Original article

V. GARALIENE1, V. BARSYS2, S. GIEDRAITIS3, R. BENETIS3, A. KRAUZE4

THE ROLE OF EXTERNAL CA2+ IN THE ACTION OF CA2+ - CHANNEL AGONISTS AND ANTAGONISTS ON ISOLATED HUMAN THORACIC ARTERIES

1Kaunas University of Technology, Kaunas, Lithuania; 2Institute of Cardiology at Medical Academy of Lithuanian University of Health Sciences, Kaunas, Lithuania; 3Department of Cardiothoracic and Vascular Surgery at Lithuanian University of Health Sciences, Kaunas, Lithuania; 4Latvian Institute of Organic Synthesis, Riga, Latvia
In systemic atherosclerosis develops the abnormal vascular tone which is associated with elevated calcium influx into smooth muscle cells and their calcification that may be proportional to the extent and severity of atherosclerotic disease. The goal of the present study was to investigate the responses of isolated human arterial samples to Ca2+-channel agonists and antagonists by varying the external Ca2+ concentration. Two dihydropyridine type calcium-channel blockers, amlodipine and cerebrocrast, were used in this study. The benzodiazepine-type calcium-channel blocker diltiazem, the benzimidazole derivative 1-acetyl-5,6-dimethoxy-2-methylthiobenzimidazole and 3,4'-bipyridine derivative milrinone were also used. Experiments were carried out on isolated human thoracic artery samples obtained from 74 patients, aged 38–88 years, during conventional myocardial revascularisation operations. The contraction of artery samples was recorded using an iFOT10 force transducer. Cumulative concentration-contraction curves of the tested agents (10-7 to 10-4 M) were established by varying the external Ca2+ concentration from 0.9 mM to 2.7 mM. Cerebrocrast, regardless of the Ca2+ concentration significantly increased arterial contraction, particularly at the lower Ca2+ (~77%). Diltiazem, the benzimidazole derivative and milrinone caused the artery samples to relax at 10-4 M concentrations by 55%, 55% and 44%, respectively, when the external Ca2+ corresponded to the physiological standard. Shifting to lower or higher Ca2+ concentrations significantly altered the response of vessel samples by increasing their contraction. In conclusion, the present study shows that the response of isolated human thoracic artery samples to both the slow calcium channel suppressant diltiazem and to agonists of that channel (milrinone and the benzimidazole derivative) is regulated by the amount of calcium present in the physiological solution. Treatment with a slow calcium channel inhibitor, the 1,4-dihydropyridine derivative cerebrocrast, resulted in a response that was independent of the external Ca2+ concentration.
Key words:
atherosclerosis, calcium antagonists, human thoracic artery, 1-acetyl-5,6-dimethoxy-2-methylthiobenzimidazole, milrinone

INTRODUCTION

Atherosclerosis disrupts not only endothelial function and endothelium-dependent responses to external stimuli but also causes noticeable changes in the histological structure of blood vessel myocytes to occur. Atherosclerotic myocytes become hypertrophied and stiffen (1). As a result of increased vascular tone, the response of smooth muscles to relaxing factors is weakened and in some cases, substantially changed, i.e., not all drugs widely used for the treatment of cardiovascular disease effectively relax blood vessels to the same degree. Besides, it is likely that agents for which the mechanism of action is endothelium-dependent can lose their efficiency. In such situations, a desirable effect will be achieved after the administration of higher doses of drugs that may cause side effects, such as cardiac arrhythmias or impaired myocardial contractile force. These factors encourage researchers and physicians to look for new techniques that enable the identification of not only the general characteristics of unbalanced vascular relaxation but also their downstream effects, which may be targeted in order to allow the treatment of patients to become more individualised.

In systemic atherosclerosis develops abnormal vascular tone, which is associated with elevated calcium influx into smooth muscle cells and their subsequent calcification. Abnormal vascular tone may be proportional to the extent and severe of atherosclerotic disease and may play an independent role in predicting the presence of vascular atherosclerosis (2-4). Ca2+-channels are the primary pathways for Ca2+ influx which trigger excitation-contraction coupling in blood vessels and use of Ca2+ channel antagonists in clinical practice may be very important. Theoretically calcium antagonists are vasodilators and tend to decrease vascular tone closely related to calcium overload of smooth muscle cells (5). Due to their properties, calcium antagonists are widely used in the antihypertensive therapy (6-8). Our previous studies have shown that treatment with some 1,4-dihydropyridine (amlodipine and cerebrocrast) compounds that possess antagonistic properties towards L-type Ca2+-channels prevents the relaxation of isolated human thoracic artery samples that were pre-contracted by phenylephrine. We suggested that this effect may be dependent upon deficiencies in free intracellular [Ca2+]i (9). Regarding the little knowledge of the impact of variable level of extracellular Ca2+, the goal of study was to investigate the responses of isolated human arterial samples to Ca2+-channel antagonists and agonists by varying the external Ca2+ concentration. We hypothesised that altered calcium homeostasis should change the vascular effects of calcium channels agonists and antagonists.

Two dihydropyridine type calcium-channel blockers, amlodipine CAS [88150-42-9] and cerebrocrast CAS [118790-71-9] were used. The benzodiazepine-type calcium-channel blocker diltiazem CAS [42399-41-7] a benzimidazole derivative 1-acetyl-5,6-dimethoxy-2-methylthiobenzimidazole and the 3,4'-bipyridine derivative milrinone CAS [78415-72-2] were also used. Milrinone is a phosphodiesterase (PDE) inhibitor that is currently approved in a number of countries for use as a short-term intravenous treatment of patients with acute decompensated heart failure. Additionally, milrinone and the benzimidazole derivative produced both positive inotropic effects, as well as vasodilation by increasing the levels of cAMP (10-13).

MATERIALS AND METHODS

Drugs and chemicals

Amlodipine (3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate), cerebrocrast (bis-2-propoxyethyl 4-(2-difluoromethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) and milrinone (2-methyl-6-oxo-1,6-dihydro[3,4']bispyridyl-5-carbonitrile) were synthesised by the Latvian Institute of Organic Synthesis, Riga, Latvia. 1-Acetyl-5, 6-dimethoxy-2-methylsulfanylbenzimidazole was synthesised in the Department of Chemistry at Vilnius University, Vilnius, Lithuania. Each of the newly synthesized and re-synthesized compound had a sertificate showing the compound's chemical structure, composition and purity of the substance.

Phenylephrine hydrochloride ((R)-3-[-1-hydroxy-2-(methylamino)-ethyl] phenol hydrochloride) and diltiazem (cis-(+)-[2-(2-dimethylaminoethyl)-5-(4-methoxyphenyl)-3-oxo-6-thia-2-azabicyclo[5.4.0]undeca-7,9,11-trien-4-yl]ethanoate) were obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany). The test compounds were prepared as stock solutions of 0.1 mM in dimethylsulfoxide.

Phenylephrine was dissolved in deionised water to create a stock solution of 0.1 mM. All stock solutions were stored at cold temperatures.

Preparation of vessel samples

Experiments were carried out on isolated human thoracic artery samples that were obtained during conventional myocardial revascularisation operations from 74 patients who underwent coronary artery bypass grafting in the Department of Cardiothoracic and Vascular Surgery at the Lithuanian University of Health Sciences in the 2012. The age of the patients ranged from 38–88 years and the average age was 67.4 ±9.4 years. Of the patients, 30% were women (n=22, 51–88 years, average age 71.0 ±7.6 years) and 70% (n=52, 38–88 years, average age 66.2 ±8.94 years) were men.

All patients signed a letter of informed consent and the study was approved by the Regional Ethics Committee of Biomedical Research on 05/11/2010, license No BE-2-64, in Kaunas, Lithuania.

For the assignment of mammary artery segments, the system obtained from the Global Town Microtechnology Company (Sarasota, FL, USA) was used. At room temperature, the vessels were gently cleaned of connective tissue and cut into 3–4 mm-long segments. These segments were hung on a vascular holder, the upper hook of which was attached to an isometric force transducer (iFOT10) and placed in 5-ml tissue baths filled with Tyrode's solution, which was composed of the following (in mM): NaCl, 137; KCl 5.4; CaCl2, 1.8; MgCl2, 0.9; Tris HCl, 10; glucose, 5 and pH=7.4. The solution was warmed to 37°C and continuously bubbled with 100% O2. The arterial segments were allowed to equilibrate for at least 60 min prior to the start of the experiment. During the investigational period, the vessels were washed every 15 min with fresh Tyrode's solution.

Protocol

In the first series of experiments, the relaxation-contraction effects of cerebrocrast (33 samples), diltiazem (33 samples), benzimidazole derivative (36 samples) and milrinone (34 samples) were studied in 136 vessel samples taken from 43 patients. Experiments examining the effects of external calcium concentration changes on vasodilatory responses after the equilibration period were conducted on arterial samples that were exposed to a Tyrode solution containing reduced (0.9 mM instead 1.8 mM) or increased (2.7 mM instead 1.8 mM) calcium [Ca2+]o concentrations.

Vasoconstriction was produced using 10-4 M phenylephrine. When the contractions had reached their steady states, the previously mentioned agents were added to the solution in a cumulative-concentration fashion. The vessel rings of the 0.9 mM Ca2+ and 2.7 mM Ca2+ groups were taken from the same patients and the measurements were carried out in parallel. The relaxation-contraction time course was recorded continuously and expressed as a percentage of the vasoconstrictor-induced pre-contraction.

In the second series of experiments investigating the preventive effects of the tested compounds on contraction response, 86 blood arterial samples were taken from 32 patients. After equilibration of the rings, the vessels were pre-treated with 50 µM amlodipine (11 samples), cerebrocrast (11 samples), diltiazem (13 samples) and the benzimidazole derivative (11 samples) for 30 min. Phenylephrine-mediated vasoconstriction was induced in a dose-dependent manner. In the control groups (40 samples), the vessel rings were taken from the same patients as in the experimental groups and pre-treatment was performed using only a physiological solution. Cumulative concentration-contraction curves for phenylephrine (10-6 to 10-4 M) were established. The contraction responses (expressed in mg) of blood vessels in the experimental and control groups were registered in parallel.

Statistical analysis

All of the results are expressed as the means ± S.E.M. Statistical analysis was performed using the SPSS software package, version 10.0. A value of p<0.05 was considered to be statistically significant.

RESULTS

External [Ca2+]o antagonists and agonists recruit calcium input to relax isolated human thoracic arteries that were pre-contracted using phenylephrine

The data presented in Fig. 1A-1D show that external Ca2+ ([Ca2+]o) concentrations modulate the influence of calcium agonists and antagonists on isolated human arterial smooth muscles in a similar fashion regardless of their distinct chemical nature and mode of action. Thus, co-administration of phenylephrine and cerebrocrast, a 4-aryl derivative of 1,4-dihydropyridine, which is characterised as a calcium channel blocker that is similar to amlodipine, induced the converse effect, irrespective of Ca2+ concentration. In physiological Tyrode solution, the contraction of artery samples significantly increased. Contraction was more pronounced when the external Ca2+ concentration was lowered and when the doses of cerebrocrast were elevated.

Figure 1
Fig. 1. Effects of different external calcium concentrations on relaxation-contraction responses induced by the 1,4-dihydropyridine derivative cerebrocrast (A), diltiazem (B), the benzimidazole derivative (C) and milrinone (D) in isolated human thoracic artery rings pre-contracted by 10-4 M phenylephrine. The results are expressed as the means ±S.E. (100% = 668 ± 71 mg, n=136).

Notably, in the 0.9 mM Ca2+ and in the 2.7 mM Ca2+ groups, contraction reached its steady state (increased by ~1.73 and ~1.5 times, respectively) at a dose of 10-5 M. This ten-fold augmentation indicated that 10 µM cerebrocrast is close to a saturating concentration for Ca2+ channel blockade.

Another calcium channel antagonist, diltiazem, considerably relaxed the artery rings in a dose-dependent manner. However, this relaxation only occurred when the [Ca2+]o concentration in the Tyrode solution corresponded to the physiological standard (1.8 mM), in which case, diltiazem-induced relaxation ranged from 26% to 55% at the 10-7 and 10-4 M doses, respectively (Fig. 1B).

Variation of the external [Ca2+]o concentration from 1.8 mM up to 2.7 mM, or down to 0.9 mM, caused the opposite effect to occur, indicating that diltiazem caused intracellular [Ca2+]i redistribution, resulting in significant increases in the isometric contraction of artery rings. For instance, in response to co-administration of diltiazem and phenylephrine, the contractile force of the vessel samples in the 0.9 mM Ca2+ group ranged from 10% to 55% at the 10-7 and 10-4 M doses, respectively. In the 2.7 mM Ca2+ group, contraction increased from 52% to 210%, respectively, at the same doses of diltiazem.

Treatment with the benzimidazole derivative and milrinone calcium agonists, whose mechanisms of action are closely associated with PDE inhibition, increased cAMP and calcium entry into myocardial and vascular cells through L-type Ca2+-channels (11-13). Their influence on contraction and relaxation events in the tested artery samples was almost identical to the influence of diltiazem (Figs. 1C and 1D). In the 1.8 mM Ca2+ group, milrinone was slightly less effective at the 10-5 and 10-4 M concentrations compared to the benzimidazole derivative. In this situation, the latter compound evoked the relaxation of the vessel rings by 47% and 55%, while milrinone relaxed the vessel rings by 34.2% and 44.2%, respectively (p>0.05). In the 0.9 mM Ca2+ and 2.7 mM Ca2+ groups, the opposite effects on artery samples were recorded in response to both of the agonists and the isometric contraction was significantly increased. The effect on isometric contraction was more pronounced in the milrinone-treated groups at all of the tested doses. For example, in the group treated with 10-4 M milrinone, the contractile force increased by 1.68 (0.9 mM Ca2+) and by 1.64 (2.7 mM Ca2+) times. Under the same conditions, treatment with the benzimidazole derivative increased the contractile force by ~1.3 and 1.08 times. These results suggest that the relaxing influence of the tested agents on the blood vessels of smooth muscle, regardless of their distinct chemical nature and mode of action, is closely related to [Ca2+]i concentrations and a negligible alteration of [Ca2+]i concentrations may evoke the opposite effect.

Prophylactic effect of amlodipine, cerebrocrast, diltiazem and the benzimidazole derivative treatment on the contraction of isolated artery samples

When arterial samples were pre-treated with 50 µM concentrations of calcium channel blockers for 30 min, the isometric contraction response to treatment with the vasoconstrictor phenylephrine was significantly decreased compared to the control (Figs. 2A-2C). In the amlodipine- and diltiazem-treated groups, this reduction was equivalent to the reduction observed at all of the phenylephrine concentrations tested. The reduction in isometric contraction was 66% (p<0.01, versus control) after treatment with the 10-6 M concentration and it was 27% (in the amlodipine-treated group) and 20% (in the diltiazem-treated group) at the 10-4 M concentration (p>0.05, versus control), indicating that increasing the concentration of the vasoconstrictor weakened the efficacy of calcium channel blockers.

Figure 2
Fig. 2. Contraction response of artery rings to phenylephrine after pre-treatment with 50 µM amlodipine, diltiazem, cerebrocrast, benzimidazole derivative and control. The results are expressed as the means ±S.E.

Prophylactic use of cerebrocrast determines its long-lasting (experiment takes more than 60 min) and permanent effect on vascular smooth muscle. The ratio of isometric contraction in the cerebrocrast and control groups fluctuated in a narrow range during the experimental period. The ratio was equal to 76% at the 10-6 M concentration and 71% at the 10-4 M concentration, suggesting that the effects of cerebrocrast do not depend on the amount of vasoconstrictor present in the physiological solution.

Pre-treatment of the vessel rings with the benzimidazole derivative, which is characterised as a calcium channel agonist (12), caused an additional increase in contractile force of 36% compared to the control at the 10-4 M concentration of phenylephrine (1185 ±209 mg and 872 ±174 mg, respectively, p>0.05).

Notably, the contractile force of artery samples in response to treatment with the vasoconstrictor phenylephrine increased pro rata in a dose-dependent manner. At the 10-4 M concentration, it reached 1339 ±203 mg (n=40), while treatment with a single dose equal to 10-4 M resulted in a two-fold lower contraction (668 ±71 mg, n=136, p<0.001).

DISCUSSION

Note: due to the low number of experimental cases the obtained results have not been differentiated neither by age, gender nor other clinical indications.

In the present study, calcium channel inhibitors (cerebrocrast and diltiazem) and stimulators (the benzimidazole derivative and milrinone) were employed to investigate the functional role of external Ca2+ in the vasodilatation of isolated human thoracic artery samples. These findings provide new evidence that (i) the dihydropyridine compound cerebrocrast, under physiological conditions, appeared to be a vasoconstrictor instead of a vasodilator and (ii) the response of the artery to treatment with the tested compounds in the presence of abnormal intracellular Ca2+ handling, which was obtained by shifting the Ca2+ concentration in a physiological solution upwards or downwards, was changed.

The contraction-relaxation cycle in smooth muscle cells involves a number of physiological processes that take place both in the endothelium as well as in the smooth muscle. A key player in this cycle is the calcium ion (14), which initiates and maintains both muscle contraction and relaxation events. Intracellular [Ca2+]i signals are the result of calcium entry from the external space, as well as intracellular release from internal stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR) (15).

We used phenylephrine, an agonist of α1-adrenoceptors, to stimulate smooth muscle contraction, which, upon binding to plasma membrane receptors, generated the second messenger IP3 (inositol 1,4,5-triphosphate) (16, 17). IP3 binds to and activates IP3 receptors (IP3Rs) in smooth muscle cells (SMSs). Numerous studies indicate that IP3R activation can elevate global (intracellular) [Ca2+]i concentrations directly through SR Ca2+ release and indirectly by stimulating plasma membrane ion channel-mediated (L-type) influx in SMSs. One of the ways to overcome the increased vascular tone is to take organic calcium channel blockers, a practice which is widely used in clinical practice. In our study, cerebrocrast, a derivative of amlodipine, a well-known calcium channel blocker, displayed unusual effects that were consistent with the behavior of calcium channel inhibitors. The isometric contraction of artery samples evoked by phenylephrine after cerebrocrast treatment continued to rise and at a concentration of 10-4 M cerebrocrast, the contraction increased to 1.5 times that of baseline (Fig. 1A). Our previous study showed that amlodipine has similar properties, resulting in an increase of 1.45 times the contraction over baseline (9). Meanwhile, the prophylactic use of 50 µM cerebrocrast and amlodipine for 30 min decreased the response of arterial smooth muscles to phenylephrine in a concentration-dependent manner. Thus, there is evidence to suggest that both tested compounds possess calcium channel blocking properties. These features are highly expressed in cerebrocrast (Fig. 2A and 2C). Naturally, the blockade of slow calcium channels leads to a myocyte [Ca2+]i reduction, thereby opposing smooth muscle contraction. However, conflicting results may be related, primarily, to the potassium channels participating in arterial relaxation (18). In addition to a global [Ca2+]i reduction, the lower levels of Ca2+ may be a result of accumulation of calcium in internal calcium sources, such as the SR. It is possible that impaired SR refilling decreases the Ca2+ spark through ryanodine sensitive calcium channels that are localised on the surface of the SR (19). Numerous studies have shown that Ca2+ spark triggers the Ca2+-mediated activation of large conductance potassium channels (BKCa) that participate in the smooth muscle relaxation process (16, 20, 21) and their blockade by iberiotoxin inhibits the relaxation of human internal thoracic artery (22). Thus, it is intriguing to suggest that the tested 1,4-dihydropyridine compounds disturb the homeostasis between intracellular [Ca2+]i and [Ca2+]SR and that the balance shifts towards the [Ca2+]i involved in the contractility of smooth muscles. This finding was indirectly confirmed by the tests carried out with external [Ca2+]o concentrations that had been decreased by half (0.9 mM instead 1.8 mM). In all of the groups, the tested compounds, regardless of their distinct modes of action, resulted in an increase in the contractile force by ~77%, 56%, 68% and 31% in the cerebrocrast-, diltiazem-, milrinone- and benzimidazole-treated groups, respectively, at 10-4 M (Fig. 1). Early et al. (23), Gonzales et al. (24) and others (25) analysed the mechanisms of Ca2+ signalling with ryanodine receptors and BKCa channels and found that the Ca2+ spark and the frequency of transient K+ currents were inhibited by lowering the external Ca2+ concentrations. Our testing was conducted on arterial samples that were taken from patients undergoing coronary artery bypass grafting. These arteries can be affected by developed systemic atherosclerosis. This view is supported by our studies with the cholinergic agonist carbachol, which indicated that the endothelium of a. thoracica was significantly injured and thus, its key function, endothelium-dependent vasodilation, was not properly performed (9, 26). Other studies have shown that in atherosclerotic vascular smooth muscle, the IP3R protein and IP3R and/or RyR-mediated Ca2+ release are reduced and the Ca2+-ATPase (SERCA2b) expression in the SR is down-regulated, suggesting that the [Ca2+]SR reduction may contribute to impaired IP3R-mediated Ca2+ release (27-29]. Taken together, the reduced [Ca2+]o levels and the inhibition of calcium channels in the cerebrocrast- and diltiazem-treated groups indicate that the amount of Ca2+ that passes into the smooth muscle cells is too low and that an insufficient amount of Ca2+ cycles in the SR. This insufficiency contributes to necessary Ca2+ spark activation of the large conductance BKCa channels and plays a key role in vascular smooth muscle dilation.

Based on the results of a previous study Zakharenko and Reznik (30) that investigated the mechanisms of vascular wall relaxation using the isometric tension method in isolated blood vessels, calcium at concentrations up to 30 mM is responsible for arterial relaxation and the relaxation of vascular smooth muscle cells is due to Ca2+-activated potassium channels. This finding suggests that the higher the [Ca2+]i level, the better is the arterial dilation. However, our data presented in Fig. 1 show that two-fold (2.7 mM instead 1.8 mM) elevations in [Ca2+]o concentrations resulted in similar vascular contractility responses to the tested compounds as those observed in the groups with lower [Ca2+]o concentrations. The isometric contraction in the groups with lower calcium concentrations was increased by 55%, 210%, 64% and 9% in the cerebrocrast, diltiazem, milrinone and benzimidazole groups, respectively. These results suggest that intracellular [Ca2+]i homeostasis and calcium distribution are notably defective. This defectiveness may include depressed Ca2+ uptake, as well as decreased storage and release from the SR. All of these events may be associated with systemic arteriosclerosis, leading to disrupted gap junctions, endothelium dysfunction and decreased myoendothelial feedback (31-33). Taken together, the shift of the external [Ca2+]o concentration from the physiological standard in one direction or another may significantly change the effect of treatment on vascular smooth muscle. In accordance with (34), the homeostasis of [Ca2+]i is critical to maintain many physiological functions, including cardiovascular events and smooth muscle relaxation.

The mechanisms of action of milrinone and the benzimidazole derivative are closely related to cAMP. Concentrations of cAMP may increase during the inhibition of PDE activity (milrinone) and/or during the stimulation of β-adrenergic receptors (benzimidazole derivative) (11, 12). Cyclic AMP is a ubiquitous intracellular second messenger that affects cell physiology by directly interacting with effector molecules such as cAMP-dependent protein kinases (PK), cyclic nucleotide-gated ion channels and hyperpolarisation of activated channels that regulate vascular tone (35).

Myosin light chain kinase (MLCK) plays a key role in the activation of smooth muscle contraction by Ca2+-calmodulin. The MLCK/Ca2+-calmodulin complex phosphorylates myosin, triggering myosin-actin interaction, myocyte contraction and, ultimately, vasoconstriction. Enhancing cAMP increases the activity of cAMP-dependent PK, which in turn phosphorylates MLCK, decreasing the affinity of MLCK for Ca2+-calmodulin and thereby decreasing myosin phosphorylation and the actin-myosin interaction, which finally causes the muscle to relax (36, 37). In addition, it is possible that the phosphorylation of phospholamban is influenced by both of the tested compounds (milrinone and the benzimidazole derivative) through the cAMP-dependent PK as well, which may increase the return of calcium ions from the intracellular space into the SR. According to previous reports (38, 39), PDE inhibitors may stimulate endothelial NO synthesis, thereby playing an essential role in blood vessel dilatation. Taken together, we suggest that these effects are specific to milrinone and the benzimidazole derivative and may lead to vascular relaxation. Furthermore, data presented in Fig. 1C and 1D indicate that both agents were significantly effective in respect to arterial sample relaxation only when the [Ca2+]o levels coincided with the physiological standard (1.8 mM). However, calcium overload (2.7 mM [Ca2+]o), as well as a lack of calcium (0.9 mM [Ca2+]o), contributed to opposite responses, particularly in the milrinone-treated group, where the contractile force of the arterial samples was raised by more than 60%. Meanwhile, in the benzimidazole group, that response was significantly lower: 31% and 9% at the 10-4 M concentration in the 0.9 mM [Ca2+]o group and the 2.7 mM [Ca2+]o group, respectively. It is possible that the benzimidazole derivative promotes better [Ca2+]i uptake at elevated [Ca2+]o concentrations. Based on previous studies (40-42), another benzimidazole derivative (1-ethyl-2-benzimidazolinone, 1-EBIO) appears to be a direct activator of KCa channels and contributes to vascular tone. Its vasodilatory action involves the opening of endothelial K+ channels and nitric oxide synthesis. However, 1-EBIO, a stimulator, requires the presence of internal [Ca2+]i for the activity of K+ channels. An increase in the apparent channel sensitivity to Ca2+ was suggested as a mechanism of action for 1-EBIO. In our study, prophylactic use of the benzimidazole derivative enhanced arterial sample contraction by 36%, suggesting that this compound increased the internal [Ca2+]i concentration involved in contractility events. Moreover, the effect of benzimidazole at the 2.7 mM [Ca2+]o concentration shows that this compound is able to better induce Ca2+ uptake compared to milrinone and that other benzimidazole derivatives can be found that selectively act on Ca2+-dependent K+ channels.

In conclusion, the present study shows that the response of isolated human thoracic artery samples to the slow calcium channel suppressant diltiazem and to channel stimulators (milrinone and the benzimidazole derivative, 1-acetyl-5,6-dimethoxy-2-methylthiobenzimidazole) is regulated by the amount of calcium in the perfused physiological solution. The 1,4-dihydropyridine derivative cerebrocrast, a slow calcium channel inhibitor, displayed a response that was independent from the external calcium concentration and its influence upon the contraction of the arterial samples was possibly increased due its blockade of several voltage-gated K+ (Kv) channels (43), as well as due cerebrocrast-bound states reflecting state-dependent block of the L-type Ca2+ channels, which is characteristic for 1,4-dihydropyridines (44).

Acknowledgements: This work has been supported by the Institute of Cardiology at the Medical Academy of the Lithuanian University of Health Sciences, PhD research programs related to medicine-07B and funded by the European Social Fund under the project "Microsensors, microactuators and controllers for mechatronic systems (Go-Smart)" (Agreement No VP1-3.1-ŠMM-08-K-01-015).

The authors would like to thank the team of American Journal Experts for the article editing by correcting the language, phrases and grammar.

Conflict of interests: None declared.

REFERENCES

  1. Bellien J, Favre J, Iacob M, et al. Arterial stiffness is regulated by nitric oxide and endothelium-derived hyperpolarizing factor during changes in blood flow in humans. Hypertension 2010; 55: 674-680.
  2. Alexopoulos A, Raggi P. Calcification in atherosclerosis. Nat Rev Cardiol 2006; 6: 681-688.
  3. Kim K, Park KU, Chun EJ, et al. A novel biomarker of coronary atherosclerosis: serum DKKI concentration correlates with coronary artery calcification and atherosclerotic plaques. J Korean Med Sci 2011; 26: 1178-1184.
  4. Soncusare S, Palade PT, March JD, Telemaque S, Pesic A, Rusch NJ. Vascular calcium channels and high blood pressure: pathophysiology and therapeutic implications. Vascul Pharmacol 2006; 44: 131-142.
  5. Trion A, Schutte-Bart C, Bax WH, Jukema JW, van der Laarse. Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination. Mol Cell Biochem 2008; 308: 25-33.
  6. Ling G, Liu AJ, Shen FM, Cai GJ, Liu JG, Su DF. Effects of combination therapy with atenolol and amlodipine on blood pressure control and stroke prevention in stroke-prone spontaneously hypertensive rats. Acta Pharmacol Sin 2007; 28: 1755-1760.
  7. Billecke SS, Marcovitz PA. Long-term safety and efficacy of telmisartan/amlodipine single pill combination in the treatment of hypertension. Vasc Health Risk Manag 2013; 9: 95-104.
  8. Sharma KK, Mathur M, Gupta R, et al. Epidemiology of cardioprotective pharmacological agent use in stable coronary heart disease. Indian Heart J 2013; 65: 250-255.
  9. Garaliene V, Barsys V, Jakuška P, Benetis R. Action of calcium antagonists and agonists on isolated human thoracic artery used for coronary bypass grafting. Pharmacol Rep 2012; 64: 733-738.
  10. Barnard MJ, Linter SPK. Acute circulatory support. BMJ 1993; 307: 35-41.
  11. Colucci WS. Cardiovascular effects of milrinone. Am Heart J 1991; 121: 1945-1947.
  12. Garaliene V, Barsys V, Jakuska P, Krauze A, Duburs G. Effect of calcium antagonists and agonists on isolated human v. saphena magna used for coronary artery bypass grafting and guinea pig's papillary muscles. Arzneimittelforshung 2011; 61: 386-392.
  13. Levy JH, Bailey JM, Deeb MG. Intravenous milrinone in cardiac surgery. Ann Thoracic Surg 2002; 73: 325-330.
  14. McGeown JG. Seeng is believing! Imaging Ca2+ -signaling events in living cells. Exp Physiol 2010; 95: 1049-1060.
  15. Hill-Eubanks DC, Werner ME, Heppner TJ, Nelson MT. Calcium signalling in smooth muscle. Cold Spring Harb Perspect Biol 2011; 3: a004549.
  16. Ledoux J, Bonev AD, Nelson MT. Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium. J Gen Physiol 2008; 131: 125-135.
  17. Narayanan D, Adebiyi A, Jagger H. Inositol triphosphate receptors in smooth muscle cells. Am Physiol Heart Circ Physiol 2012; 302: H2190-H2210.
  18. Dick GM, Tune DJ. Role of potassium channels in coronary vasodilation. Exp Biol Med (Maywood) 2010; 235: 10-22.
  19. Lannet JT. Ryanodine receptor physiology and its role in disease. Adv Exp Med Biol 2012; 740: 217-234.
  20. Latore R, Branchi S. Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage. Biol Res 2006; 39: 385-401.
  21. Ledoux J, Taylor MS, Bonev AD, et al. Functional architecture of inositol 1,4,5-triphosphate signalling in restricted spaces of myoendothelial projections. Proc Nat Acad Sci USA 2008; 105: 9627-9632.
  22. Malinowski M, Deja MA, Janusiewicz P, Golba KS, Roleder T, Wos S. Mechanisms of vasodilatatory effect of perivascular tissue of human internal thoracic artery. J Physiol Pharmacol 2013; 64: 309-316.
  23. Earley S, Heppner TJ, Nelson MT, Brayden JE. TRPV4 forms a novel Ca2+ signalling complex with ryanodine receptors and BKCa channels. Circ Res 2005; 97: 1270-1279.
  24. Gonzales AL, Amberg GC, Early S. Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells. Am J Physiol Cell Physiol 2010; 299: C279-C288.
  25. Koide M, Nystoriak MA, Krishnamoorthy G, et al. Reduced Ca2+ spark activity after subarachnoid hemorrhage disables BK channel control of cerebral artery tone. J Cereb Blood Flow Metab 2011; 31: 3-16.
  26. Kohler R, Ruth P. Endothelial dysfunction and blood pressure alterations in K+-channel transgenic mice. Pflugers Arch 2010; 459: 969-976.
  27. Adachi T. Modulation of vascular sarco/endoplasmic reticulum calcium ATPase in cardiovascular pathophysiology. Adv Pharmacol 2010; 59: 165-195.
  28. Massaeli H, Austria JA, Pierce GN. Chronic exposure of smooth muscle cells to minimally oxidized LDL results in depressed inositol 1,4,5-triphosphate receptor density and Ca2+ transients. Circ Res 1999; 85: 515-523.
  29. Massaeli H, Austria JA, Pierce GN. Overexpression of SERCA2 ATP-ase in vascular smooth muscle cells treated with oxidized low density lipoprotein. Mol Cell Biochem 2000; 207: 137-141.
  30. Zakharenko SS, Reznik AV. Mechanism of vascular wall calcium relaxation. Membr Cell Biol 1988; 12: 441-451.
  31. Kerr PM, Tam R, Ondrusova K, et al. Endothelial feedback and myoendothelial projection. Microcirculation 2012; 19: 416-422.
  32. Lompre AM, Hajjar RJ, Harding SE, Kranias EG, Lohse MJ, Marks AR. Ca2+ cycling and new therapeutic approaches for heart failure. Circulation 2010; 121: 822-830.
  33. Sokoya EM, Burns AR, Setiawan CT, Coleman HA, Parkington HC, Tare M. Evidence for the involvement of myoendothelial gap junctions in EDHF-mediated relaxation in the rat middle cerebral artery. Am J Physiol Circ Physiol 2006; 291: H385-H393.
  34. Choi KC, An BS, Yang H, Jeung EB. Regulation and molecular mechanisms of calcium transport genes: do they play role in calcium transport in the uterine endometrium? J Physiol Pharmacol 2011; 62: 499-504.
  35. Bedner P, Niessen H, Odermatt B, Kretz M, Willecke K,Harz H. Selective permeability of different connexin channels to the second messenger cyclic AMP. J Biol Chem 2006; 28: 6673-6681.
  36. Earley S, Nelson MT. Central role of Ca2+ -dependent regulation of vascular tone in vivo. J Appl Physiol 2006; 101: 10-11.
  37. Shen O, Rigor RR, Pivetti CD, Wu MN, Yuan SY. Myosin light chain kinase in microvascular barrier function. Cardiovasc Res 2010; 87: 272-280.
  38. Griffith TM, Chaytor AT, Taylor HJ, Giddings BD, Edwards DH. cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions. Proc Nat Acad Sci USA 2002; 99: 6392-6397.
  39. Zhang XP, Tada H, Wang Z, Hintze TH. cAMP signal transduction, a potential compensatory pathway for coronary endothelial NO production after heart failure. Arterioscler Thromb Vasc Biol 2002; 22: 1273-1278.
  40. Adeagbo AS. 1-Ethyl-2-benzimidazolinone stimulates endothelial K(Ca) channels and nitric oxide formation in rat mesenteric vessels. Eur J Pharmacol 1999; 379: 151-159.
  41. Chadha PS, Liu L, Rikard-Bell M, et al. Endothelium-dependent vasodilation in human mesenteric artery is primarily mediated by myoendothelial gap junctions intermediate conductance calcium-activated K+ channel and nitric oxide. J Pharmacol Exp Ther 2011; 336: 701-708.
  42. Dutta AK, Khimji A, Sathe M, et al. Identification and characterization of the intermediate-conductance Ca2+ -activated K+ channel (IK-1) in biliary epithelium. Am J Physiol Gastrointest Liver Physiol 2009; 297: G1009-G1018.
  43. Zhang XL, Gold MS. Dihydropyridine block of voltage-dependent K+ currents in rat DRG neurons. Neuroscience 2009; 161: 184-194.
  44. Baylie RL, Cheng H, Langton PD, James AF. Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol. J Physiol Pharmacol 2010; 61: 543-550.
R e c e i v e d : July 27, 2013
A c c e p t e d : December 30, 2013
Author’s address: Dr. Vida Garaliene, Institute of Cardiology, 17 Sukileliu Street, Kaunas, LT-50009, Lithuania. e-mail: vida.garaliene@gmail.com