effect of this substance. This rules out that EDRF is involved in the mechanisms discussed here for coronary arteries. This is obviously not the case in rat aorta, where EDRF seems to contribute to the vasodilating action of midazolam . As indomethacin, an inhibitor of prostanoid synthesis, failed to influence the concentration-response curve of midazolam, it can be concluded that prostanoids (e.g. PGI2) do not play a role in the midazolam-induced vasorelaxation. The combined effect of indomethacin and L-NNA also left the response of midazolam unchanged. EDHF or another endothelial factor may be a possible candidate to explain why midazolam acts, in part, endothelium dependently. As the chemical nature of EDHF is unknown, as yet, evidence can only be indirect. One of these is the fact that rings precontracted with KCl were dilated to a lower degree compared to PGF2α contracted segments. Similar effects were seen when KCl was compared with norepinephrine as a constrictor . KCl acts as a depolarizing compound by increasing extracellular K+ concentration. The K+ gradient over the smooth muscle cell membrane is lowered by adding KCl to the solution. Hence a possible hyperpolarizing effect , elicited by midazolam, may be inhibited in the presence of KCl. Hyperpolarization can be achieved by activating K+ channels, resulting in K+ efflux out of the smooth muscle cell. To identify possible pathways of action, different K+ channels were blocked. Glibenclamide, an inhibitor of KATP channel did not alter the vasodilating effect of midazolam. TEA, an inhibitor of the Ca2+-sensitive K+ channel BKCa, dose dependently attenuated the relaxing effect of midazolam. This gives rise to the suspicion that midazolam may activate the K+ channel BKCa, hereby possibly causing hyperpolarization of the cell membrane and reduction of the smooth muscle tone in the vessel wall. However, since the membrane potential was not recorded this conclusion is speculative and suggests at best a potential contribution of this mechanism to the vasodilating effect of midazolam. It is noteworthy that midazolam causes hyperpolarization, and augments Ca2+-mediated K+ conductance, in hippocampal neurons . Recently, Jacob and White  showed that diazepam, another benzodiazepine, opens BKCa channels in single myocytes isolated from porcine coronary arteries, thereby promoting vessel relaxation.
In summary, midazolam was shown to relax coronary arteries in a concentration-dependent manner in concentrations beyond those used in clinical practice. In a former study, we were able to demonstrate that midazolam acts by both endothelium-dependent and -independent mechanisms. The present investigation suggests that midazolam may act as an activator of the K+ channel BKCa, which is possibly involved in hyperpolarization of the smooth muscle cell and consecutive relaxation of the vessel wall.
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