Catharanthine is a constituent of anticancer vinca alkaloids. Its cardiovascular effects have not been investigated. This study compares the in vivo hemodynamic as well as in vitro effects of catharanthine on isolated blood vessels, vascular smooth muscle cells (VSMCs), and cardiomyocytes. Intravenous administration of catharanthine (0.5–20 mg/kg) to anesthetized rats induced rapid, dose-dependent decreases in blood pressure (BP), heart rate (HR), left ventricular blood pressure, cardiac contractility (dP/dtmax), and the slope of the end-systolic pressure-volume relationship (ESPVR) curve. Catharanthine evoked concentration-dependent decreases (Imax >98%) in endothelium-independent tonic responses of aortic rings to phenylephrine (PE) and KCl (IC50 = 28 µM for PE and IC50 = 34 µM for KCl) and of third-order branches of the small mesenteric artery (MA) (IC50 = 3 µM for PE and IC50 = 6 µM for KCl). Catharanthine also increased the inner vessel wall diameter (IC50 = 10 µM) and reduced intracellular free Ca2+ levels (IC50 = 16 µM) in PE-constricted MAs. Patch-clamp studies demonstrated that catharanthine inhibited voltage-operated L-type Ca2+ channel (VOCC) currents in cardiomyocytes and VSMCs (IC50 = 220 µM and IC50 = 8 µM, respectively) of MA. Catharanthine lowers BP, HR, left ventricular systolic blood pressure, and dP/dtmax and ESPVR likely via inhibition of VOCCs in both VSMCs and cardiomyocytes. Since smaller vessels such as the third-order branches of MAs are more sensitive to VOCC blockade than conduit vessels (aorta), the primary site of action of catharanthine for lowering mean arterial pressure appears to be the resistance vasculature, whereas blockade of cardiac VOCCs may contribute to the reduction in HR and cardiac contractility seen with this agent.
Catharanthus roseus, also known as Vinca rosea or Madagascar periwinkle, is a tropical shrub containing a wide spectrum of bioactive constituents such as alkaloids, terpenoids, bioflavonoids and tannins (van Der Heijden et al., 2004; Sertel et al., 2011). The crude extract of the plant, popular in folklore medicine, has been purported to possess hypotensive, cerebral vasodilator, antidiabetic, anti-inflammatory, wound healing, and diuretic properties (Lans, 2006; Nayak et al., 2007; Ara et al., 2009; Rasineni et al., 2010). However, the mechanisms or the active constituents that contribute to these claims are currently unknown. Several oral formulations of Madagascar periwinkle extracts are present as homeopathic or herbal medication in Western countries (Madagascar Periwinkle Monograph; http://naturaldatabase.therapeuticresearch.com/nd/Search.aspx?cs=&s=ND&pt=100&sh=1&id=637). The major bioactive constituents are the indole ring containing catharanthine and the terpenoid-based vindoline alkaloids. Together, these constitute anticancer agents such as vincristine, vinblastine, vinorelbine, and vinflunine, which belong to the class of vinca alkaloids (van Der Heijden et al., 2004; Sertel et al., 2011). Vincristine (0.8–2 mg/m2) and vinblastine (0.1–20 mg/m2) are administered by the i.v. route in milligram dose ranges for the management of solid tumors in cancer patients. Hypotension is reported to be either an immediate or long-term effect (onset in hours to days) [see Yeh et al. (2004); Vincristine Monograph, 2008; http://www.bccancer.bc.ca/NR/rdonlyres/A06F1DF4-79C5-4215-BBED-57F73F357E61/28173/Vincristinemonograph_1Mar08.pdf; Vinorelbine Monograph, 2008; http://www.bccancer.bc.ca/NR/rdonlyres/7267649C-7408-497B-AF85-0EBE7CA6E8F5/28176/Vinorelbinemonograph1Mar08.pdf]. We also recently showed that l-tryptophan ethyl ester (l-Wee), an indole ring containing amino acid analog (but not l-tryptophan), exerts transient vasodilation in third-order branches of the superior rat mesenteric artery (MA) by acting as an voltage-operated L-type calcium channel (VOCC) blocker in vascular smooth muscle cells (VSMCs) (Jadhav et al., 2012). The responses arising from such small MAs are known to contribute to the regulation of total peripheral resistance (Christensen and Mulvany, 1993). We also suggested that inhibition of VOCCs on VSMCs may contribute to reductions in arterial pressure seen after administration of l-Wee. Therefore, we asked whether catharanthine, an indole ring containing alkaloid, might also show a similar blood pressure (BP)–lowering effect like l-Wee, via blockade of VOCCs on VSMCs. It is not presently known whether catharanthine affects cardiovascular function via regulation of VOCCs on the heart in addition to its effect on the vasculature. The present study addresses all of these issues by examining the effects of catharanthine on vascular tissues and heart by a series of in vivo and in vitro experiments.
We found that catharanthine dose-dependently lowered BP, heart rate (HR), and cardiac contractility (dP/dtmax) as well as force generation of preconstricted rat aortae (conduit vessel) and small MAs with either phenylephrine (PE) or a depolarizing buffer containing high potassium chloride (KCl). These effects of catharanthine were associated with vessel dilatation and reduced cytosolic free calcium ([Ca2+]i) levels in isolated MAs as well as inhibition of VOCCs in freshly dispersed cardiomyocytes and VSMCs isolated from MAs.
Materials and Methods
These studies were conducted in 13-week-old male Sprague-Dawley rats (300–350 g) obtained from Charles River Laboratories (St. Constant, QC, Canada). The experimental protocols approved by the respective animal care committees at both universities conformed to the Guide for the Care and Use of Laboratory Animals stipulated by the Canadian Council on Animal Care and the National Institutes of Health. The rats (n = 14) anesthetized with isoflurane gas (5% induction and 2% maintenance) were maintained on heating pads at approximately 37°C. A thermometer inserted in the rectum before and during the experimental procedure assessed the body temperature. The maximum fluid administered was kept at a level of <1.2 ml over a 1-h period. Eye drops were administered to protect the eyes from drying up during surgical catheterization and BP measurement. Rats were also anesthetized with thiopental sodium (100 mg/kg i.p.; n = 18). These animals were used to isolate blood vessels or heart for in vitro studies.
Acetylcholine chloride (ACh), bovine serum albumin (BSA), dithiothreitol, EGTA, fura-2 acetoxymethyl ester, indomethacin, PE hydrochloride, papain, as well as all of the salts used in the preparation of various buffers were of analytical grade obtained from either Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) or Bishop & Firkin (Burlington, ON, Canada). Collagenase type II and elastase were obtained from Worthington Biochemical Inc. (Lakewood, NJ). Thiopental sodium was obtained from Abbott Laboratories Ltd. (Saint-Laurent, QC, Canada). Catharanthine was extracted and purified to 99%. This was confirmed by nuclear magnetic resonance and mass spectrometry characterization at the Plant Biotechnology Institute of the National Research Council of Canada. It was used as hydrochloride salt.
Measurement of BP, HR, Intracardiac Pressure, and Cardiac Contractility.
Detailed methodology for the measurements of BP, HR, dP/dtmax, and left ventricular blood pressure performed in rats that were anesthetized with isoflurane was as previously described (Jadhav et al., 2012; Papageorgiou et al., 2012). Either catharanthine (0.5–20 mg/kg) or saline (vehicle) was administered (in volumes <0.3 ml/kg) via a catheter inserted in the femoral vein. A pressure transducer attached to a catheter (PE50) inserted in the femoral artery recorded BP and HR (Jadhav et al., 2012). The studies performed at the University of Saskatchewan determined the changes occurring in BP and HR after administration of either saline or increasing doses of catharanthine in the same rat. The total volume of vehicle (saline) or catharanthine administered to each rat was kept to a minimum (<1.2 ml) and no more than six doses were given to each rat over a period of 60 minutes. A dose of catharanthine >20 mg/kg was not attempted since it produced a profound fall in BP and HR that did not return to baseline. On the basis of these findings, further studies conducted at the University of Toronto determined the changes in intracardiac pressures [left ventricular systolic blood pressure (LVSBP)] and cardiac contractility (dP/dtmax) by advancing pressure-volume catheter (SPR-838; Millar Instruments, Houston, TX) through a cannula inserted into the right common carotid artery into the left ventricle. From the data gathered, it was feasible to calculate the changes in left ventricular volume and the slope of the end-systolic pressure-volume relationship (ESPVR) curves before and after the administration of catharanthine at 1, 3, and 10 mg/kg (Papageorgiou et al., 2012).
Rat Aortic Rings.
The detailed methodology for the measurement of vasodilator responses in endothelium-denuded rat aortic rings was provided earlier (Hopfner et al., 1998; Jadhav et al., 2012). Briefly, isolated rat thoracic aortic rings were set up in organ baths containing 10 ml of Krebs buffer [in mM: 120 NaCl, 4.8 KCl, 1.2 MgCl2, 1.8 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 11 glucose (pH 7.4 gassed with 95% O2, 5% CO2 at 37°C)] maintained under a resting preload tension of 2 g. Steady-state tension responses were elicited to either PE (1 µM) or a depolarizing solution of KCl (100 mM with equimolar NaCl reduced in the bathing buffer) that elicited a submaximal response (approximate EC80–90 level). Once a steady tonic response was reached, cumulatively increasing concentrations of catharanthine (500 nM–160 µM) were added in such a way that the next concentration was added only after the response to the previous concentration had plateaued. Since the presence or the absence of endothelium did not affect the responses to catharanthine, all studies were performed in endothelium-denuded vessels as previously described. The tension responses were recorded in millinewtons on a chart program (Chart V5.0.1) using a Powerlab/8SP data acquisition system (AD Instruments Pvt. Ltd., Sydney, NSW, Australia).
Third-Order Branches of Superior MA.
The vessels were isolated (i.d., <200 µm) and suspended between a micropositioner and force transducer with stainless steel wires (40 µm diameter) in a myograph chamber, model 610M Multi Wire Myograph System (Danish Myotechnology, Aarhus, Denmark). Resting tension (2 mN) was fixed and the rings were maintained for initial equilibration period of 1 hour in Krebs buffer having the same composition and conditions of incubation as described for aortic rings. The changes in force developed were recorded as the increase in millinewtons on a Powerlab data acquisition system. Endothelium was removed by scratching the intimal layer by passing a human hair a few times through the isolated small MA before setting it up in a wire myograph apparatus. It was considered as denuded if the dilator response to ACh was ≤10% in PE-constricted MA (10 µM, approximately EC80–90 concentration) (Mishra et al., 2008; Jadhav et al., 2012). The concentration-inhibitory response relationships for cumulative additions of catharanthine (50 nM–40 µM) were determined under steady-state tonic responses elicited by either PE (10 µM) or KCl (100 mM).
The steady-state tension responses elicited by a depolarizing buffer containing high KCl (100 mM) was prepared with appropriate reduction in NaCl concentration in the buffer to a level of 24 mM with all other salts being present at the same level as indicated above for the normal Krebs buffer. We also determined the responses to depolarizing KCl either in the presence or absence of phentolamine (10 µM) to rule out a role for depolarization-induced norepinephrine release from the sympathetic nerve endings contributing to the altered tension response. It was ascertained in preliminary experiments that the presence or absence of phentolamine did not affect the steady-state tension response to KCl (100 mM). Moreover, the steady tension responses elicited by either PE or high KCl or the vasodilator effects of catharanthine were not significantly different when the glucose concentration in the Krebs buffer was reduced to a level of 5.5 mM from 11.0 mM.
Vessel Wall Diameter and [Ca2+]i Changes in Third-Order Branches of MA.
Isolated MA vessels mounted in a pressure myography chamber under isobaric conditions were subjected to fura-2 acetoxymethyl ester (2 µM) loading in 4-morpholinepropanesulfonic acid buffer containing indomethacin (3 µM) for a period of 2 hours to ensure sufficient fura-2 loading. Simultaneous measurement of changes occurring in outer vessel wall diameter and [Ca2+]i levels were determined using a video dimension analyzer and a monochromator that measured changes in the fura-2 fluorescence (340/380 nm excitation) ratio (Bolz et al., 2000). For each vessel, diameter and calcium time control curves (20-minute duration) were generated after the addition of PE (10 µM), in the absence of catharanthine (PE alone), after which time PE was washed out. The vessels were allowed to recover to their baseline tone diameters (15–20 minutes). The effect of catharanthine was then examined after re-addition of 10 µM PE in the presence of increasing concentrations of catharanthine (0.1–160 µM) in the perfusion medium (PE+CATH) over a similar time period. The changes in diameter are expressed as the percentage decrease from PE induced constriction using the following equation: (Dconc – DPE)/(DBL – DPE) × 100, where Dconc refers to the diameter after catharanthine administration or equivalent time control diameter value in the absence of catharanthine, DPE is the minimum diameter after PE (10 µM) administration, and DBL refers to the initial baseline diameter. Importantly, there were no differences in baseline diameter or DPE between the control and PE + CATH conditions. In vessels undergoing vasomotor responses, the mean diameter over one constriction-relaxation cycle was used as the Dconc value. Fold decrease in Ca2+ was calculated as (Fconc – FPE)/FPE, where Fconc refers to the Ca2+ 340/380 ratio at the concentration or equivalent time control and FPE refers to the peak Ca2+ 340/380 ratio after PE administration. There were no differences in baseline or PE-induced peaks in the Ca2+ 340/380 nm fura-2 fluorescence excitation ratio between the PE alone and PE+CATH conditions. Vessels with unusually low constrictor and calcium responses before catharanthine addition were excluded from the study.
Isolation of Single VSMCs.
Rat mesenteric arteries were dissected in ice-cold Hanks' balanced salt solution [in mM: 140 NaCl, 4.2 KCl, 1.2 MgCl2, 1.2 KH2PO4, 10, HEPES, and 6 glucose (pH 7.4 adjusted with NaOH)] and stripped of connective tissue under a dissecting microscope. Arteries were placed in Hanks’ solution at room temperature for 10 minutes and then transferred into Hanks’ solution, containing 0.2 mg/ml BSA, 1 mg/ml papain, and 0.85 mg/ml dithiothreitol and kept at 4°C for 50 minutes and then at 37°C for 15 minutes. Arteries were transferred into Hanks’ solution containing 1 mg/ml BSA, 1 mg/ml collagenase type II, and 0.5 mg/ml elastase and kept at 37°C for 30 minutes. Arteries were then washed in fresh Hanks’ solution at room temperature for 10 minutes. The third- to fifth-degree small branches were cut off and put into Hanks’ solution to release single VSMCs by gentle trituration through a fire-polished Pasteur pipette. Cell suspension was kept at 4°C and used for experiments within 8 hours of dissociation (Liang et al., 2009; Jadhav et al., 2012).
Recording of Ba2+ Currents VOCCs.
Activities of VOCC were measured by recording Ba2+ currents from single MA VSMCs using the whole-cell patch-clamp technique (Axopatch 200B; Axon Instruments, Foster City, CA) at 22°C. VSMCs were placed in a perfusion chamber (volume 0.5 ml) on a microscope stage (IX50; Olympus Inc., Center Valley, PA) and superfused with a solution containing the following (in mM): 137 NaCl, 5.4 CsCl, 1.0 MgCl2, 5.0 BaCl2, 10 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH). The pipettes had a resistance of 2.0–2.5 MΩ when filled with a solution containing the following (in mM): 115 CsCl, 20 TEA-Cl, 1.0 MgCl2, 10 EGTA, 10 HEPES, 0.2 Na2GTP, 5 Mg-ATP (pH 7.2 adjusted with CsOH). Series resistance was compensated by 80–90%. VSMCs were held at –60 mV and stepped to –40 mV for 300 milliseconds before voltage steps from −60 to +50 mV for 300 milliseconds to examine VOCC currents (Liang et al., 2009; Jadhav et al., 2012). Currents at 0 mV were recorded every 10 seconds during catharanthine perfusion. The mean ± S.E.M. cell capacitance values of VSMCs were 12.9 ± 1.8 pF (n = 8 VSMCs isolated from five rats).
Isolation of Rat Ventricular Myocytes.
Single ventricular myocytes were isolated from rat hearts using a protocol we previously described for the mouse (Liang et al., 2010). Hearts were isolated and perfused in a retrograde manner for 5 minutes with nominally Ca2+-free Tyrode solution containing the following (in mM): 136 NaCl, 5.4 KCl, 0.5 Na2HPO4, 10 HEPES, 1 MgCl2, and 10 glucose (pH 7.40 adjusted with NaOH). After perfusion with a Ca2+-free Tyrode solution containing collagenase (1 mg/ml, type II; Worthington) and protease (0.028 mg/ml, type XIV; Sigma-Aldrich) for 7–10 minutes, the left ventricle was dissected, cut into small pieces, and gently triturated to release single myocytes. Isolated myocytes were stored in Kraft-Brühe (KB) solution, containing the following (in mM): 100 potassium glutamate, 10 potassium aspartate, 2.5 KCl, 10 KH2PO4, 2 MgSO4, 5 HEPES, 20 glucose, 20 taurine, 5 creatine, 0.5 EGTA, and 0.1% albumin (pH 7.2, adjusted with NaOH). The cells were used for the study within 8 hours of isolation.
Recording of VOCC Currents from Ventricular Myocytes.
VOCC currents were recorded from single ventricular myocytes at 22°C using whole-cell patch-clamp technique as described above. The external recording solution contained the following (in mM): 137 NaCl, 5.4 CsCl, 1 MgCl2, 1.2 CaCl2, 10 HEPES, and 10 glucose (pH 7.35 adjusted with NaOH). The pipettes solution contained (in mM): 115 CsCl, 20 TEA-Cl, 1 MgCl2, 10 EGTA, 10 HEPES, 0.2 Na2-GTP, and 5 Mg-ATP (pH 7.2 adjusted with CsOH). Myocytes were held at –85 mV and Na+ currents were inactivated by applying a 300-millisecond voltage step to –40 mV before voltage steps from −60 to +50 mV for 400 milliseconds to examine VOCC currents (Liang et al., 2010).
For in vivo study, the percentage (%) maximum fall in BP and HR attained from basal value after administration of each dose of catharanthine was determined in each rat and the pooled values are expressed as mean ± S.E.M. (n = 7 rats). The data indicated as mean ± S.E.M. were analyzed for statistical significance using one-way analysis of variance (ANOVA) as the same variable (BP or HR change) was compared in the same animal before and after the additions of each dose of catharanthine, followed by Tukey’s post hoc test. These results were presented as line graphs. The closest P value obtained is given in the Results. The mean ± S.E.M. data were subjected to statistical significance for differences between means using one-way ANOVA followed by Tukey’s post hoc test when the same variable was compared in the same animal/tissue/cell before and after the administration of catharanthine. The data for comparison of the mean values for the inhibitory effects of catharanthine on PE or KCl-evoked responses were analyzed by two-way ANOVA followed by Tukey’s post hoc test. Statistical comparisons in left ventricular performance parameters performed at the University of Toronto were analyzed using the Sigma Plot program (version 11.0; Systat Software Inc., San Jose, CA). The differences between means was considered significant when P < 0.05.
Intravenous administration of vehicle did not affect the basal BP or HR for a period of 1 hour (Fig. 1). In contrast, administration of catharanthine (0.5–20 mg/kg i.v.) evoked dose-dependent reductions in both BP and HR. The data from a representative tracing are shown in Fig. 2, A and B. At low doses (0.5–5 mg/kg), catharanthine evoked rapid, transient reductions in BP and HR (lasting <2 minutes), whereas at higher doses (10 and 20 mg/kg), the BP and HR reductions were sustained. The maximal reductions in BP and HR were reached after about 10–30 seconds postinjection, respectively. The responses at all concentrations reached stable levels after 5 minutes. The data from several experiments confirmed that BP decreased (P < 0.01) by more than 50% from 101 ± 12 mmHg (control value) to 48 ± 7 mm Hg after the administration of the highest dose (20 mg/kg) of catharanthine (Fig. 2C). Similarly, HR decreased (P < 0.05) from 399 ± 12 beats/min in control conditions to 297 ± 14 beats/min after the administration of 20 mg/kg catharanthine (Fig. 2D).
It is clear that catharanthine causes sustained dose-dependent reductions in both BP and HR. To further explore the action of catharanthine on the heart, we measured intraventricular pressure-volume relationships using an impedance catheter. The representative data from a single experiment for the fall in LVSBP and the dP/dtmax attained after a single dose of catharanthine (10 mg/kg) administration are shown in Fig. 3A. Consistent with the BP results, peak LVSBPs were reduced after administration of increasing doses (1, 3, 10 mg/kg dose i.v.) of catharanthine in a concentration-dependent manner by values of 10 ± 1 mm Hg, 17 ± 2 mm Hg, and 23 ± 1 mm Hg at nadir (approximately 30 seconds postinjection), and by 5 ± 1 mm Hg, 8 ± 2 mm Hg, and 9 ± 1 mm Hg at steady state (approximately 5 minutes postinjection), respectively (Fig. 3B). Catharanthine dose-dependently decreased the maximal time derivative of the left ventricular pressure (i.e., dP/dtmax) with a pronounced transient effect 30 seconds after catharanthine administration, which reached a stable level approximately 5 minutes postinjection (Fig. 3, A and C). In addition to decreasing dP/dtmax, catharanthine also dose-dependently decreased the slope of the ESPVR from 0.18 ± 0.01 mm Hg/µl in the absence of drug to 0.06 ± 0.01 mm Hg/µl (measured approximately 5 min postinjection of 10 mg/kg catharanthine), further demonstrating that catharanthine impairs cardiac contractility (Fig. 4).
Although the effects of catharanthine on heart function (HR and cardiac contractility) could be responsible for the profound reductions in BP, it is conceivable that this agent could also have vascular effects. The addition of Krebs buffer in organ baths failed to affect the steady-state sustained tonic response elicited by a fixed concentration of either PE or KCl in both aortic and small MA rings (Figs. 5 and 6). In contrast, we found that addition of cumulatively increasing concentrations of catharanthine caused progressive reductions in tension of endothelium-denuded preconstricted isolated aortic vessels (Fig. 7) and MAs (Fig. 8). Similar results were observed in both vessels with intact endothelium. It should be noted that the inhibitory effects were slower and generally higher levels of catharanthine were required to inhibit the steady-state tension responses in aortae (IC50 = 28 ± 2 µM for PE and IC50 = 34 ± 3 µM for KCl) compared with MA (IC50 = 3 ± 1 µM for PE and IC50 = 6 ± 2 µM for KCl). However, the next concentration of catharanthine was added only after the response plateaued for the previous concentration and as the time scale was crunched to accommodate all of the additions, it is not distinctly clear in the case of aortic rings. However, this is not the case with MA because the inhibitory effect for each concentration was reached quickly and the concentration-response curve showed a clear step-wise ladder pattern. These data revealed that MA was more sensitive (P < 0.01) to catharanthine blockade than aortic rings.
To explore the cellular basis for force inhibition in arterial vessels, we measured the vessel diameter and [Ca2+]i level in PE-constricted (10 µM) MA. The data from a single experiment for the decrease in vessel wall diameter (vasoconstriction), along with the increase in fura-2 fluorescence ratio followed by the inhibitory effects on these responses exerted by the addition of increasing concentrations of catharanthine, are shown (Fig. 9, A and B). The concentration-dependent increases in vessel diameter and fura-2 fluorescence ratio after the addition of catharanthine in PE-constricted vessels were calculated from several experiments. These data were compared over the same time course for the responses to PE determined in the absence of catharanthine (Fig. 9, C and D). Catharanthine evoked vasodilatation (IC50 = 9.8 ± 1.4 µM) along with parallel decreases in [Ca2+]i (IC50 = 15.8 ± 2.3 µM) in PE-constricted MA. Interestingly, at lower catharanthine concentrations (i.e., < 5 µM), the oscillatory vasomotor activity typically seen in the vessel diameter and [Ca2+]i of MA that have been pretreated with PE are eliminated (Fig. 9, A and B), suggesting that catharanthine could affect vascular tone at lower concentration ranges below those required for global vasodilation and BP-lowering effects encountered at higher concentrations.
On the basis of the above results, we hypothesized that a common mechanism for the actions of catharanthine on arterial vasculature and heart is the inhibition of VOCC. Consistent with our hypothesis, catharanthine inhibited Ba2+ currents (P < 0.01) through VOCCs measured in isolated single VSMCs of the MA (Fig. 10, A and B). Specifically, the Ba2+ current density at 0 mV (–3.0 ± 0.3 pA/pF) was reduced (P < 0.01; n = 8 cells) to −1.62 ± 0.3 pA/pF at 3 µM, −0.93 ± 0.2 pA/pF at 10 µM, and −0.26 ± 0.1 pA/pF at 30 µM catharanthine. These data support the conclusion that the BP-lowering and vasodilatory effects of catharanthine occur as a result of inhibition of VOCCs in VSMCs of the MA. Catharanthine also dose-dependently inhibited (P < 0.05) VOCCs in cardiomyocytes (Fig. 11, A and B). Specifically, Ca2+ currents through cardiomyocytes at 0 mV were reduced from −4.0 ± 0.5 pA/pF to −2.3 ± 0.5 pA/pF at 100 µM, −1.3 ± 0.3 pA/pF at 300 µM, and −0.45 ± 0.2 pA/pF at 1 mM catharanthine. The inhibitory effect of catharanthine on VOCC currents was less potent (P < 0.01) in cardiomyocytes (IC50 = 224 ± 80 µM; Fig. 11C) than in VSMCs from MA (IC50 = 8.4 ± 2.5 µM; Fig. 10C).
Extracts from C. roseus plants are used to treat many conditions, including cancer and diabetes (Lans, 2006; Nayak et al., 2007; Ara et al., 2009; Rasineni et al., 2010; Madagascar Periwinkle Monograph; http://naturaldatabase.therapeuticresearch.com/nd/Search.aspx?cs=&s=ND&pt=100&sh=1&id=637). Our studies revealed that catharanthine, a major constituent of C. roseus plants, lowers BP, reduces HR, and impairs cardiac contractility in normotensive rats. The hemodynamic and cardiac effects of catharanthine injection were biphasic, characterized by a potent rapid response (<30 seconds) followed by sustained effects at its higher concentrations.
Although the reductions in BP could be partially related to the cardiac actions of catharanthine, we found that catharanthine induces endothelium-independent vasodilatation of MA (IC50 = 3–6 µM) and aortic rings (IC50 = 28–34 µM) with different degrees of sensitivity. The relaxation effects of catharanthine correlated with the inhibition of VOCCs (IC50 = 8.4 µM) in freshly dispersed VSMCs isolated from MAs as well as the reductions in [Ca2+]i (IC50 = 15.8 µM) in MA rings. Taken together, these findings are consistent with the conclusion that the dose-dependent drop in BP evoked by catharanthine is caused, at least in part, by endothelium-independent inhibition of smooth muscle contraction in small blood vessels such as MAs as a result of reductions in [Ca2+]i arising from the blockade of VOCCs.
Catharanthine also had cardiac effects that may contribute to the BP-lowering effects of this agent. Specifically, we observed that the injection of catharanthine caused dose-dependent reductions in HR, despite the reductions in BP that should normally cause reflex elevations of HR. The drop in HR was accompanied by a concomitant dose-dependent decrease in cardiac contractility as indicated by reductions in dP/dtmax as well as the flattening of the slope of the ESPVR. Since the VOCCs in VSMCs contain the same pore-forming subunit, α1C or CaV1,2 like the majority of VOCCs in the heart, we anticipated that catharanthine might exert its effects via blockade of VOCCs (Liao et al., 2005). Indeed, we found that catharanthine blocked VOCC in ventricular cardiomyocytes and VSMC isolated from MA, but the potency of blockade was approximately 22-fold higher in the VSMCs compared with cardiomyocytes. This difference in drug potency of catharanthine on ventricular myocytes versus VSMCs is similar to that seen with other well known VOCC blockers such as the phenylalkylamines (like verapamil) and dihydropyridines (McDonald et al., 1994). With phenylalkylamines and dihydropyridines, blockade is voltage dependent, leading to a potency that depends strongly on the resting membrane potential of the target cells. Thus, since smooth muscle cells are relatively depolarized (resting membrane potential of approximately −60 mV) compared with ventricular cardiomyocytes (resting membrane potential of approximately 85 mV), the potency of VOCCs is far higher for smooth muscle cells compared with ventricular cardiomyocytes. These observations are consistent with the conclusion that catharanthine inhibits VOCCs via a voltage-dependent mechanism similar to that seen with the conventional VOCC blockers. Further studies will be required to assess the molecular basis for the inhibition of VOCCs by catharanthine.
At first glance, the observation that catharanthine preferentially blocks VOCC in smooth muscle cells with a much higher sensitivity compared with ventricular myocytes suggests that the potency of the actions of catharanthine on BP might be dramatically different from its effects on the cardiac contractility. In addition, i.v. administration of catharanthine might have blocked VOCCs regulating sino-atrial (SA) and atrio-ventricular nodes leading to decreased conduction and thus lowered the HR dose dependently at concentration ranges between 0.5 and 20 mg/kg. Our in vitro data focused on ventricular myocytes; thus, it would be difficult to extrapolate the data from our in vitro findings to the in vivo setting. Future studies aimed at characterizing the VOCC blockade of SA nodal cells by catharanthine will resolve this issue. Thus, although we did not measure the Ca2+ currents through VOCCs in SA nodal myocytes, one could anticipate that catharanthine would more potently block VOCCs in the SA node to account for its lowering of the HR.
We recently showed that the amino acid analog, l-Wee, an agent that possesses an indole ring, also exerts endothelium-independent vasodilatation of preconstricted MA (Jadhav et al., 2012). It is of interest that catharanthine is an alkaloid containing an indole ring. It is thus conceivable that some of the cardiovascular actions of catharanthine may be related to the actions of the indole ring. However, catharanthine showed a far greater potency than l-Wee on aortic rings (2 mM l-Wee versus catharanthine: IC50 = 28–34 µM) and MA (17 µM l-Wee versus catharanthine: IC50 = 3–6 µM). On the other hand, catharanthine and l-Wee showed a similar rank order of tissue selectivity for blocking VOCCs (MA > aorta > cardiomyocyte), suggesting a similar mechanism of action. Consistent with a role for the indole ring in, at least partially, mediating the effects of catharanthine, several previous studies have reported on the actions of indole-containing alkaloids on the cardiovascular tissues. For example, ajmalicine and hirsutine (which are also present in C. roseus) block vascular VOCCs in rat aortic rings (Yano et al., 1991). Fumigaclavine C (from Aspergillus fumigatus) blocks the tension responses to KCl in rat aortic rings (Ma et al., 2006). Geissocchizine methyl ester (from Uncariae ramulus et Uncus) induces vasodilatation in rat aortic rings (Yuzurihara et al., 2002). MB-101 (2-hydroxyacetophenone; derivative of isatin or a derivative of 1H-indole-2,3 dione) blocks the tension responses to agonists and KCl in rat aorta and cardiac papillary muscle preparations (Gabriel et al., 2011). Calindol (a 1H-indole derivative) acts as a Ca2+ channel antagonist in MA rings (Thakore and Ho, 2011). These observations further suggest that the indole ring in catharanthine is a factor in the ability of catharanthine to block VOCC. In this regard, a previous study concluded that the structurally related alkaloid, tetrandine, inhibits VOCCs by binding to the phenylalkylamine [verapamil and methoxyverapamil (D-600)] binding site (King et al., 1988). Clearly, more studies are required to establish the role of drugs and alkaloids such as catharanthine that possesses an indole moiety in altering hemodynamic functions via their actions on the heart and the vasculature.
Herbal extracts of C. roseus in concentration range (milligram doses) are reported to have a diverse range of beneficial effects (Lans, 2006; Nayak et al., 2007; Ara et al., 2009; Rasineni et al., 2010; Madagascar Periwinkle Monograph, 2011; http://naturaldatabase.therapeuticresearch.com/nd/Search.aspx?cs=&s=ND&pt=100&sh=1&id=637). Despite this, little is known regarding their effects on hemodynamics or the mechanism of action of catharanthine, a major purified compound extracted from these plants. Our results establish that catharanthine induces hypotension and reductions in HR combined with impairment of cardiac contractility. Thus, the indiscriminate use of these compounds could lead to serious cardiovascular complications that need to be considered. Indeed, the effects of catharanthine could also explain the cardiotoxicity seen in >20% of patients being treated with vinca alkaloid for cancer (Pai and Nahata, 2000). Our results provide further impetus to undertake detailed examination of the effects of other vinca alkaloids including vinflunine for their potential to contribute to cardiovascular toxicity, possibly via their effects on VSMCs and cardiac VOCCs (Pai and Nahata, 2000; Holwell et al., 2001; Kruczynski et al., 2006; Honore et al., 2008; Schutz et al., 2011; Vinflunine, 2011). In other studies (unpublished data), we have also observed that catharanthine reduces the tension responses to both ACh and KCl in isolated rat ileum and colon (IC50 5–10 µM), which can explain the ileus, constipation, and cardiac ischemia seen in cancer patients treated with vinca alkaloids (Schutz et al., 2011; Vinflunine, 2011). Finally, our studies showed that, at very low doses below those required for the induction of hypotension, catharanthine eliminated cyclical vasomotor activity and the associated calcium oscillations. It is conceivable that this action of catharanthine might be useful for the treatment of arteriolar vasospasm that is linked to stroke or other ischemic events such as angina (Inzitari and Poggesi, 2005).
Participated in research design: Jadhav, Gopalakrishnan, Heximer, Backx.
Conducted experiments: Jadhav, Liang, Papageorgiou, Levy.
Contributed new reagents or analytic tools: Balsevich.
Performed data analysis: Jadhav, Liang, Papageorgiou, Levy.
Wrote or contributed to the writing of the manuscript: Jadhav, Shoker, Kanthan, Heximer, Backx, Gopalakrishnan.
- Received August 31, 2012.
- Accepted January 24, 2013.
This research was supported by grants-in-aid from the Canadian Institutes of Health Research [Grants MOP-67060 (to V.G.), MOP-153103 (to P.H.B.), and MOP-152797 (to S.H.)]. P.H.B. is a career investigator of the Heart & Stroke Foundation of Ontario, Canada. W.L. was a recipient of the Heart & Stroke Foundation of Canada doctoral award.
- acetylcholine chloride
- analysis of variance
- blood pressure
- bovine serum albumin
- cytosolic free calcium
- cardiac contractility
- end-systolic pressure-volume relationship
- heart rate
- l-tryptophan ethyl ester
- left ventricular systolic blood pressure
- superior mesenteric artery
- voltage-operated L-type Ca2+ channel
- vascular smooth muscle cell
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics