Direct Inhibitory Effect of Chlorpromazine on Smooth Muscle of the Porcine Pulmonary Artery

The lungs of adult pigs were obtained from a local slaughterhouse immediately after the pigs had been killed and were transported to our laboratory in preaerated ice-cold physiologic saline solution (PSS). The intrapulmonary arteries were dissected from lungs and cut longitudinally. The endothelium was removed by rubbing the inner surface with a cotton swab, and the adventitia was trimmed away using a microscope. The muscle sheets were transversely cut into strips measuring 5 mm in length, 1 mm in width, and approximately 0.1 mm in thickness. All tissue preparations were performed in oxygenated (95% Oxygen²and 5% CO²) PSS.

The strips were then loaded with fura-2, in the form of acetoxymethyl ester (fura-2/AM). The strips were incubated in Dulbecco-modified Eagle's medium that contained 25 micro Meter fura-2/AM dissolved in dimethyl sulphoxide and 5% fetal bovine serum [11,12]for 4 h at 37 degrees Celsius. After loading with fura-2, the strips were rinsed with normal PSS to remove the dye in the extracellular space and to equilibrate them for at least 60 min at 37 degrees Celsius before starting the measurements.

The fura-2-loaded strips were mounted vertically in a quartz organ bath and connected to a strain gauge (TB-612T, Nihon, Koden, Japan). During the 60-min fura-2 equilibration period, the strips were stimulated with 80 mM Potassium sup + -depolarization every 15-20 min, and the resting tension was increased in a stepwise manner to obtain the maximal force development. The appropriate resting tension level obtained by this procedure was approximately 300-400 mg. The responsiveness of each strip to 80 mM Potassium sup + was recorded before starting the experimental protocol. The developed force was expressed in percentage, assigning the values in normal (5.9 mM Potassium sup +) PSS and steady state of 80 mM Potassium sup + -PSS to be 0 and 100%, respectively.

Changes in the fluorescence intensity of the fura-2-Calcium sup 2+ complex were monitored by using a front-surface fluorometer specifically designed by us for fura-2 fluorometry (model CAM-OF) with the collaboration of Japan Spectroscopic (Tokyo, Japan). [11,12]In brief, dual wavelength excitation light (340 and 380 mm) was obtained from a spectroscope from a xenon light source. By using a chopper wheel, the excitation light was alternately (400 Hz) guided through quartz optic fibers arranged in a concentric inner circle (diameter = 3 mm) and which directly illuminated the entire vascular strip. The surface fluorescence of the strip was collected by glass optic fibers arranged in an outer circle (diameter = 7 mm) and introduced through a 500 plus/minus 10 nm band-pass filter into a photomultiplier. The ratio (F sub 340 /F³⁸⁰) of the fluorescence intensities at 340 nm excitation (F³⁴⁰) to those at 380 nm excitation (F³⁸⁰) was monitored and expressed as a percentage, assigning the values at rest in normal (5.9 mM Potassium sup +) and in depolarization with 80 mM Potassium sup + -PSS to be 0% and 100%, respectively. The percent values of the fluorescence ratios were used for the statistical analysis of [Calcium sup 2+]ⁱ. As for the reference, the absolute value of [Calcium²+]ⁱwas determined as follows. The minimum and maximum fluorescence ratios were determined by the addition of 25 micro Meter ionomycin to Calcium²+ -free PSS that contained 2 mM ethyleneglycol-bis (beta-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), followed by replacement with normal PSS, respectively. The absolute value of [Calcium²+]ⁱwas then calculated in a separate measurement using the equation of Grynkiewicz et al. [13]The calculated [Calcium²+]ⁱin normal PSS (0%) and steady state of 80 m MK sup + -PSS (100%) were 86.6 plus/minus 9.8 and 508 plus/minus 67.3 nM (n = 10) at 37 degrees Celsius, respectively.

To examine the effects of chlorpromazine on the high Potassium sup + -, norepinephrine-, or U46619 (a thromboxane A²analogue)-induced changes in [Calcium²+]ⁱand force development, various concentrations of chlorpromazine were applied 10 min before and during the high Potassium sup + -, norepinephrine-, or U46619-induced contraction. To examine the effects of chlorpromazine on the intracellular Calcium²+ release, the strips were treated in Calcium²+ -free solution that contained 2 mM EGTA and stimulated by U46619 in the presence or absence of chlorpromazine. In the current study, we used chlorpromazine at the concentration ranging from 10 nM to 1 micro Meter, which could be regarded as the possible therapeutic concentration. [14,15] 

For each preparation of aortic membranes, 5-6 porcine thoracic aortas were obtained from a local slaughterhouse immediately after the pigs had been killed and were transported to our laboratory in ice-cold buffer composed of 0.25 M sucrose, 10 mM morpholinopropanesulfonic acid, and 0.05% bovine serum albumin (pH 7.4). The aortas were opened longitudinally, the first intima layer scraped off, and the media layer stripped from the adventitia. The microsomal fraction was prepared as described previously. [16]The trimmed media layers were finely minced and homogenized in the buffer with a Polytron PT 10 homogenizer. The homogenate was centrifuged at 900 x g for 10 min, the supernatant was centrifuged at 9,000 x g for 20 min, and then again at 108,000 x g for 45 min. The resultant pellet was resuspended in 0.25 M sucrose solution that contained 10 mM morpholinopropanesulfonic acid (pH 7.4), recentrifuged at 9,000 x g for 10 min, and the supernatant centrifuged at 178,000 x g for 45 min. The resultant pellet (microsomal fraction) was suspended in 50 mM Tris buffer (pH 7.4) for the binding study. The protein was assayed according to the method of Markwell et al., [17]using bovine serum albumin as a standard.

The dissociation constant (Kd) and the maximum binding (B max) values determined by the Scatchard analysis in microsomal fraction were 0.069 nM and 273 fmol/mg protein, respectively, as reported previously. [16]In the displacement study, 100 micro gram protein of the aortic microsome and 0.1 nM [sup 3 Hydrogen] prazosin were incubated with various concentrations of the indicated drugs at 25 degrees Celsius for 30 min in a total volume of 1 ml that contained 50 mM Tris and 5 mM MgCl²(pH 7.4), with or without 10 micro Meter phentolamine, to determine the nonspecific or total binding, respectively. Binding was terminated by adding 5 ml ice-cold buffer and filtered onto Whatman GF/C glass fiber filters, with 3 x 5 ml washes with ice-cold buffer. Specific binding was defined as the total binding minus nonspecific binding. All binding assays were carried out either in duplicate or triplicate.

All data for the simultaneous measurements of [Calcium²+]ⁱand force were collected with a computerized data acquisition system (MacLab; Analog Digital instruments, Castle Hill, Australia; and Macintosh, Apple Computer, Cupertino, CA). The data for the representative traces shown in this report were directly printed from a computer to a laser printer (LaserWriter II NTX-J, Apple Computer). The measured values were expressed as the means plus/minus SE (n = number of the experiments). For each experiment, a strip from a different animal (3-8) was used. Unpaired Student's t test was used to determine the statistical significance. Analysis of variance (ANOVA) was used to determine the concentration-dependency of effects of chlorpromazine. P values less than 0.05 were considered significant.

All data for binding study were analyzed as follows. The inhibition constant (Ki) was determined from the formula of Cheng and Prusoff, [18]Ki = IC⁵⁰/(1 + A/Kd), where A = radioligand concentration, Kd = dissociation constant, and IC⁵⁰= concentration of competitive ligand that inhibits radioligand binding by 50%. The IC sub 50 values were determined from the competition curves by the 4-parameter logistic equation of DeLean et al. [19]The data are expressed as the means plus/minus SE. The group mean values were compared using the two-tailed Student's t test.

Normal PSS was of the following composition (in mM): NaCl 123, KCl 4.7, NaHCO³15.5, KH²PO⁴1.2, MgCl²1.2, CaCl²1.25, and D-glucose 11.5. High Potassium sup + PSS was identical to normal PSS, except for an equimolar substitution of KCl for NaCl. Physiologic saline solution was bubbled with 95% Oxygen²and 5% CO², with a resulting pH of 7.4 at 37 degrees Celsius. Fura-2 AM, U46619, and EGTA were purchased from Dojindo (Kumamoto, Japan). Chlorpromazine was from Wako (Osaka, Japan). Phentolamine was a gift from Ciba-Geigy (Osaka, Japan). [sup 3 Hydrogen] prazosin (specific activity = 80.9 Ci/mmol) was purchased from New England Nuclear (Boston, MA). All other agents were purchased from Sigma Chemical (St. Louis, MO).

(Figure 1(A)) shows the representative recordings of the changes in the intensity of 500 nm fluorescence at 340 (F³⁴⁰) and 380 nm (F³⁸⁰) excitation, the ratio (F³⁴⁰/F³⁸⁰) and force induced by 80 mM Potassium sup + -PSS of the porcine pulmonary arterial strips. Depolarization with 80 mM Potassium sup + -PSS induced a rapid increase in F³⁴⁰and a decrease in F³⁸⁰as a mirror image, which ensures the specificity of fura-2 signal. After a rapid increase, the ratio ([Calcium²+]ⁱ) reached a peak, and then declined to reach a plateau level. The force also rapidly elevated to reach a plateau level. In all the fura-2 measurements, we monitored F sub 340 and F³⁸⁰routinely and confirmed that F³⁴⁰and F³⁸⁰showed a mirror image. Except in Figure 1(A), only the fluorescence ratio was illustrated to indicate [Calcium²+]ⁱ. When the strips were pretreated with various concentrations of chlorpromazine during the resting state in normal PSS, no significant changes in [Calcium²+]ⁱand force were observed (data not shown). Figure 1(B and C) show the effects of various concentrations of chlorpromazine on the increases in [Calcium²+]ⁱand force induced by 80 mM Potassium sup + -PSS. Chlorpromazine inhibited the elevation of [Calcium²+]ⁱand force induced by 80 mM Potassium sup + -depolarization in a concentration-dependent manner (P < 0.05, by two-way ANOVA). Chlorpromazine at the concentration of 1 micro Meter inhibited 80 mM Potassium sup + -induced increases in [Calcium²+] sub i and force to 60.6 plus/minus 4.6% and 66.5 plus/minus 6.2% (n = 5), respectively, while assigning the value during depolarization with 80 mM Potassium sup + PSS to be 100%. Therefore, chlorpromazine at the highest concentration examined (1 micro Meter) could only partially reduce the elevated [Calcium²+]ⁱand force induced by 80 mM Potassium sup + -depolarization. After washing out chlorpromazine with normal PSS, 80 mM Potassium sup + -PSS induced the same extent of response in [Calcium²+]ⁱand force as the control value (data not shown), which indicated that the effect of chlorpromazine is reversible.

When 1 micro Meter norepinephrine was applied in normal PSS, [Calcium²+]ⁱrapidly increased to form a peak (first phase) and then [Calcium²+]ⁱgradually declined, but remained at a level higher than the prestimulation level (second phase). The force also developed rapidly to reach a peak and then declined gradually (Figure 2(A)). The [Calcium²+]ⁱand force observed at maximum level were 106.5 plus/minus 6.8% and 116.3 plus/minus 9.6%, respectively, of those induced by 80 mM Potassium sup + -depolarization. Therefore, the maximum levels of [Calcium²+]ⁱand force observed in the contraction induced by norepinephrine were either similar or slightly higher than those induced by 80 mM Potassium sup + -depolarization. The [Calcium²+]ⁱand force observed at the second phase (10 min after the application) were 63.1 plus/minus 5.4% and 97.9 plus/minus 15.4%, respectively (n = 3). Figure 2(B and C) show the effects of various concentrations of chlorpromazine on norepinephrine-induced increases in [Calcium²+]ⁱand force in normal PSS. Chlorpromazine inhibited the elevation of [Calcium²+] sub i and force in a concentration-dependent manner (P < 0.05, by two-way ANOVA). Both the first and the second phases of the norepinephrine-induced [Calcium²+]ⁱincrease were suppressed in parallel. Differing from the case of contraction induced by 80 mM Potassium sup + -depolarization, chlorpromazine at the concentration of 1 micro Meter almost completely inhibited the increases in [Calcium²+]ⁱand force induced by norepinephrine (9.1 plus/minus 6.2% for [Calcium²+]ⁱand 11.6 plus/minus 7.9% for force; n = 3). Chlorpromazine preferentially decreased the norepinephrine-induced increases in [Calcium²+]ⁱand force, compared with those induced by 80 mM Potassium sup + -PSS (P < 0.05, by two-way ANOVA).

When 1 micro Meter U46619 was applied in normal PSS, [Calcium sup 2+]ⁱrapidly increased to form a peak (first phase), and thereafter declined, but still remained at a higher level than the prestimulation level (second phase). In the case of U46619-induced contractions, force also developed rapidly and was maintained for at least 10 min (Figure 3(A)). The [Calcium²+]ⁱobserved at maximum and steady state level were 115.7 plus/minus 8.0% and 63.8 plus/minus 11.2% of those induced by high Potassium sup + -depolarization, respectively. The force observed at the second phase, 10 min after application, was equal to that at a maximum level, which accounted for 126.3 plus/minus 6.8% of that induced by high Potassium sup + -depolarization (n = 3). Figure 3(B and C) show the effects of various concentrations of chlorpromazine on the increases in [Calcium sup 2+]ⁱand force induced by U46619 in normal PSS. Under the treatment with 1 micro Meter chlorpromazine, the [Calcium²+]ⁱobserved at maximum and steady state level were 102.6 plus/minus 18.7% and 54.3 plus/minus 5.9% of those induced by high Potassium sup + -depolarization, respectively. The force observed at steady state level was equal to that at a maximum level, which accounted for 135.1 plus/minus 1.5% of that induced by high Potassium sup + depolarization (n = 3). Chlorpromazine had no significant effect on the increase in [Calcium²+]ⁱand force induced by U46619 (by two-way ANOVA).

The effect of chlorpromazine on the intracellular Calcium²+ release mechanism was determined in particular. When the strips were incubated in Calcium²+ -free PSS that contained 2 mM EGTA, [Calcium sup 2+]ⁱgradually reduced to reach a steady state, whereas the force remained at the resting level. When 1 micro Meter U46619 was applied at the steady state in Calcium²+ -free PSS, an increased elevation in [Calcium²+]ⁱassociated with a sustained force development occurred (Figure 4(A)). When the strips were pretreated with 1 micro Meter chlorpromazine in Calcium²+ -free PSS, no significant change in [Calcium²+]ⁱor force was observed (Figure 4(B)). Chlorpromazine at the concentration of 1 micro Meter had no effect on the transient increases in [Calcium²+]ⁱor force at the concentration of 1 micro Meter induced by U46619 in the absence of extracellular Calcium²+ (Figure 4(B) and Figure 5, by unpaired Student's t test), which indicated that chlorpromazine does not inhibit the intracellular Calcium²+ release mechanism, which is presumably mediated by inositol 1,4,5 trisphosphate.

Because the data obtained so far indicated that the potent mechanism for chlorpromazine-induced vasorelaxation would be due to the alpha-adrenergic blocking action, we measured the affinity of chlorpromazine to vascular alpha-adrenergic receptors by using [sup 3 Hydrogen] prazosin binding to the porcine aortic membranes. For comparison, we also investigated the alpha-adrenergic blocking action of a typical alpha-adrenergic blocking agent, phentolamine, and other neuroleptics, including trifluoperazine (another phenothiazine derivative), haloperidol (butyrophenone derivative), and imipramine (tricyclic antidepressant). In the displacement study, as shown in Figure 6, chlorpromazine, haloperidol, phentolamine, trifluoperazine, and imipramine inhibited the specific binding of [sup 3 Hydrogen] prazosin binding to the porcine aortic membranes, in this order of potency. The slope factor and the Ki values are shown in Table 1. The slope factors of the competition curves of these compounds were near unity, which indicated that the binding was to a single population of binding sites. These data clearly showed that these neuroleptics, including chlorpromazine, bind to vascular alpha¹-adrenergic receptors.

The purpose of the current study was to clarify the mechanism for the vasorelaxing effects of chlorpromazine on vascular smooth muscle. The major findings of the current study are as follows: (1) Chlorpromazine has a calcium antagonistic action, because it decreases the [Calcium²+]ⁱand force of the contraction induced by high Potassium sup + -depolarization; (2) Chlorpromazine also has an alpha-adrenergic blocking action; (3) As a mechanism for chlorpromazine-induced vasorelaxation, the alpha-adrenergic blocking action plays a major role than calcium antagonistic action; (4) Chlorpromazine has almost no effect on U46619-induced contraction or U46619-induced intracellular Calcium²+ release, which indicates that it has a negligible effect on the intracellular signal transduction; (5) Several neuroleptics, including chlorpromazine, induce an inhibition of [sup 3 Hydrogen] prazosin binding to vascular alpha¹-adrenergic receptors.

Chlorpromazine suppressed the increases in [Calcium²+] sub i and force induced by 80 mM Potassium sup + -depolarization in a concentration-dependent manner (Figure 1(B and C)). The most plausible explanation for this observation may be that chlorpromazine inhibits the influx of extracellular Calcium²+ through a voltage-dependent Calcium²+ channel. Other possibilities, such as the activation of cyclic adenosine monophosphate or cyclic guanosine monophosphate-dependent protein kinases, the acceleration of Calcium²+ extrusion, and inhibition of intracellular Calcium²+ release mechanisms, could be ruled out, because chlorpromazine had almost no effect on U46619-induced increases in [Calcium²+]ⁱ(Figure 3(B and C)) or on U46619-induced intracellular Calcium²+ release (Figure 4and Figure 5). Consistent with this conclusion, Flaim et al. [9]reported that neuroleptic drugs, including chlorpromazine, attenuate calcium influx and tension development in rabbit aorta by measuring ^[45]Calcium influx rate. Schaeffer et al. [10]reported chlorpromazine had high affinity for [sup 3 Hydrogen]d-cis-diltiazem binding site on rat cerebral cortex membrane. Although few reports have been published that indicate the chlorpromazine-induced inhibition of the smooth muscle contraction induced by high Potassium sup + -depolarization, [9,10]our data directly showed that chlorpromazine decreases [Calcium²+]ⁱduring activation by high Potassium sup + -depolarization in intact vascular smooth muscle.

Chlorpromazine inhibited the increases in [Calcium²+] sub i and force induced by norepinephrine in a concentration-dependent manner (Figure 2(B and C)). The site of action of chlorpromazine-induced inhibition for the norepinephrine-induced contraction is located on the receptor agonist association process, but not on the intracellular signal transduction system, because chlorpromazine had little effect on the U46619-induced contraction in the presence or absence of extracellular Calcium²+ (Figure 3, Figure 4, and Figure 5). In vascular smooth muscle, norepinephrine and U46619 share much of the intracellular signal transduction systems. The conclusion obtained by this pharmacologic study was confirmed by the biochemical study (i.e., radioligand binding study). We concluded, therefore, that chlorpromazine has an alpha-adrenergic blocking action.

As to the relative importance of the calcium antagonistic action and an alpha-adrenergic blocking action for the chlorpromazine-induced vasorelaxation, the current results indicated that the alpha-adrenergic blocking action might play a major role, because the calcium antagonistic action may require a much higher concentration of chlorpromazine than the alpha-adrenergic blocking action. The therapeutic blood concentrations of chlorpromazine clinically used range from 30-350 ng/ml for adults and 40-80 ng/ml for children. [14,15]These concentrations are approximately 100 nM-1 micro Meter. Considering the fact that chlorpromazine highly binds to protein, [20]the concentrations less than 100 nM in the current study could, therefore, be possible in vivo. As shown in the results, 100 nM chlorpromazine inhibited norepinephrine-induced contraction to approximately 50% of the control (Figure 2), although it showed less of an effect in suppressing the Potassium sup + -induced contraction (Figure 3). In addition, because we used a higher concentration of norepinephrine to stimulate the effect in pulmonary arterial strips, such a higher concentration of chlorpromazine might also be required to observe its pharmacologic effects in situ. Therefore, chlorpromazine could demonstrate an ability to act as an alpha-blocking agent at relatively high concentrations within the therapeutic range. At higher concentrations, calcium antagonistic property might also play a role in the induction of hypotension in vivo. In addition, although several neuroleptics are known as calmodulin antagonists pharmacologically, the concentrations needed to inhibit calmodulin activity is more than 10 micro Meter, which is far higher than the available therapeutic concentrations. [21]Therefore, the anticalmodulin activity, if any, may have little effect on the mechanism of neuroleptics-induced vasorelaxation.

Chlorpromazine has almost no effect on U46619-induced contraction or U46619-induced intracellular Calcium²+ release, which indicates that chlorpromazine does not block TXA²receptor or intracellular signal transduction system coupled to TXA²receptor activation. This observation potentially helps to evaluate the effect of chlorpromazine in laboratory use, because chlorpromazine is known to block many kinds of receptors, including alpha-adrenergic, histamine, serotonin, and dopamine. [7]In addition, U46619 induces a sustained contraction of the porcine pulmonary artery even in the absence of the extracellular Calcium²+, although the [Calcium²+]ⁱlevel is below the resting level. The exact mechanism for this "Calcium²+ sensitization," however, has not yet been elucidated.

Although it has been well established that many neuroleptics block peripheral alpha-adrenergic receptors, [1,22]this idea is based on the observation that many neuroleptics inhibit the radioligand binding to alpha-adrenergic receptors of the brain membrane preparations. [4-8]In the current study, we showed, for the first time, that several neuroleptics induce the inhibition of [sup 3 Hydrogen] prazosin binding to vascular alpha¹-adrenergic receptors (Figure 6and Table 1). Among the several neuroleptics tested, chlorpromazine showed the highest affinity for alpha¹-adrenergic receptors, which was even higher than that of typical alpha-blocker, phentolamine. These results are in agreement with those of a previous study done in the brain membrane preparations. [7]Because one of the derivatives of butyrophenone, haloperidol, also has a high affinity for vascular alpha¹-adrenergic receptors, and is used in neurolept analgesia, [23]it, too, may cause symptomatic hypotension, as seen with phenothiazines. In addition, the alpha-adrenergic blocking action of trifluoperazine also should be taken into account, when it was used as a calmodulin antagonist in laboratory use. However, it should be noted that there was a discrepancy between the Potassiumⁱvalue obtained by the binding study and the potency of chlorpromazine-induced inhibition of contraction. This discrepancy could be explained by the differences in the affinity constants obtained by the binding study in the cell-free in vitro experiments at 25 degrees Celsius and those obtained by the in situ functional study at 37 degrees Celsius.

In summary, we examined the direct effects of chlorpromazine on vascular smooth muscle. Chlorpromazine caused vasorelaxation through an alpha-adrenergic blocking action as well as a calcium antagonistic action, and the former action is considered as having a major role in this vasorelaxation. We provided, therefore, for the first time, evidence that many neuroleptics, including chlorpromazine, strongly block vascular alpha-adrenergic receptors.

The authors thank Dr. B. T. Quinn for critical comments. They also thank K. Kajishima for secretarial services.