Introduction
Mastoparan-7 (mas-7) is a basic tetradecapeptide isolated from wasp venom, which activates guanine nucleotide-binding regulatory proteins (G-proteins) by catalyzing the guanosine 5′-diphosphate/guanosine 5′-triphosphate (GDP/GTP) exchange. Thus, this compound mimics the action of activated G-protein-coupled receptors. Mas-7 has been shown to stimulate phospholipase C (PLC) in several cellular compartments, such as rat mast cells, rat hepatocytes and HL-60 human leukaemia cells. By contrast, the inhibition of PLC by mastoparan has been demonstrated in SH-SY5Y human neuroblastoma cells and in human astrocytoma cells (1). Recent studies suggested the possibility of programmed cell death stimulation in different types of cells (2–4).
The pertussis toxin (PT) catalyzes the adenosine 5′-diphosphate-ribosylation of the α subunits of the heterotrimeric Gi, Go and Gt proteins. This prevents the G-protein heterotrimers from interacting with their receptors, thus blocking their coupling and activation. Since the Gα subunits remain in their GDP-bound, inactive state, they are unable to inactivate adenylyl cyclase or open K+ channels (5). PT is commonly used in several models of signaling pathways.
Calcium (Ca2+) ions play a central role in the life of the cell. Accordingly to pathological factors, Ca2+ concentration changes occur in various cell compartments, which may induce apoptosis. Prolonged Ca2+ ions concentration changes in the cytoplasm, nucleus or mitochondria, may initiate the cascade of events that lead to cell death. Following stimulation by pathological factors, Ca2+ ions are released from the endoplasmic reticulum and bind to several molecules, such as calpain or calcineurin. Calpain belongs to the cysteine protease family, which activates BH3 interacting-domain death agonist and Bcl-2-associated X protein and it promotes their transport to the mitochondria. In addition, Ca2+ excess in the mitochondria leads to release of proapoptotic proteins located in the intracellular space, such as the second mitochondria-derived activator of caspases/Diablo and cytochrome c(6,7).
The main aim of this study was to evaluate the physiological effect of the direct stimulation of the G-proteins in comparison to the typical stimulation of α-adrenergic receptors and vasopressin receptor type 1 in vascular smooth muscle cells (VSMCs).
Materials and methods
Animals
The experiments were performed on the isolated and perfused tail artery of Wistar rats (weight, 250–270 g). The animals were housed under a 12-h light/dark cycle and had unlimited access to food and water. The rats were narcotized by intraperitoneal injection of 120 mg urethane per 1 kg body weight and were sacrificed by stunning and cervical dislocation. The study protocol was approved by the local Ethics Committee. All the studies were performed in accordance with the United States NIH guidelines [Guide for the Care and Use of Laboratory Animals (1985), DHEW Publication No. (NIH) 85-23: Office of Science and Health Reports, DRR/NIH, Bethesda, MD, USA].
Drugs and solutions
Mas-7 was used as a G-protein activator and mastoparan-17 (mas-17) was used as negative control. The Krebs solution consisted of NaCl (71.8 mM/l), KCl (4.7 mM/l), CaCl2 (1.7 mM/l) NaHCO3 (28.4 mM/l), MgSO4 (2.4 mM/l), KH2PO4 (1.2 mM/l) and glucose (11.1 mM/l). All the reagents were purchased from Sigma-Aldrich Chemical Co. (Poznań, Poland). Study design and conduction. Following dissection from the surrounding tissues, a 2–3-cm long segment of a rat tail artery was cannulated and connected to a perfusion device. The distal part was weighted with a 500-mg weight and the tail was placed in a 20-ml container filled with oxygenated Krebs solution at 37°C (pH 7.4). The perfusion pressure was continuously measured. The perfusion solution flow was gradually increased using a peristaltic pump to 1 ml/min, until the optimum perfusion pressure of 2–4 kPa (8,9).
Data analysis and statistical procedures
The investigations were performed on the TSZ-04 system (Experimetria, Ltd., Balatonfüred, Hungary). The perfusion pressure was measured on BPR-01 and BPR-02 devices and the vascular smooth muscle tension was measured on an FSG-01 transducer. All the transducers used in our experiments were provided by Experimetria, Ltd. The peristaltic pump was provided by Zalimp, Warsaw, Poland.
The concentration response curves (CRCs) were calculated according to the van Rossum method. The maximum response of the tissue (Emax) was calculated as the percentage of the maximal response for phenylephrine. The half maximal effective concentration (EC50) was estimated using classical pharmacological methods with pD2, the negative logarithm of the EC50. We used CRC and Emax in all the calculations estimating statistical significance. Mas-17 was used as negative control.
The results are presented as mean values ± standard deviation. The statistical analysis was performed using the analysis of variance for multiple comparisons of the means. P<0.05 was considered to indicate a statistically significant difference.
Discussion
In the performed experiment, vascular contraction was induced by mas-7, an activator of G-proteins. The vasoconstriction triggered by mas-7 exhibited a slower increase compared to that simulated by phenylephrine or vasopressin. In response to the stimulation of α1-adrenergic, vasopressin or angiotensin receptors, vasoconstriction was observed within a few seconds, whereas the maximum response to mas-7 appeared after 30–40 min. The present study demonstrated that PT exerted no inhibitory effect on the vasoconstriction stimulated by mas-7. Kanagy and Webb (10) previously investigated spiral cutting fragments of the common carotid artery and demonstrated a measureable response after 10 min and a maximum response after 30 min following the application of mas-7 (10−5 M/l) (11). Furthermore, in rats with hypertension, the arterial reactivity was significantly higher compared to that of controls. PT and the phospholipase A2 inhibitor indomethacin did not affect the response to mas-7. Nifedipine at a concentration of 10−5 M/l was shown to inhibit the contraction of VSMCs induced by mas-7 at a concentration of 10−7 M/l, revealing a correlation between voltage-dependent Ca2+ channels and mas-7-induced vasoconstriction. The lack of complete reversal by nifedipine at higher concentrations of mas-7 (10−5 M/l) suggests that an additional mechanism may be activated at higher concentrations (10).
In this study, the effect of the G-protein inhibitor PT on mas-7-induced contraction was investigated. We observed a notable inhibition of VSMC contraction triggered by G-protein activation and a proportional perfusion pressure reduction caused by intra- and extracellular Ca2+ influx.
The vasoconstriction induced by the activation of metabotropic receptors, such as α-adrenergic receptors, vassopresin receptors (V1) or angiotensin II receptors type 1, is conditioned upon the activation of G-proteins. Subsequently, G-proteins activate PLC, leading to the the hydrolysis of phosphatidylinositol 4,5-bisphosphate and increased intracellular concentration of inositol-1,4,5-triphosphate (11–13). Mas-7 penetrates through biological barriers and binds to the ligand-binding site of the G-protein-coupled receptor, stimulating G-proteins in a way similar to an activating receptor. As demonstrated by biochemical studies, the affinity of mastoparan for individual G-protein types is significantly different. Mas-7 exhibits a higher affinity for Gi and Go compared to the Gs protein (14). PT does not affect the VSMC contraction induced by mas-7 by inhibiting the function of Gi and Go, indicating that the target in this process may be Gq. Secondarily to the activation of Gq and PLC, the metabolism of membrane phospholipids may be increased (10). Ca2+ channel blockers directly inhibit Ca2+ influx, thereby decreasing the efficiency of the contraction induced by mas-7, highlighting the role of Ca2+ channels in this process (8,10,15,16). A previous study conducted by Perianin and Snyderman (15) demonstrated that mas-7 may increase Ca2+ ion concentration in the cytoplasm through mechanisms which are unrelated to the production of inositol triphosphate and diacylglycerol.
The affinity for the Gq-protein has not been specified thus far; however, functional investigations were performed on the process of G-protein activation with mas-7 in the VSMCs of the carotid artery in rats (10). The results demonstrated that mas-7 activates Gq-proteins in VSMCs, leading to the increase of Ca2+ ion concentration in the cytoplasm and resulting vasoconstriction. Moreover, in rats with genetically determined hypertension, the contraction of VSMCs was significantly more prominent compared to the control group (10). Mas-7 may activate phospolipase A2 at a concentration of 5×10−5 M/l, leading to the degranulation of mast cells (17). In VSMCs, the process of prostanoid production does not modify the contraction triggered by mas-7, as determined by experiments performed in the presence of indomethacin. No significant effect of indomethacin was demonstrated in those studies (10,15). Mas-7 at a concentration of 10−5 M/l may affect vasoconstriction through additional mechanisms, such as Ca2+ channel modulation and voltage-independent Ca2+ channels (15). Mas-7 also exerts a direct effect on PLC. At low concentrations (<3×10−6 M/l), PLC activation was inhibited by mas-7, although at higher concentrations (>5×10−6 M/l) direct activation was observed (18,19). In our study, a lower concentration of mas-7 (3×10−10-10−6 M/l) was used, which was not sufficiently high to affect elements of signaling pathway other than the G-proteins. The vasoconstriction induced by mas-7 depends on the intra- and extracellular Ca2+ pool, which may also affect apoptosis. The results of this process were higher values of perfusion pressure.
The constriction of VSMCs induced by mas-7 was significantly lower in comparison to that induced by phenylephrine and vasopressin, as was previously confirmed by Kanagy and Webb (10). The effects observed for mas-7 were similar to the effect observed following activation by partial receptor agonists, rather than full agonists such as phenylephrine or vasopressin. Similar perfusion pressure values were reported with the α2-receptor agonist clonidine (20).
In conclusion, our results suggest that mas-7 significantly induces VSMC contraction. The binding site for mas-7 is different from that for PT; therefore, PT does not affect VSMC contraction. The tissue effect of this stimulation is comparable to the effect of stimulation with partial receptor agonists. Our current knowledge regarding the apoptosis pathway demonstrates the significance of Ca2+ ions involved in this process. Thus, mas-7 may induce apoptosis through an increase in the cytoplasmic Ca2+ concentration; however, the use of this mechanism in anticancer therapy must be preceded by a molecule modification that eliminates the vasoconstrictive effect.