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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

Potential L-Type Voltage–Operated Calcium Channel Blocking Effect of Drotaverine on Functional Models

Zoltán Patai, András Guttman and Endre G. Mikus
Journal of Pharmacology and Experimental Therapeutics December 2016, 359 (3) 442-451; DOI: https://doi.org/10.1124/jpet.116.237271

Abstract

Drotaverine is considered an inhibitor of cyclic-3′,5′-nucleotide-phophodiesterase (PDE) enzymes; however, published receptor binding data also support the potential L-type voltage– operated calcium channel (L-VOCC) blocking effect of drotaverine. Hence, in this work, we focus on the potential L-VOCC blocking effect of drotaverine by using L-VOCC–associated functional in vitro models. Accordingly, drotaverine and reference agents were tested on KCl-induced guinea pig tracheal contraction. Drotaverine, like the L-VOCC blockers nifedipine or diltiazem, inhibited the KCl-induced inward Ca2+- induced contraction in a concentration- dependent fashion. The PDE inhibitor theophylline had no effect on the KCl-evoked contractions, indicating its lack of inhibition on inward Ca2+ flow. Drotaverine was also tested on the L-VOCC–mediated resting Ca2+ refill model. In this model, the extracellular Ca2+ enters the cells to replenish the emptied intracellular Ca2+ stores. Drotaverine and L-VOCC blocker reference molecules inhibited Ca2+ replenishment of Ca2+-depleted preparations detected by agonist-induced contractions in post–Ca2+ replenishment Ca2+-free medium. Theophylline did not modify the Ca2+ store replenishment after contraction. It seems that drotaverine, but not theophylline, inhibits inward Ca2+ flux. The addition of CaCl2 to Ca2+-free medium containing the agonist induced inward Ca2+ flow and subsequent contraction of Ca2+-depleted tracheal preparations. Drotaverine, similar to the L-VOCC blockers, inhibited inward Ca2+ flow and blunted the slope of CaCl2-induced contraction in agonist containing Ca2+-free medium with Ca2+-depleted tracheal preparations. These results show that drotaverine behaves like L-VOCC blockers but, unlike PDE inhibitors using L-VOCC associated in vitro experimental models.

Introduction

Two families of drugs that inhibit PDE activity, methylxanthines and isoquinolines, are used clinically in two distinct therapeutic areas involving smooth muscle function. Methylxanthines, such as theophylline, are frontline asthma medications, whereas the isoquinolines, for example, papaverine, are used to ameliorate the symptoms of visceral smooth muscle spasm and associated pain. Neither of them, however, is active if used in the opposite field: Why is that? Based on indirect experimental evidence, the difference may be associated with their differing activity on the voltage-operated calcium channel (L-VOCC).

The natural isoquinoline alkaloide of Papaver somniferum, papaverine, and its synthetic derivate drotaverine have been broadly used as antispasmodic agents in human medicine for decades. It has been published that drotaverine binds to the L-VOCC on pregnant rat uterine membranes (Tömösközi et al., 2002), and Ca2+ activated potassium channels and L-VOCCs may be involved in papaverine-induced vascular relaxation in rat basilar artery (Han et al., 2007). Moreover, it has been shown that both molecules inhibit cyclic-3′,5′-nucleotide-phophodiesterase (PDE) enzymes with concentration-dependent specificity (Kukovetz and Pöch,1970; Triner et al.,1970; Pöch and Kukovetz, 1971; Kukovetz et al., 1976). However, the detailed molecular mechanism of action and associated function of drotaverine on airway smooth muscle have not yet been systematically investigated. Specifically, the interaction of drotaverine with the Ca2+ flux essential for the contraction-relaxation machinery of airway smooth muscle has not been tested. Both these cellular mechanisms play a fundamental regulatory role in smooth muscle function. In airway smooth muscle, bronchodilatory agents increase the intracellular cAMP level (e.g., by the inhibition of the PDEs) and cause the relaxation of precontracted (post-agonist administration) preparations (Frossard et al., 1981; Torphy and Undem, 1991); however, these agents are less effective in the inhibition of the agonist-induced contraction (pre-agonist administration) (Bilcíková et al., 1987). The airway smooth muscle contractile mechanism is considered to be triggered either by release of intracellularly sequestered Ca2+ or by the increased influx of extracellular Ca2+. In airway smooth muscle, Ca2+ release from the intracellular stores predominates over inward Ca2+ flux through L-VOCCs after receptor activation-induced airway smooth muscle contraction (Pelaia et al., 2008). Although operation of L-VOCCs can induce some relaxation of agonist-induced precontracted airway smooth muscle (Fanta et al., 19-qu82), they are not effective before agonist administration (Foster et al., 1984), which may explain why L-VOCC blockers have no principal therapeutic benefit for the treatment of bronchial asthma (Hirota et al., 2003). Airway smooth muscle receptor–mediated activation uses intracellular Ca2+ stores, whereas vascular smooth muscle is more dependent on Ca2+ influx; however, the L-VOCCs may play a role in the Ca2+ balance necessary for maintenance of the physiologic airway smooth muscle function. In this context, the principal role of L-VOCCs may be regulation of the Ca2+ influx responsible for refilling of the intracellular Ca2+ stores that are partly depleted during agonist-induced contraction in airway smooth muscle (Bourreau et al., 1993). It has been demonstrated that L-VOCC blockers are able to inhibit the refill of the depleted sarcoplasmic Ca2+ stores in resting conditions (Bourreau et al., 1991; Liu and Farley, 1996), whereas their impact on receptor activation–associated inward Ca2+ currents and subsequent contractions in normal Ca2+ containing medium is minimal (Cheng and Townley, 1983). We have hypothesized that the L-VOCCs may also play an important role in receptor-mediated inward Ca2+ current associated contractions when Ca2+-depleted preparations in Ca2+-free medium were supplemented with extracellular Ca2+. Our aim with this study was to generate functional data supporting the potential L-VOCC blocking effect of drotaverine using proven L-VOCC-dependent tracheal models (e.g., KCl depolarization- induced contractions (Foster et al., 1983), resting Ca2+-refill linked contractions, and receptor- mediated inward Ca2+-induced contractions).

Methods

Chemicals.

The ingredients of the Krebs-Henseleit (KH) solution are as follows: KCl, CaCl2, 2H2O, KH2PO4, NaHCO3, MgSO4, and 7H2O, obtained from Merck Inc. (Darmstadt, Germany). NaCl was from Reanal ZRt (Budapest, Hungary). The drotaverine HCl was synthesized in Chinoin Co. Ltd. (Budapest, Hungary). EGTA, indomethacin, nifedipine, theophylline, papaverine HCl, diltiazem HCl, acetyl-β-methylcholine chloride (methacholine), histamine dihydrochloride, and d-(+)-glucose were purchased from Sigma (St. Louis, MO).

Animals.

We used male guinea pigs (body weight, 300–350 g; Harlan MD), distributed by INNOVO Ltd., (Gödöllő, Hungary). All experiments were performed in accordance with the Institutional Ethical Codex, Hungarian Act of Animal Care and Experimentation (1998, XXVIII, section 243/1998) and the European Union guidelines (directive 2010/63/EU). The animals were housed in open cages in a temperature-controlled and ventilated environment (21–23°C) with a 12-hour light/dark cycle. Water and standard ascorbic acid containing guinea pig chow (Altromin, Lage, Germany) were provided ad libitum. The animals were tissue donors; therefore, approval of the experimental protocol by the Hungarian Governmental Animal Ethics Committee was not mandatory.

Isolation of the Trachea and Organ Bath Preparation.

On the day of the experiments, the guinea pigs were euthanized by an overdose of pentobarbital sodium. The trachea was rapidly removed and placed in KH solution of the following composition: 119 mM NaCl, 25 mM NaHCO3, 2.5 mM CaCl2, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 11.1 mM glucose, and 5 · 10−6 M indomethacin. The cartilaginous rings from the distal part of the trachea were opened longitudinally and mounted in a 20-ml organ bath containing KH solution, maintained at 37.4°C, and continuously bubbled with 95% O2 and 5% CO2 to give a pH of 7.4 ± 0.1. The preparations were preloaded with 5 millinewton (mN; approximately 0.5 g). The tissues were then allowed to equilibrate for at least 1 hour, during which time they were washed approximately every 20 minutes with fresh KH solution. Eight tracheal strip preparations were used from the same guinea pigs on the same day. Four preparations were used for the control groups, and the other four preparations were used for the test compounds. Earlier studies showed no difference in reactivity to histamine or methacholine with respect to the tracheal ring location from this part of the trachea (data not shown). The strength of the isometric contractile responses was measured (mN) using a force displacement transducer and preamplifier (MDE Co. Ltd., Budapest, Hungary). The experimental data were collected and evaluated by a computer-aided data acquisition system (SpelIso v3.2 software; MDE Co. Ltd.).

Owing to small variations in the baselines and the acclimation of the tissue preparations to the organ bath environment, the second contraction force was in many cases higher than the first (but not as high as the third contraction force). Consequently, the second contraction was used as the control in all the experimental protocols.

KCl-Induced Contraction Protocol.

Contractions of the eight mounted tracheal preparations in normal KH solution were evoked by consecutive applications of 20, 30, and 50 mM KCl, separated by a washout (Fig. 1A). The contraction force was continuously registered. The contraction force derived from the second concentration-response curve was used as the control value (100% contraction). Once the control value had been established, four of the eight preparations were treated with10−7 M of the test molecules; the other four were treated with the vehicle of the test molecule (control preparations). After a 15-minute incubation, another concentration-response curve to KCl was constructed for all eight preparations. After a washout, the experiment was repeated using 10−6 M (or vehicle) and 10−5 M (or vehicle) test molecule, with a washout between increasing doses of test molecule. The experimental design is shown in Fig. 1A. The percentage of inhibition was calculated for each dose of the test molecule at every KCl concentration. The test molecule dose causing a 50% inhibition of KCl contraction was calculated in each case when feasible.

Fig. 1.
Fig. 1.

(A) The experimental design for demonstrating the effect of test molecules on KCl depolarization-induced contractions. Consecutive 20, 30, and 50 mM KCl-induced contractions were evoked twice in normal KH solution. The value of the second contraction at each KCl concentration was taken as 100%, followed by three cumulative KCl concentration-response curves in the presence of increasing concentrations of the test molecule (or vehicle) with a washout (W) between each. Please note that this is an illustration of the method, not a real trace. W, washout. (B) The experimental design for investigating the intracellular Ca2+ store depletion associated decrease in contraction and subsequent calcium reload-dependent contraction recovery. Agonist-induced contractions were evoked twice in normal KH solution, followed by three consecutive agonist stimulations (separated by W) in Ca2+-free KH medium. After the third stimulation and W, the medium is changed to normal KH solution containing the test molecule. After a 30-minute incubation, the medium was again changed to Ca2+-free KH solution and three agonist-induced contractions were evoked; each was separated by a washout. Please note that this is an illustration of method, not a real trace. C1, control contraction; C2, contraction after test molecule. (C) Experimental protocol for investigating the effect of test molecules on CaCl2-induced contraction. Agonist-induced contractions are evoked twice in normal KH solution, followed by three consecutive agonist stimulations, each separated by W in Ca2+-free KH medium. After the final W, the medium was changed to Ca2+-free KH buffer containing the agonist, and 2.75 mM CaCl2 was added to the buffer to evoke the Ca2+ infux associated contraction. Please note that this is an illustration of method, not a real trace. C1, contraction without test molecule C2, contraction after test molecule.

Ca2+ Reload after Agonist-Induced Contractions in Ca2+-Free Medium (Resting Refill).

The intracellular Ca2+-dependent contractions were tested using two mediators that induce contraction of airway smooth muscle, histamine (3 × 10−6 M) and methacholine (5 × 10−7 M). The concentrations chosen were the calculated EC50 values for these mediators from previous experiments in our laboratory (data not shown). Histamine and methacholine were used in separate experiments. After the equilibration period, two consecutive contractions were evoked with either histamine or methacholine separated by a washout. The second contraction was considered the control (100%) contraction. After washing out the agonist, 2.5 mM Ca2+ containing KH solution was changed to a Ca2+-free medium. Next, three consecutive contractions were evoked by the constrictor agents, each separated by a washout to deplete the sarcoplasmic Ca2+ stores. The third agonist stimulation usually evoked a minimal, if any, contraction, indicating depletion of the intracellular calcium stores (Table 2, fifth contraction column). After the last washout in Ca2+-free medium, test compounds (10−5 M) were added to four of the eight organ baths. The other four preparations served as controls, with only the vehicle added to the baths. After a 15-minute incubation, the solution in the bath was changed to normal KH solution (2.5 mM Ca2+) containing 10−5 M test molecule (or vehicle). The preparations were then incubated for 30 minutes without any agonist stimulation. Previous studies have shown this was sufficient time for the Ca2+ refill of intracellular calcium stores. The normal KH solution was then changed to the Ca2+-free KH solution in all eight organ baths, after which three consecutive contractions were evoked by constrictor agonists, each separated by a washout period. The contraction force was measured (in mN), and the percentage contraction values were calculated using the second agonist-induced contraction as 100% contraction. The experimental design is shown in Fig. 1B.

CaCl2-Induced Contraction in Agonist Containing Ca2+-Free Medium (Receptor-Operated Refill).

The experimental protocol was identical to the “resting refill” protocol, up to the intracellular Ca2+ depletion step in Ca2+-free medium. As before, the third agonist stimulation evoked only limited, if any, contraction denoting the depleted Ca2+ content of the intracellular calcium stores (Table 2, fifth contraction column). At this point, 10−5 M test molecule was added to four of the organ baths; the other preparations were treated with the vehicle as control preparations. After 15-minute incubation, 2.75 mM CaCl2 was added to all eight organ baths. The experimental design is presented in Fig. 1C. In this experimental protocol, both the maximum contraction and the slope of the contractions were evaluated. The percent decrease of the contraction force was also calculated.

Statistics.

Statistical comparison of the treated versus the nontreated (control) groups was conducted by using Student’s t test (GraphPad Software, GraphPad Prism v6.0, La Jolla, CA). Differences among groups were considered statistically significant when the P < 0.05.

Results

KCl Depolarization.

KCl caused contraction of the guinea pig tracheal preparation in a concentration-dependent fashion. The contraction force generated by 20 mM, 30 mM, and 50 mM was 4.0 ± 2.2, 9.5 ± 2.5 and 13.9 ± 3.1 mN (n = 122), respectively. The KCl responses were reproducible in control preparations each time they were repeated for constructing drug dose-response curves (data not shown). L-VOCC blockers (e.g., nifedipine and diltiazem) decreased the KCl-induced contractions in a concentration-dependent manner. 10−5 M nifedipine or diltiazem practically abolished the KCl-induced contractions (Fig. 2), proving that the applied experimental protocol was suitable to test the functional consequences of L-VOCC blockade. The two isoquinoline derivatives also decreased KCl-induced contractions in a concentration-dependent fashion (Fig. 2), supporting the proposed functional L-VOCC blocking activity of both drotaverine and papaverine; however, the potency and efficacy of both drotaverine and papaverine were weaker than that of the reference L-VOCC blockers (Fig. 2; Table 1). Unlike the L-VOCC blockers, the PDE inhibitor theophylline did not modify the KCl-induced contractions, indicating that the cAMP/PDE system does not play a significant role. The mixture of the equimolar concentrations of nifedipine and theophylline behaved more like a L-VOCC blockers alone in this experimental mode (Fig. 2; Table 1). Interestingly, the inhibitory efficacy of the nifedipine and theophylline combination was significantly higher than that of nifedipine alone. This observation will be explored in future studies.

Fig. 2.
Fig. 2.

The effect of test molecules on KCl-induced tracheal contractions: (A) 20 mM; (B) 30 mM; (C) 50 mM. The experiments were carried out in normal KH solution supplemented with the appropriate concentration of KCl. Values were expressed as mean ± S.D. (n = 4–8). Student’s t test was used to compare the test molecule treated organs to the vehicle treated control ones. * P < 0.05; **P < 0.01; or ***P < 0.001.

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TABLE 1

EC50 (μM) values of the test molecules

The EC50 values of the test compounds calculated at 20, 30, and 50 mM KCl-induced tracheal contractions.

Ca2+ Reload after Agonist-Induced Contractions in Ca2+-Free Medium (Resting Refill).

Both 3 × 10−6 M histamine and 5 × 10−7 M methacholine in normal KH solution induced tracheal smoot h muscle contractions of similar strength (11.9 ± 3.2 mN; n = 43 and 10.4 ± 2.8 mN n = 32, respectively). With consecutive administration of histamine and methacholine, the contraction force decreased gradually in calcium-free KH medium to 70.7% ± 14.8%, 22.9% ± 25.1%, and 3.0% ± 6.3% (n = 43); and 76.2% ± 12.4%, 13.3% ± 16.8%, and 0.4% ± 1.1% (n = 32), respectively, of the original reference contraction (Table 2). After agonist-provoked calcium depletion, the preparations were put into normal KH solution (reload solution) and incubated for 30 minutes to allow the depleted calcium stores to refill (resting refill). The organ bath solution was replaced with a calcium-free medium (postreload), after which the preparations were able to contract again with agonist stimulation, indicating refilling of their intracellular calcium stores (Table 2, sixth contraction column). L-VOCC blockers like nifedipine (Fig. 3C and 4C), or diltiazem (Fig. 3D and 4D) added to the calcium reload medium were able to reduce the agonist-induced contractions (sixth contraction) in the postreload Ca2+-free medium. This observation indicates that both L-VOCC blockers may inhibit the resting Ca2+ refill of emptied intracellular calcium stores regardless of the constrictor mediator used (Table 2).

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TABLE 2

Tracheal smooth muscle contraction

Histamine- (3 × 10−6 M) or methacholine- (5 × 10−7 M) induced guinea pig isolated tracheal contractions in normal (first contraction and second contraction) and calcium free Krebs-Henseleit (KH) solution before (third contraction, fourth contraction, and fifth contraction), and after incubating for 30 minutes in normal KH solution (sixth contraction and seventh contraction).a

Fig. 3.
Fig. 3.

Histamine-induced guinea pig isolated tracheal contractions in normal and calcium free Krebs-Henseleit (KH) solution before and after a 30-minute incubation in normal KH solution (Ca2+ reload) with or without 10−5 M drotaverine (A), papaverine (B), nifedipine (C), diltiazem (D), theophylline (E), or theophylline + nifedipine (F). Values represent mean ± S.D. (n = 4–12 preparations). Student’s t test was used for the comparison of control and test molecule treated groups. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.
Fig. 4.

Methacholine-induced guinea pig isolated tracheal contractions in normal and calcium free Krebs-Henseleit (KH) solution before and after 30 minutes of incubation in normal KH solution (Ca reload) with or without 10−5 M drotaverine (A), papaverine (B), nifedipine (C), diltiazem (D), theophylline (E), or theophylline + nifedipine (F). Values represent mean ± S.D. (n = 4–12 preparations). Student’s t test was used for the comparison of control and test molecule–treated groups. *P < 0.05; **P < 0.01; ***P < 0.001.

The nonspecific phosphodiesterase inhibitor theophylline was also tested on the resting calcium refill model; however, as can be seen in Fig. 4E, theophylline, unlike L-VOCC blockers, enhanced, rather than inhibited, the histamine-induced contraction. The combination of 10−5 M theophylline with 10−5 M nifedipine in the Ca2+ refill medium inhibited the agonist-induced contraction in the postreload calcium-free medium (Figs. 3F and 4F).

Drotaverine and papaverine were also tested on this model (Figs. 3A and 4A and Figs. 3B and 4B). Both drotaverine and papaverine blocked the resting Ca2+refill–associated contractions at 10−5 M concentration, making these two isoquinoline derivatives more similar to the L-VOCC blockers than to the PDE inhibitors.

CaCl2-Induced Contraction in Agonist Containing Ca2+-Free Medium (Receptor-Operated Refill).

Administration of 2.75 mM CaCl2 to the calcium-depleted tracheal preparation incubated with histamine (3 × 10−6M) or methacholine (5 × 10−7M) in Ca2+-free (0.25 mM EGTA) buffer induced a contraction as strong as that produced by the same concentration of agonists in normal (2.5 mM Ca2+) KH solution at the start of the experimental protocol. Neither the maximal contraction force nor the slope (Table 3) of the contraction of the control preparations differed markedly between the two experimental conditions. So the mechanical response to the agonist-induced contraction in normal KH solution is identical to the contraction developed by adding CaCl2 to the Ca2+-free KH solution containing the agonist.

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TABLE 3

Contraction force (mN) and slope values of different protocols

Comparison of the contraction force (mN) and slope values of the contractions were obtained using four experimental protocols. Histamine (3 × 10-6 M; protocol 1) and methacholine (5 × 10-7 M; protocol 3)-induced tracheal contraction in normal Krebs-Henseleit (KH) solution. CaCl2-induced contraction of intracellular calcium predepleted tracheal preparations in histamine (protocol 2) or methacholine (protocol 4) containing Ca2+-free KH solution.a b

The L-VOCC blockers nifedipine or diltiazem (10−5 M) decreased the slope of the CaCl2-induced contraction force. This effect was not agonist-specific (Table 3) as they blunted the force development slope in the presence of both histamine and methacholine.

In the same model, both drotaverine and papaverine decreased the slope of the CaCl2-induced contraction (Table 4), and in this context, the two tested isoquinoline derivatives behaved like the L-VOCC blockers; however, the PDE inhibitor theophylline had no effect on the contraction slope with either agonist in the organ bath. The combination of theophylline and nifedipine produced the same result as nifedipine alone. There were a variety of potencies among the test compounds with respect to maximum contraction. The rank order for decreasing the contraction amplitude in the presence of histamine was diltiazem < theophylline < drotaverine = nifedipine = nifedipine + theophyilline < papaverine, whereas in the presence of methacholine, the rank order was theophylline = drotaverine < nifedipine = nifedipine + theophylline < diltiazem < papaverine (Fig. 5). Based on these results, it has been suggested that in the case of diltiazem, sufficient Ca2+ penetrated the cells to develop contractions as strong as histamine- or methacholine-induced contractions in normal KH solution. This did not appear to be the case with other test molecules because in addition to decreasing the contraction slope, they modified the maximum CaCl2-induced contraction force as well. Theophylline alone had no effect on either slope or contraction maximum, and the nifedipine and theophylline combination acted like nifedipine alone.

View this table:
TABLE 4

Slopes of CaCl2-induced contractions

The CaCl2-induced contraction slope values for intracellular calcium predepleted tracheal preparations in histamine (3 × 10−6 M) or methacholine (5 × 10−7 M) containing Ca2+-free KH solution.a b

Fig. 5.
Fig. 5.

The maximum CaCl2-induced contraction force of intracellular calcium predepleted tracheal preparations in 3 × 10−6 M histamine (A) or 5 × 10−7 M methacholine (B) containing Ca2+-free KH solution. The percent change of the contraction force is also indicated on the figures. Values represent the mean ± S.D. (n = 4–8). Student’s t test was used to compare the CaCl2-induced contraction in nontreated to the treated organs. *P < 0.05; **P < 0.01; ***P < 0.001.

Discussion

Phosphodiesterdase inhibitors have long been used therapeutically. Methylxanthines (e.g., theophylline) are frontline treatments of asthma, and isoquinolines (e.g., papaverine) ameliorate the symptoms of visceral smooth muscle spasm and associated pain. The medical use of these structurally dissimilar PDE inhibitors is based on bedside experience rather than on a rational knowledge of their molecular mechanism of action. The question to be considered is what molecular mechanisms support the different clinical uses for these two types of PDE inhibitors? Based on indirect experimental evidence, the difference may be associated with their differing activity on the L-VOCC.

It has been published that drotaverine increases intracellular cAMP levels by the inhibition of PDEs, and it may also have an allosteric L-VOCC-regulating effect, as proved by the displacement of [H3]-nitrendipine (Tömösközi et al., 2002) from its binding site on pregnant rat uterine membranes. Two other isoquinoline derivatives, papaverine and ethaverine, also inhibit PDEs and bind to the L-VOCC (Wang and Rosenberg, 1991; Iguchi et al., 1992), suggesting a common cellular mechanism of action for these structurally similar derivatives; however, neither the interaction of drotaverine with the L-VOCC binding site(s) of guinea pig airway smooth muscle nor the functional L-VOCC blocking effect of drotaverine has been investigated to date. Therefore, our aim was to provide functional data that support the L-VOCC blocking effect of isoquinoline derivatives using proven L-VOCC dependent tracheal models like the KCl depolarization-induced contraction model, the resting Ca2+ refill–linked contraction model, and the receptor activation-associated inward Ca2+-induced contraction model.

Traditional L-VOCC blockers and papaverine are able to inhibit the KCl-induced contraction through the inhibition of depolarization triggered inward calcium current (Cerrina et al., 1983; Cheng and Townley, 1983; Ohashi and Takayanagi, 1983; Foster et al., 1984; Baersch and Frölich, 1995). Our results showed that both papaverine and drotaverine, but not theophylline, inhibited the KCl-induced contractions in a dose-dependent fashion. Others (Small et al., 1989) also demonstrated the lack of inhibition by theophylline on the amplitude of KCl-induced tracheal contractions. A combination of theophylline with nifedipine produced the same effect as nifedipine alone, indicating that cAMP/PDE-related mechanism was not related to L-VOCC function on KCl-induced contraction on guinea pig isolated tracheal preparations. Despite widespread use of the KCl model for testing L-VOCC blockers, it is not truly physiologic as it is not the high extracellular concentration of KCl that is responsible for membrane depolarization and the subsequent smooth muscle contraction. Instead, airway smooth muscle contraction is considered Ca2+ dependent, either by the release of intracellularly sequestered Ca2+ or by an increase in influx of extracellular Ca2+. In airway smooth muscle, Ca2+ release from intracellular stores favors inward Ca2+ flux through L-VOCCs after receptor activation–induced airway smooth muscle contraction. This mechanism is indirectly supported by the relative ineffectiveness of L-VOCC blockers on agonist-induced tracheal contractions described by others (Drazen et al., 1983; Advenier et al., 1984; Ahmed et al., 1985; Baersch and Frölich, 1995). Repeated agonist stimulation in the presence of L-VOCC blockers results in gradual but only moderately decreasing maximal contraction force (Flores-Soto et al., 2013), which indicates that the airway smooth muscle is able to contract in a condition when the inward Ca2+ current via L-VOCCs is blocked. So the intracellular Ca2+ stores are the primary Ca2+ sources for contractions (Creese and Denborough, 1981), and the partly emptied sarcoplasmic Ca2+ stores are able to be replenished before a new contraction, even if the L-VOCCs are blocked. In this case, the receptor-operated and other calcium channels are responsible for the inward Ca2+ current (McFadzean and Gibson, 2002). Contrary to the results obtained in normal KH solution, consecutive constrictor mediator stimulation in a Ca2+-free medium resulted in gradually decreasing contractions (Creese and Denborough, 1981; Noguera et al., 1994). If the Ca2+-depleted preparation is then transferred to a normal Ca2+-containing buffer, the intracellular Ca2+ stores are refilled (Noguera et al., 1995). It is highly probable that inward Ca2+ flux through the L-VOCC is the main mechanism for the postcontraction Ca2+ refill (Bourreau et al., 1991, 1993; Dessy and Godfraind, 1996; Hirota and Janssen, 2007; Flores-Soto et al., 2013). This is called resting Ca2+ refill because the agonist is not present between two stimulations when the Ca2+ refill takes place. In our studies, repeated histamine or methacholine administration in a Ca2+-free medium elicited gradually decreasing contractions, indicating possible depletion of the internal Ca2+ stores. Changing the Ca2+-free medium to a normal 2.5 mM Ca2+-containing KH buffer (reload medium), the agonists were again able to evoke contraction in the postreload Ca2+-free medium. The observed reaction was mediator-independent since both the mast cell mediator histamine and the muscarinic M3 receptor agonist methacholine produced the same result. Similarly to the L-VOCC blockers (e.g., nifedipine or diltiazem), isoquinolines introduced to the Ca2+ reload medium inhibited the histamine or methacholine-induced contraction in the postreload Ca2+-free solution, suggesting that the observed mechanism is linked to L-VOCC function. In contrast, using the PDE inhibitor theophylline in the Ca2+ reload medium did not block, and rather increased, the magnitude of the constrictor mediator–induced contraction. These observations suggest that the Ca2+ refill model is L-VOCC dependent and is not inhibited by blocking the cAMP/PDE system.

Receptor activation induces the depolarization of airway smooth muscle resulting in inward Ca2+ flux via opening of both receptor-operated calcium channels s and L-VOCCs (Cuthbert et al., 1994; Flores-Soto et al., 2013) with subsequent receptor-operated calcium channel stores through both receptor-operated calcium channels ROCCs and L-VOCCs. The Ca2+ depleted tracheal preparation in an agonist containing Ca2+ free medium responded with as strong a contraction by the addition of CaCl2 to the organ bath, as the same dose of agonist in normal KH solution. There was also no difference in the development speed of the contraction (slope) indicating that with the added CaCl2, Ca2+ penetrates rapidly into the Ca2+ depleted smooth muscle and evokes a contraction in the presence of either histamine or methacholine. The receptor activated refill process was significantly decreased by L-VOCC blockers or isoquinoline derivatives (e.g., drotaverine or papaverine) as shown by the reduced slope of the CaCl2 contraction curve, but they only moderately affected the amplitude of the contraction.. This indicates that the speed of the Ca2+ entry is reduced after L-VOCC blockade but after a while sufficient Ca2+ becomes accessible to the contractile apparatus to allow maximal contraction. So, unlike the resting Ca2+ refill model, the L-VOCCs are not the only channel type where the Ca2+ entry into the smooth muscle takes place. It seems that L-VOCCs are responsible for rapid Ca2+ entry, and other channels allow a slower entry. The PDE inhibitor, theophylline, had no effect on either the contraction slope or on the magnitude of contraction, indicating that the mechanism is independent of the cAMP/PDE system. This hypothesis is further supported by the result that mixed equimolar concentrations of nifedipine and theophylline behaved more like L-VOCC blockers.

In conclusion, the experiments we have performed demonstrate a mechanistic rationale why the two classes of PDE inhibitor successfully treat separate clinical indications but are not interchangeable.

Acknowledgments

The authors gratefully acknowledge the valuable work of Neil Fitch for revising the manuscript.

Authorship Contributions

Participated in research design: Mikus.

Conducted experiments: Patai.

Contributed new reagents or analytic tools: Mikus.

Performed data analysis: Mikus, Patai.

Wrote or contributed to the writing of the manuscript: Mikus, Guttman.

Footnotes

Abbreviations

KH
Krebs-Heneleit
L-VOCC
L-type voltage–operated calcium channel
PDE
phosphodiesterase receptor-operated calcium channels

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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

Functional Calcium Channel Blocking Effect of Drotaverine

Zoltán Patai, András Guttman and Endre G. Mikus
Journal of Pharmacology and Experimental Therapeutics December 1, 2016, 359 (3) 442-451; DOI: https://doi.org/10.1124/jpet.116.237271
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