Picotamide inhibits a wide spectrum of agonist‐induced smooth muscle contractions in porcine renal interlobar and coronary arteries

Abstract

Picotamide is a thromboxane A₂ (TXA₂) receptor antagonist and TXA₂ synthase inhibitor. In clinical studies, it has been considered as a platelet aggregation inhibitor and improved renal function. In vitro studies suggested inhibition of smooth muscle contraction by picotamide, which is poorly understood. Here, we examined effects of picotamide on contractions of renal interlobar and coronary porcine arteries, induced by different vasoconstrictors. Contractions were induced in an organ bath by agonists or electric field stimulation (EFS). Picotamide inhibited EFS‐induced contractions of interlobar arteries around 50% using concentrations of 100 and 300 µM. In interlobar arteries, concentration response curves for contractions induced by three different α₁‐adrenoceptor agonists were shifted to the right by picotamide (2–10‐fold increases in EC₅₀). In coronary arteries, α₁‐adrenergic contractions were inhibited without right shift (approx. 50%). Contractions induced by two different cholinergic agonists in coronary arteries were inhibited by picotamide (≥50%) withouth right shift. Inhibition of serotonin‐induced contractions by picotamide showed features of a right shift, whereas contractions induced by the TXA₂ analog U46619, angiotensin‐II, and endothelin‐1 were inhibited by picotamide in interlobar and coronary arteries without right shifts and to different degree. Picotamide inhibits a wide spectrum of vasoconstrictor‐induced contractions in porcine interlobar and coronary arteries. Inhibition of vasocontraction may contribute to beneficial effects of picotamide in the cardiovascular system and kidney.

In addition to thromboxane A₂‐induced contractions, the thromboxane A₂ receptor antagonist and thromboxane A₂ synthase inhibitor picotamide inhibits contractions induced by panel of other vasoconstrictors in porcine renal interlobar and coronary arteries.

Abbreviations

DMSO

dimethylsulfoxide

EFS

electric field stimulation

TXA₂

thromboxane A₂

1. INTRODUCTION

Picotamide is known as a combined thromboxane A₂ receptor antagonist and thromboxane synthase inhibitor, which inhibits thromboxane A₂‐induced platelet aggregation. ¹ , ² It has been tested as an antiplatelet drug in clinical trials, resulting in approval as a platelet aggregation inhibitor in Italy. ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ Recent meta‐analyses have considered applications in prevention and prophylaxis of cardiovascular disease. ⁹ , ¹⁰ However, clinical and preclinical studies suggested that effects of picotamide are not limited to antiplatelet activity, or to inhibition of thromboxane A₂‐mediated functions.

In patients with type 2 diabetes, picotamide improved renal function and increased the intrarenal vascular resistance and perfusion. ³ Although inhibition of intrarenal vasocontraction rather than platelet aggregation was proposed to account for beneficial effects of picotamide on kidney function, this has never been fully explained. Preclinical studies pointed to thromboxane A₂ receptor‐independent effects, in particular in smooth muscle contraction. Thus, besides platelet aggregation, picotamide inhibited vascular, prostate, and trigone smooth muscle contractions in vitro. ¹¹ , ¹² , ¹³ In addition to an expectable, competitive inhibition of thromboxane A₂ receptor‐induced contractions, inhibition of α₁‐adrenoceptor‐ and serotonin‐induced contractions with non‐competitve features was observed in rabbit aortic rings. ¹³ In rat aortic rings, picotamide inhibited acetylcholine‐induced contractions. ¹⁴ Similarly, effects on contractions on other agonists than thromboxane A₂ have been reported from lower urinary tract smooth muscle. ¹¹ , ¹² Application of picotamide to human tissues suggested inhibition of α₁‐adrenergic, neurogenic and endothelin‐induced smooth contractions in the prostate, and of cholinergic, α₁‐adrenergic and neurogenic smooth muscle contractions in the bladder trigone, besides inhibition of thromboxane A₂‐induced contractions. ¹¹ , ¹²

Together, properties and actions of picotamide are obviously still incompletely understood, despite its applications in clinical studies and as an antiplatelet drug. Acknowledging the importance of smooth muscle contraction for cardiovascular hemoostasis and diseases, but also considering potential applications in treatment of lower urinary tract symptoms, where cardiovascular side effects may be critical, ¹⁵ adequate understanding of vascular picotamide effects may be clinically relevant. Evidence for actions of picotamide on vascular smooth muscle contractions other than thromboxane A₂ induced is to the best of our knowledge limited to phenylephrine‐, serotonin‐, and acetylcholine‐induced contractions in rabbit and rat aortic rings, ¹³ , ¹⁴ and did not include other vessel types, other species, or neurogenic contractions. Apart from thromboxane A₂, α₁‐adrenoceptors, and serotonin, the spectrum of vasoconstrictors inducing receptor‐dependent contractions of vascular smooth muscle includes angiotensin‐II and endothelin‐1, besides others. To obtain a broader understanding of vascular picotamide effects, we here examined the effects of picotamide on neurogenic and agonist‐induced vascular smooth muscle contractions of porcine interlobar kidney and coronary arteries.

2. MATERIALS AND METHODS

2.1. Pig arteries

Kidneys and hearts from pigs were obtained from a local slaughterhouse, where animals were killed during night. Subsequently, organs were picked up by a local butcher, transported and stored at 4°C, and transferred from the butcher's shop (Metzgerei Brehm, Planegg, Germany) to the laboratory in the morning. Here, interlobar arteries were prepared from kidneys, and middle sections of left anterior descending arteries were prepared from hearts. Preparation was started approximately 15 min following pickup of organs from the butcher. Following preparation and dissection of adipose and connective tissues, vessels were cut into rings, transferred to Custodiol^® solution (Köhler, Bensheim, Germany), and stored at 4°C in Custodiol solution until use. Experiments were started not later than three hours following preparation of vessels.

2.2. Organ bath studies

Vessel rings (i.e., segments cutted from vessels) were mounted in tissue baths with four chambers (Danish Myotechnology, Aahus, Denmark). Rings had a length of 2–3 mm, with diameters ranged between 3 and 4 mm for renal interlobar arteries, and around 5 mm for coronary arteries. Each chamber contained 10 ml aerated (95% O₂ and 5% CO₂) Krebs–Henseleit solution (37°C, pH 7.4). For pretension, rings of interlobar arteries were stretched to 9.8 mN, whereas rings of left anterior descending arteries were stretched to 19.6 mN. In the initial phase of the equilibration period, spontaneous decreases in tone are usually observed. Therefore, tension was adjusted three times during the equilibration period, until a stable resting tone of 9.8 or 19.6 mN, respectively, was attained. After the equilibration period, maximum contraction induced by 80 mM KCl was assessed. As soon as a plateau contraction induced by KCl was obtained, chambers were washed three times with Krebs–Henseleit solution for a total of 30 min, and picotamide or an equivalent amount of dimethylsulfoxid (DMSO, used as solvent for picotamide) was added for controls. Cumulative concentration response curves for agonists, or frequency response curves for electric field stimulation (EFS), were constructed 30 min after addition of picotamide or DMSO. Application of EFS simulates action potentials, resulting in the release of endogenous neurotransmitters. Picotamide was examined in final concentrations of 30, 100, and 300 µM in experiments with EFS‐induced contractions, and 300 µM in experiments with agonist‐induced contractions. A stock solution of 300 mM was applied to organ bath chambers, so that final concentrations of DMSO amounted to 0.1‰, 0.33‰, and 1‰ for in EFS experiments, and 1‰ for in agonist experiments.

In each experiment, all four chambers of one organ bath were filled with rings from the same vessel, and only one concentration or frequency response curve was recorded with each chamber. From these four channels, two were examined with picotamide, and two with DMSO, with changing allocations of picotamide and DMSO channels between different experiments. Each of these independent experiments was repeated in indicated numbers (n), with arteries from n different animals, resulting indicated numbers of experiments in each series. Consequently, control and picotamide groups in each series (i.e., each diagram in figures) were obtained using the same vessels, and single experiments were based on double determinations.

For calculation of agonist‐ and EFS‐induced contractions, tensions were expressed as percentage of 80 mM KCl‐induced contractions, to correct for inter‐strip variabilities. In fact, normalization to KCl allows to examine possible alterations of receptor responsiveness, whereas correlations between agonist‐induced force and ring weight, length, or cross‐sectional area are weak or lacking in organ bath experiments using vessel segements. ¹⁶

E_max values, EC₅₀ values for contractile agonists, and frequencies (f) inducing 50% of the maximum EFS‐induced contraction (Ef₅₀) were calculated by curve fitting using GraphPad Prism 6 (Statcon, Witzenhausen, Germany), and analyzed as described below. As presentation of single values in scatter plots was intended, automatic curve fitting was performed separately for each single experiment, to obtain single values for each independent experiment. Sigmoidal concentration response curves were fitted by non‐linear regression, without predefined constraints for bottom, top or EC₅₀ values, by ordinary fit, without weighting, and without chosing automatic outlier elimination. However, and as recommended in the “GraphPad Curve Fitting Guide“ (GraphPad Software Inc., San Diego, CA, USA), resulting values were checked for plausibility, and settings were adapted as follows if error messages occured. Thus, curve fitting of concentration response curves for serotonin was performed using sigmoidal parts of curves, i.e. including the concentration range from 0.1 to 100 µM (0.1 to 10 µM in one case), but excluding steady‐state or downhill portions at high concentrations, as fitting with all concentrations (up to 1 mM) resulted in error messages („not converted“) in 3 out of 6 experiments. Similarly, the concentration range from 30 to 100 µM was excluded in one carbachol experiment, as maximum conctractions occured at low concentrations in this experiment, resulting in otherwise non‐plausible results. Results from curve fitting were flagged as “ambigous” in one methoxamine experiment in renal arteries, so that curve fitting was performed using the concentration range of 0.1–10 µM. Finally, and despite these procedures, one outlier occured, which was handled as described in Section 2.4.

2.3. Materials, drugs, and nomenclature

Picotamide [4‐methoxy‐N,N′‐bis(pyridin‐3‐ylmethyl)isophtalamide] is a combined antagonist of the thromboxane A₂ receptor and inhibitor of thromboxane A₂ synthase. ¹⁷ Stock solutions (300 mM) were prepared with DMSO and kept at −20°C until use. The applied concentrations of picotamide were based on previous studies performed with prostate and platelets. ¹ , ¹¹ , ¹² , ¹⁸ U46619 ((Z)‐7‐[(1S,4R,5R,6S)‐5‐[(E,3S)‐3‐hydroxyoct‐1‐enyl]‐3‐oxabicyclo[2.2.1]heptan‐6‐yl]hept‐5‐enoic acid) is an agonist of the TXA₂ receptor, ¹⁹ and was dissolved in ethanol. As thromboxane A₂ is highly unstable, U46619 is commonly used as a thromboxane A₂ receptor agonist. ²⁰ , ²¹ Stock solutions (10 mM) were stored at −80°C until use. Phenylephrine ((R)‐3‐[−1‐hydroxy‐2‐(methylamino)ethyl]phenol) and methoxamine (α‐(1‐Aminoethyl)‐2,5‐dimethoxybenzyl alcohol) are α₁‐selective adrenoceptor agonists. ¹⁹ Carbachol (carbamoylcholin) and methacholine are muscarinic acetylcholine receptor agonists. ¹⁹ , ²² Adenosine‐5'‐triphosphate (ATP) is the endogenous ligand of purinergic P2X receptors, whereas α,β‐methylene‐adenosine 5'‐triphosphate (α,β‐methylene‐ATP) is a full P2X1 and P2X3 agonist, but may also cause rapid desensitization and consequent inhibition of P2X1‐ and P2X3‐mediated effects. ²³ Aqueous stock solutions (10 mM) of noradrenaline, phenylephrine, methoxamine, carbachol, methacholine, and serotonin (5‐hydroxytryptamine) were freshly prepared before each experiment. ATP (500 mM) were freshly perpared before each experiment and stored on ice until application to organ bath chambers. Stock solutions (10 mM) of α,β‐methylene‐ATP were prepared with water and stored stored at −20°C until used and before further dilution with water. Aqueous stock solutions of endothelin‐1 (0.75 mM), angiotensin‐II (1 mM), and [lys⁸]‐vasopressin (500 µM) were stored at −20°C as small aliquots, so that repeating freezing and thawing cycles were avoided. Picotamide, U46619, and endothelin‐1 were obtained from Enzo Life Sciences (Lörrach, Germany). Noradrenaline, phenylephrine, methoxamine, carbachol, methacholine, serotonin, and ATP disodium salt were obtained from Sigma‐Aldrich (Munich, Germany). α,β‐Methylene‐ATP and vasopressin were obtained from Tocris (Bristol, UK).

2.4. Data and statistical analyses

Data in concentration and frequency response curves are presented as means ± standard deviation (SD), with the indicated number (n) of independent experiments. Tissue from one animal was used for one independent experiment, so that the indicated numbers of experiments also reflect the number of animals, on which a given series is built up. For presentation of E_max and EC₅₀ values, single values from all experiments are shown in scatter plots. Maximum contractions are reported as means with 95% confidence intervals (CIs). Effect sizes become obvious from concentration and frequency response curves. Nevertheless, some exemplary values are additionally reported as mean differences (MD) with 95% CI, e.g. for inhibitions at maximum contractions or for maximum effects. One‐way analysis of variance (ANOVA) was used for comparison of whole concentration/frequency response curves, and two‐way ANOVA was used for comparison of contractions at single concentrations or frequencies. For comparison of E_max and EC₅₀ values, a paired Student's t‐test was applied. Tests for concentration response curves and calculation of MD with 95% CI were performed using the SPSS^® version 20 (IBM SPSS Statistics, IBM Corporation). Tests for paired comparison of E_max and EC₅₀ values were performed using GraphPad Prism 6. p < .05 were considered significant. However, the present study and analyses were designed to be exploratory, but not designed to test a pre‐specified statistical null hypothesis. ²⁴ Apart from a lacking hypothesis, recently defined features imparting the character of hypothesis‐testing study design were lacking in our study, including a clear preset study plan, blinding, or biometric calculation of groups sizes. ²⁴ Therefore, p values reported here should be considered as descriptive and not as hypothesis‐testing. ²⁴ In line with recent recommendations, p values were used sparingly, and the focus in our study is on effect sizes. ²⁴

According to recent guidelines for analyzing and reporting data, procedures and statistical methods, our procedures are described in detail as follows. ²⁴ As a calculation of (descriptive) p values was intended, the minimum number of experiments and group sizes in organ bath experiments was pre‐planned as n = 5/group for each series. Data were analyzed, after at least five experiments of a series were performed. Following this analysis, series were discontinued if it became obvious that no effect could be expected on this data basis, or if descriptive p < .05 were obtained in concentration/frequency response curves (at single frequencies/agonist concentrations, and/or between whole groups). If these initial results appeared ambigous, i.e. did not reveal p < .05, but suggested that an effect could be expected, series were continued and analyzed again. This procedure was possible due to the explorative character of this study, and as it is reported here in detail. ²⁴ In fact, flexible group sizes have been recommended by guidelines for experimental design and analysis in experimental pharmacology, if data are characterized by large variations, what applies here. ²⁵ , ²⁶ However, interim analyses were limited to concentration and frequency response curves and did not include E_max and EC₅₀ values, which were calculated by curve fitting following completion of series. According to the paired design (allocation of samples from each tissue to the control and inhibitor groups), groups being compared with each other by statistical tests showed identical group sizes. No data or experiments were excluded from analyses, apart from one value in a series addressing effects of DMSO and picotamide on phenylephrine‐induced contractions of interlobar arteries. Thus, in the control group of one single experiment of this series, curve fitting revealed an EC₅₀ value ranging >1500‐fold higher than the highest of all other values in this group. Consequently, this value was regarded as an obvious outlier and excluded from diagrams and calculations, what is again mentioned in the legend to figures.

2.5. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in https://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, ²⁷ and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. ¹⁹ , ²³ , ²⁸

3. RESULTS

3.1. Maximum contractions

Effects of picotamide were examined on contractions induced by the thromboxane A₂ analog U46619, EFS, by three different α₁‐adrenoceptor agonists (noradrenaline, phenylephrine, methoxamine), two cholinergic agonists (methacholine, carbachol), serotonin, the purinerigic agonists ATP and α,β‐methylene‐ATP, angiotensin‐II, endothelin‐1, and vasopressin. EFS induced frequency‐dependent contractions in interlobar arteries (Figure 1A), but not in coronary arteries (data not shown). All three α₁‐adrenergic agonists induced concentration‐dependent contractions in interlobar arteries, whereas only phenylephrine and methoxamine, but not noradrenaline induced contractions in coronary arteries (Figure 1A). Both cholinergic agonists induced concentration‐dependent contractions in coronary arteries (Figure 1A), but were not applied to interlobar arteries. Both purinergic agonists induced contractions in interlobar, but not in coronary arteries (Figure 1A). Vasopressin induced no obvious contractions in both vessel types (Figure 1A). The remaining agonists induced contractions in both vessel types (Figure 1A), which were concentration‐dependent for serotonin and angiotensin‐II, but not for endothelin‐1 in the applied concentration range.

FIGURE 1.

Maximum and highmolar KCl‐induced contractions of porcine renal interlobar and coronary arteries. In (A), values are maximum contractions following application of EFS and agonists as indicated in control groups, regardless at which frequency or agonist concentration they occured (for concentrations, see Table 1). In (B), contractions induced by highmolar KCl are expressed in mN. EFS‐ and agonist‐induced contractions in all other diagrams have been calculated as percentage of contractions induced by 80 mM KCl. KCl‐induced contractions were assessed before application of DMSO (in controls) or picotamide. Thus, contractions shown for controls and picotamide were in fact assessed under the same conditions, i.e. withouth DMSO or picotamide, but here shown separately to demonstrate that overall contractions were similar in picotamide and control groups. Shown are all single values from all experiments performed for frequency response curves and concentration response curves in Figures 2, 3, 4, 5 together with means (=bars). Contractions were obtained under the presence of DMSO (controls, solvent for picotamide). As three concentrations of picotamide were examined for EFS‐induced contractions, final concentrations of DMSO in organ bath chambers amounted to 0.1‰, 0.33‰, and 1 ‰ for EFS, and 1‰ in experiments with agonists.

In interlobar arteries, strongest contractions were observed for α₁‐adrenergic agonists and endothelin‐1, all ranging roughly around threefold of EFS‐induced contractions (Figure 1A). Serotonin‐ and U46619‐induced contractions in interlobar arteries were in similar ranges as EFS‐induced contractions (Figure 1A). Purinergic and angiotensin‐II induced the lowest contractions, which were in similar ranges (Figure 1A). Vasopressin induced no contractions (Figure 1A). In coronary arteries, contractions were strongest following application of U46619 or endothelin‐1 (Figure 1A). α₁‐Adrenergic contractions in coronary arteries were lower compared with interlobar arteries, whereas serotonin‐ and angiotensin‐II‐induced contractions were comparable between both vessel types (Figure 1A). Cholinergic contractions exceeded α₁‐adrenergic contractions in coronary arteries (Figure 1A). Maximum contractions in control groups (shown in Figure 1A) occured at different frequencies and agonist concentrations (Table 1).

TABLE 1.

Distribution of maximum EFS‐ and agonist‐induced contractions in porcine renal interlobar and coronary arteries accross frequencies (EFS) and agonist‐concentrations. Indicated are concentrations and frequencies inducing the maximum response in construction of concentration and frequence response curves, in at least one independent experiment, together with the number of experiments in which this concentration/frequence induced the maximum response and with the total number of experiments in each series.

	Renal interlobar	Coronary
U46619	Total: n = 5 1 µM: n = 1 3 µM: n = 5	Total: n = 5 100 nM: n = 1 300 nM: n = 2 10 µM: n = 1 30 µM: n = 1
EFS	Total: n = 16 16 Hz: n = 2 32 Hz: n = 3 64 Hz: n = 11	—
Noradrenaline	Total: n = 5 10 µM: n = 5	—
Phenylephrine	Total: n = 6 3 µM: n = 1 10 µM: n = 1 30 µM: n = 4	Total: n = 6 1 µM: n = 2 10 µM: n = 2 100 µM: n = 2
Methoxamine	Total: n = 5 10 µM: n = 3 100 µM: n = 1 300 µM: n = 1	Total: n = 5 100 µM: n = 1 300 µM: n = 1 1 mM: n = 3
Methacholine	—	Total: n = 5 1 µM: n = 1 3 µM: n = 3 10 µM: n = 1
Carbachol	—	Total: n = 5 1 µM: n = 1 10 µM: n = 2 30 µM: n = 1 100 µM: n = 1
Serotonin	Total: n = 6 1 µM: n = 3 3 µM: n = 2 10 µM: n = 10	Total: n = 5 3 µM: n = 1 10 µM: n = 1 30 µM: n = 3
α,β‐methylene‐ATP	Total: n = 5 3 µM: n = 1 10 µM: n = 1 30 µM: n = 3	Total: n = 5 100 nM: n = 1 10 µM: n = 4
ATP	Total: n = 5 0.3 µM: n = 1 3 µM: n = 2 30 µM: n = 2	—
Angiotensin‐II	Total: n = 6 10 nM: n = 1 100 nM: n = 5	Total: n = 5 100 nM: n = 2 1 µM: n = 3
Endothelin−1	Total: n = 10 10 nM: n = 1 30 nM: n = 6 300 nM: n = 3	Total: n = 8 30 nM: n = 1 100 nM: n = 3 300 nM: n = 1 1 µM: n = 1 3 µM: n = 2

On average, contractions induced by 80 mM KCl amounted to 13.6 mN [12.0 to 15.1] in renal interlobar arteries, and to 28.8 mN [25.0 to 32.5] in coronary arteries. KCl‐induced contractions were assessed before application of DMSO (in controls) or picotamide, and did not differ between groups allocated subsequently to control and picotamide groups (Figure 1B).

3.2. U46619‐induced contractions

U46619‐induced contractions were inhibited by picotamide (300 µM) in interlobar and coronary arteries, without right shifts and without clearly increased EC₅₀ values (p < .002 for controls vs. picotamide in renal interlobar arteries; p<0.002 in coronary arteries) (Figure 2A,B). Exemplarily, submaximum contractions induced by 100 nM U46619 were inhibited by 28% [9.7 to 25.5] in interlobar arteries, and by 24% [−16.7 to 63.9] in coronary arteries. E_max values for U46619 calculated by curve fitting of concentration response were reduced by trend by picotamide, whereas EC₅₀ values of U46619 were not clearly changed (Figure 2A,B).

FIGURE 2.

Effects of picotamide on U46619‐, EFS‐, and noradrenaline‐induced contractions in renal interlobar (A, C–F) and coronary (B) arteries. Contractions were induced by cumulative concentrations of U46619, different frequencies of EFS, or by cumulative concentrations of noradrenaline in interlobar arteries, 30 min following administration of picotamide or solvent (controls). Shown are means ± SD in frequency response curves (#p < .05 for control vs. picotamide by two‐way ANOVA, and p values for whole groups from one‐way ANOVA in inserts), and all single E_max values and frequencies inducing 50% of the maximum EFS‐induced contraction (Ef₅₀) or EC₅₀ values for noradrenaline from all experiments (calculated by curve fitting) in scatter plots (p value from paired Student's t‐test), from experiments using tissues from n = 5 animals in A, B and D‐F, and n = 6 animals in C. For each single experiment, tissue from one animal was allocated to the control and the picotamide group, which were examined in the same experiment.

3.3. EFS‐induced contractions

Effects of picotamide on EFS‐induced contractions of interlobar arteries were examined in three independent series, where three different concentrations of picotamide and corresponding amounts of solvent were applied. EFS‐induced contractions were inhibited by 100 and 300 µM of picotamide (Figure 2C,D). Inhibitions occurred at different frequencies, and were confirmed by comparisons of whole frequency response curves to controls (p < .02 for 100 µM picotamide, p < .005 for 300 µM picotamide) (Figure 2C,D). Exemplarily, average maximum contractions in frequency response curves were inhibited by 32% [−4.6 to 68.3] at 64 Hz by 100 µM picotamide, and by 48% [7.8 to 88.1] at 16 Hz by 300 µM picotamide. E_max values calculated by curve fitting were reduced by trend, whereas frequencies inducing 50% of the maximum EFS‐induced contraction were not affected (Figure 2C,D): 30 µM picotamide did not change EFS‐induced contractions (Figure 2E).

3.4. α₁‐Adrenergic contractions

In interlobar arteries, picotamide (300 µM) shifted concentration response curves for noradrenaline to the right, so that contractions were reduced at submaximal concentrations for noradrenaline, but recovered at high noradrenaline concentrations (Figure 2F). Accordingly, picotamide increased EC₅₀ values for noradrenaline from 0.5 µM [−0.006 to 0.952] in controls to 1.9 µM [0.7 to 3.1] (MD 1.44 µM [−0.366 to 2.518], whereas E_max values calculated from curve fitting remained similar (Figure 2F).

In interlobar arteries, picotamide (300 µM) shifted concentration response curves for phenylephrine and methoxamine to the right, so that contractions were reduced at submaximal agonist concentrations, but recovered at high agonist concentrations (Figure 3A,B). Accordingly, picotamide increased EC₅₀ values for phenylephrine from 0.4 µM [0.1 to 0.7] in controls to 3.1 µM [1.2 to 5] (MD 2.7 [0.9 to 4.6]), and EC₅₀ values for methoxamine from 1.1 µM [−0.5 to 2.6] to 9.0 µM [2.3 to 15.8] (MD 8.0 [2.2 to 13.7]) (Figure 3A,B). E_max values of both agonists were not affected by picotamide (Figure 3A,B).

FIGURE 3.

Effects of picotamide on α₁‐adrenergic and cholinergic contractions in renal interlobar and coronary arteries. Contractions were induced by cumulative concentrations of α₁‐adrenergic agonists (phenylephrine, methoxamine) or cholinergic agonists (methacholine, carbachol) in interlobar arteris (A, B) or coronary arteris (C‐F), 30 min following administration of picotamide (300 µM) or solvent (controls). Shown are means ±SD in concentration response curves (# p < .05 for control vs. picotamide by two‐way ANOVA, and p values for whole groups from one‐way ANOVA in inserts), and all single E_max and EC₅₀ values for single experiments (calculated by curve fitting) in scatter plots (p value from paired Student's t‐test), from experiments using tissues from n = 5 animals in B, D, E and F, and n=6 animals in A and C. For each single experiment, tissue from one animal was allocated to the control and the picotamide group, which were examined in the same experiment. In one of six experiments performed for the series in (A), curve fitting revealed an EC₅₀ value of 756 µM for phenylephrine in the control group, which has been omitted from the diagram and calculations. For each single experiment, tissue from one animal was allocated to the control and the picotamide group, which were examined in the same experiment.

In coronary arteries, picotamide inhibited phenylephrine‐ and methoxamine‐induced contractions, without consistent changes of EC₅₀ values and without recovery in the examined concentration range (phenylephrine: p < .05 for controls vs. picotamide; methoxamine: p < .01) (Figure 3C,D). Exemplarily, average maximum phenylephrine‐induced contractions (at 1 mM phenylephrine) were inhibited by 58.3% [−25.6 to 97.6], and average maximum methoxamine‐induced contractions (at 300 µM methoxamine) by 46.1% [22.8 to 69.5] by picotamide (Figure 3C,D). In contrast to interlobar arteries, no rightshifts and no increases of EC₅₀ values by picotamide were observed for phenylephrine or methoxamine in coronary arteries (Figure 3C,D). Noradrenaline did not induce contractions of coronary arteries (data not shown).

3.5. Cholinergic contractions

In coronary arteries, picotamide (300 µM) inhibited contractions induced by methacholine and carbachol, without consistent changes of EC₅₀ values and without recovery in the examined concentration range (methacholine: p < .001 for controls vs. picotamide; carbachol: p < .006) (Figure 3E,F). Average maximum methacholine‐induced contractions (at 3 µM methacholine) were inhibited by 82% [76.9 to 87.6], and average maximum carbachol‐induced contractions (at 10 µM carbachol) by 70% [46.7 to 92.6] by picotamide (Figure 3E,F). E_max values calculated from frequency response curves were reduced by trend (Figure 3E,F).

3.6. Serotonin‐induced contractions

Serotonin‐induced contractions were inhibited by picotamide (300 µM) in interlobar and coronary arteries (p < .03 for controls vs. picotamide in renal interlobar arteries; p < .001 in coronary arteries) (Figure 4A,B). Average maximum contractions occured at 3 µM serotonin in interlobar arteries, which were inhibited by 72% [41.8 to 103] in interlobar arteries, and at 30 µM in coronary arteries, which were inhibited by 52% [−3.2 to 107.7]. E_max values for serotonin calculated by curve fitting of concentration response were reduced by trend by picotamide (Figure 4A,B). EC₅₀ values for serotonin were increased by picotamide from 0.248 µM [−0.196 to 0.692] in controls to 10.9 µM [−9.6 to 31.3] (MD 10.6 [−7.2 to 28.4]) in interlobar arteries, and from 0.605 µM [−0.422 to 1.633] to 9.584 µM [3.285 to 15.88] (MD 8.98 µM [3.677 to 14.28]) in coronary arteries (Figure 4A,B).

FIGURE 4.

Effects of picotamide on serotonin‐induced and purinergic contractions in renal interlobar and coronary arteries. Contractions were induced by cumulative concentrations of serotonin (A, B) and α,β‐methylene‐ATP and ATP (C, D) in interlobar arteries (A, C) or coronary arteries (B, D), 30 min following administration of picotamide (300 µM) or solvent (controls) (with the exception of (D), where not picotamide was included in experiments with α,β‐methylene‐ATP). Shown are means ±SD in concentration response curves (#p < .05 for control vs. picotamide by two‐way ANOVA, and p values for whole groups from one‐way ANOVA in inserts), and (with one exception) all single E_max and EC₅₀ values for single experiments (calculated by curve fitting) in scatter plots (p value from paired Student's t‐test), from experiments using tissues from n = 5 animals in B‐D, and n = 6 animals in A.

3.7. Purinergic contractions

α,β‐Methylene‐induced contractions were inhibited by picotamide (300 µM) in interlobar arteries (p < .03 for controls vs. picotamide), at the two highest applied concentrations of α,β‐methylene‐ATP (Figure 4C). Thus, contractions induced by 10 µM α,β‐methylene‐ATP were inhibited by 65% [13 to 116], whereas contractions induced by 30 µM were inhibited by 22% [−94 to 138]. ATP‐induced contractions were weak and hardly lower in the presence of picotamide, althouth statistical testing suggested an inhibition (p < .04) (Figure 4C). Calculation of conclusive E_max and EC₅₀ values was not possible, as curve fitting was not possible for several single experiments using α,β‐methylene‐ATP, and a sigmoidal character was not given, and as clear contraction–concentration relationships were lacking for ATP (Figure 4D). No contractions were observed in response to both agonists in coronary arteries (Figure 4D). ATP even induced relaxations of basal tone in coronary arteries, which was not changed by picotamide (Figure 4D).

3.8. Angiotensin‐II‐induced contrations

Angiotensin‐II‐induced contractions were inhibited by picotamide (300 µM) in interlobar and coronary arteries (p < .008 for controls vs. picotamide in renal interlobar arteries; p < .04 in coronary arteries) (Figure 5A,B). Average maximum contractions occured at 100 nM angiotensin‐II in interlobar arteries, which were inhibited by 42% [0.4 to 82.9] in interlobar arteries, and at 1 µM in coronary arteries, which were inhibited by 60% [38.3 to 80.8]. E_max values for angiotensin‐II calculated by curve fitting of concentration response were reduced by trend by picotamide (Figure 5A,B). EC₅₀ values for angiotensin‐II were not clearly affected by picotamide (Figure 5A,B).

FIGURE 5.

Effects of picotamide on angiotensin‐II‐, endothelin‐1‐, and vasopressin‐induced contraction in renal interlobar and coronary arteries. Contractions were induced by cumulative concentrations of angiotensin‐II (A, B), endothelin‐1 (C, D), or vasopressin (E, F) in interlobar arteries (A, C, E) or coronary arteries (B, D, F), 30 min following administration of picotamide (300 µM) or solvent (controls), with the exception of (E). Shown are means ± SD in concentration response curves (#p < .05 for control vs. picotamide by two‐way ANOVA, and p values for whole groups from one‐way ANOVA in inserts), and all single E_max and EC₅₀ values for single experiments (calculated by curve fitting) in scatter plots (p value from paired Student's t‐test), from experiments using tissues from n = 5 animals in B, E, and F, n = 6 animals in A, n = 8 animals in D, and n = 10 animals in C. For each single experiment, tissue from one animal was allocated to the control and the picotamide group, which were examined in the same experiment.

3.9. Endothelin‐1‐induced contractions

Endothelin‐1‐induced contractions were inhibited by picotamide (300 µM) in interlobar and coronary arteries (p < .008 for controls vs. picotamide in renal interlobar arteries; p < .002 in coronary arteries) (Figure 5C,D). Due to lacking concentration‐dependent relationships of endothelin‐1‐induced contractions, curve fitting was not possible (Figure 5C,D). Exemplarily, contractions induced by 1 µM endothelin‐1 were inhibited by 24% [10.6 to 37.9] by picotamide in interlobar arteries, and by 44% [25.3 to 62.2] in coronary arteries.

3.10. Vasopressin‐induced contractions

Vasopressin did not induce contractions in interlobar and coronary arteries (Figure 5E‐F). Consequently, results obtained under the presence of picotamide in interlobar arteries are inconclusive (Figure 5E), and no curves with picotamide were recorded in coronary arteries (Figure 5F).

4. DISCUSSION

Previous studies addressing inhibition of smooth muscle contraction suggested effects of the combined thromboxane A₂ receptor antagonist and thromboxane A₂ synthase inhibitor picotamide on smooth muscle contractions, induced by agonists other than thromboxane A₂. Besides thromboxane A₂‐induced contractions, pictoamide inhibited α₁‐adrenergic, cholinergic, and serotonin‐induced contractions of rodent aortic tissues. ¹³ , ¹⁴ Data for other vasoconstrictors, other vessel types, or other species were to the best of knowledge not available. Here, we examined effects of pictoamide on a broad panel of vasoconstrictors and on neurogenic contractions in renal interlobar arteries, and in coronary arteries. We observed that picotamide inhibited contractions induced by a wide spectrum of vasoconstrictors and by neurogenic stimulation in both vessel types. The pattern of inhibition differed between vasoconstrictors and vessel types (Table 2).

TABLE 2.

Effects of picotamide on agonist‐ and EFS‐induced contractions of porcine renal interlobar and coronary arteries, summary and simplification of present finding

	Renal interlobar	Coronary
U46619	Reduced contraction in concentration response curves. EC50 and Emax unchanged	Reduced contraction in concentration response curves. EC50 and Emax unchanged
EFS	Inhibited by 100 and 300 µM picotamide, but not 30 µM	—
Noradrenaline	Right shift of concentration response curve. EC50 increased, Emax unchanged	—
Phenylephrine	Right shift of concentration response curve. EC50 increased, Emax unchanged	Reduced contraction in concentration response curves. EC50 not increased
Methoxamine	Right shift of concentration response curve. EC50 increased, Emax unchanged	Reduced contraction in concentration response curves. EC50 not clearly increased
Methacholine	—	Reduced contraction in concentration response curves. EC50 increased, Emax reduced
Carbachol	—	Reduced contraction in concentration response curves. Emax slighly reduced
Serotonin	Reduced contraction in concentration response curves, possible right shift. EC50 increased	Reduced contraction in concentration response curves, possible right shift. EC50 increased
α,β‐methylene‐ATP	Reduced contractions at high agonist concentrations. No curve fitting	Contractions very low, <10% of KCl in control group
ATP	Contractions very low, <10% of KCl in control group	—
Angiotensin‐II	Reduced contractions at high agonist concentrations. EC50 not clearly changed	Reduced contractions at high agonist concentrations. EC50 not clearly changed
Endothelin‐1	Reduced contraction in concentration response curves. No curve fitting	Reduced contraction in concentration response curves. No curve fitting

Regarding the inhibition of U46619‐induced contractions, the lacking right shift may be surprising. As picotamide has been originally described as a thromboxane A₂ receptor antagonist, a corresponding inhibition pattern should be expected, including right shifts of concentration response curves and increases of EC₅₀ values for U46619, as described for rabbit aortic rings. ¹³ On the other hand, inhibition of U46619‐induced contractions without patterns of competitive agonists has been reported from human trigone tissues, and partially from human prostate tissues. ¹¹ , ¹² It has been suggested, that features of competitve antagonism may be covered by non‐competitive antagonism, if both occur together. In fact, non‐competitive inhibition of vasoconstrictor effects, and irreversible and insurmountable antagonism of thromboxane A₂ receptors by picotamide have been previously proposed, in addition to competitive antagonism. ¹⁸ , ²⁹ Such indisplaceable binding may result from stable interactions between picotamide and thromboxane A₂ receptors, what was observed in platelets following incubation periods exceeding 20 min. ¹⁸ , ²⁹ Irreversible, insurmountable antagonism, occuring alone or together with competitive antagonism, may be characterized by reduced E_max values, and was supposed to account for effects of picotamide on receptors other than thromboxane A₂ receptors. ¹² , ³⁰ , ³¹ Insurmountable antagonism may have occurred in our experiments, as we applied picotamide for 30 min. However, certain identification of true competitive and non‐competitive antagonism requires extended experimental settings and is not possible on the basis of our data. ³⁰ , ³¹ , ³²

In interlobar arteries, picotamide shifted concentration response curves for contractions induced by α₁‐adrenergic agonists to the right, resulting in increased EC₅₀ values for agonists without changes in maximum contractions. This pattern was confirmed using three different α₁‐adrenergic agonists. Although these are features of competitive antagonism, our data are not fully conclusive to this regard. Binding studies were not performed here, but would be required to confirm competitive antagonism of α₁‐adrenoceptors by picotamide. In coronary arteries, phenylephrine‐ and methoxamine‐induced contractions were inhibited by picotamide as well, but without features of competitive antagonism. Partially, this is in line with previous findings suggesting inhibition of α₁‐adrenergic contractions by picotamide without right shift in rabbit aortic rings. ¹³ In addition to vasocontraction, inhibition of α₁‐adrenergic smooth muscle contraction by picotamide has been reported from human prostate and trigone tissues. ¹² However, these studies did not include agonist concentrations higher than 100 µM, which may be required to observe recovery and curve shifts in competitive antagonism.

Apart from thromboxane A₂ and α₁‐adrenoceptors, vascular smooth muscle contraction can be induced by serotonin, angiotensin‐II, and endothelin‐1. All of these contractions were inhibited by picotamide, although to different extent and by divergent patterns (Table 2). Our data for serotonin are characterized by high variations of contraction levels between experiments using interlobar arteries, and are limited by the applied concentration range in coronary arteries. With all due caution, a right shift may be assumed considering our concentration response curves for serotonin. Regarding speculations about a competitive antagonism, the same limitations as for α₁‐adrenoceptor‐agonists explained above apply here again. For endothelin‐1, a clear relationship between concentration and contraction was lacking in our applied concentration range. In line with our current findings, inhibition of endothelin‐induced contractions has been reported from human trigone tissues. ¹²

Although vasorelaxation is the predominant outcome of cholinergic regulation in blood vessels, cholinergic vasocontraction occurs in vessels with defective or lacking endothelium and has been repeatedly reported from coronary arteries. We induced cholinergic contractions of coronary arteries using methacholine and carbachol, which were both inhibited by picotamide. In isolated aortic rings from spontaneously hypertensive rats, where the release of nitric oxide was inhibited using a nitric oxide inhibitor, acetylcholine‐induced contractions were inhibited by picotamide. ¹⁴

Picotamide inhibited EFS‐induced contractions using concentrations of 100 and 300 µM, but not 30 µM. Previous studies reported varying K_D, IC_50, and EC₅₀ values of picotamide, which were obtained using different models. It has been proposed, that picotamide binding to receptors stabilizes over time, resulting in turnover from displaceable to non‐displaceable binding, and in a high range of K_D, IC_50, and EC₅₀ values. ¹⁸ In radioligand binding assays in human platelets, thromboxane A₂ receptor agonists were replaced by picotamide with K_D values of 0.38 or 1.5 µM. ¹ , ¹⁸ In vitro, U46619‐induced platelet aggregation was inhibited with time‐dependent IC₅₀ values, ranging from 4.7 to 450 µM. ¹⁸ Other studies addressing effects of picotamide on platelet aggregation used concentrations of 100–500 µM and reported IC₅₀ values in these ranges. ¹³ , ¹⁷ , ²⁹ , ³³ , ³⁴ In cultured human and rat aortic smooth muscle cells, picotamide inhibited migration and growth factor‐induced proliferation in one to low two‐digit ranges. ³⁵ In the same study, U46619‐induced proliferation was not inhibited by picotamide concentrations up to 50 µM, but by 500 µM. ³⁵ In addition to thromboxane A₂ receptor antagonism, picotamide acts as an inhibitor of thromboxane A₂ synthase, ² and consequently reduces plasma thromboxane A₂ levels in patients. ³⁶ In vitro, picotamide inhibits thromboxane A₂ formation by platelets, with IC₅₀ values ranging from 0.43 to 54 µM ¹⁸ or up to 140 µM. ¹⁷ According to these previous reports, we applied picotamide in a concentration of 300 µM in our previous studies, which inhibited contractions of human prostate and trigone tissues. ¹¹ , ¹² In our present study, 100 and 300 µM appeared equally effective, whereas 30 µM were without effect on EFS‐induced contractions. Together, our findings are in line with previous studies suggesting that functional picotamide effects in vitro require concentrations in a three‐digit micromolar range. Our current and previous observations contrast K_D values for picotamide binding to thromboxane A₂ receptors and clinical observations. Thus, oral administration of a standard dose of picotamide (300 mg) results in peak plasma levels of around 5.4 µM. ³⁷ However, antiplatelet activity and inhibition of TXA₂ administration by picotamide in patients do not correlate with plasma concentrations of picotamide. ³⁷ Administration is safe and possible in daily cumulative doses up to 1500 mg per day. ³³ , ³⁸

Inhibition of thromboxane A₂ synthase may be involved as well in our findings, and may account for inhibition of different vasoconstrictors without off‐target effects. Biosynthesis of thromboxane A₂ involves formation of arachidonic acid by phospholipase A₂ (PLA₂), which is processed to prostaglandins and finally to thromboxane A₂ by thromboxane A₂ synthase. ² Activation of PLA₂ isoforms by contractile agonists and agonist‐induced formation of arachidonic acid or thromboxane A₂ have been reported from different smooth muscle types. α₁‐Adrenoceptors, angiotensin‐II, and serotonin activate PLA₂ and cause arachidonic acid formation in vascular smooth muscle cells, what may contribute to receptor‐mediated vasocontraction. ³⁹ , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ PLA₂ activation by muscarinic receptors, followed by arachidonic acid formation and subsequent smooth muscle contraction by thromboxane A₂ has been suggested for airway and esophageal smooth muscle. ⁴⁴ , ⁴⁵ Endothelin‐1 may activate PLA₂ in a variety of cell types, including iris sphincter smooth muscle cells and glomerular smooth muscle cells, ⁴⁶ , ⁴⁷ but is obviously not involved in vasocontraction. ⁴⁸ , ⁴⁹ Consequently, inhibition of contractions in our study must not necessarily reflect off‐target effects of picotamide, but may (at least partially) result from inhibition of vasoconstrictor‐induced thromboxane A₂ synthesis. In line with this, the dual thromboxane A₂ receptor antagonist and thromboxane A₂ synthase inhibitor picotamide previously inhibited EFS‐induced contractions of prostate smooth muscle, what was not observed using pure thromboxane A₂ antagonists. ¹¹

Assuming that agonist‐induced release of thromboxane A₂ is involved in contractions of some vasoconstrictors, it follows that portions exceeding U46619‐induced contractions can not be mediated by thromboxane A₂ release. In interlobar arteries, α₁‐adrenergic and endothelin‐1‐induced contractions clearly exceeded U46619‐induced contractions. Consequently, the exceeding portion is most likely not mediated by thromboxane A₂‐release and occurs thromboxane A₂‐independently. Whether the other, non‐exceeding components are in fact mediated by thromboxane A₂ release, cannot be confirmed on the basis of our data. None of the other agonists, and no agonist in coronary arteries induced stronger contractions than U46619. Some contractions were weaker than U46619‐induced contractions, so that the amount of any released thromboxane A₂ (if involved at all) is not sufficient to induce a full thromboxane A₂‐induced contraction.

Our findings obtained from interlobar arteries are in line with clinical observations. In patients with congestive heart failure, picotamide improved renal perfusion and renal function, which was explained by decreased renal vascular resistance. ³ Chronic congestive heart failure includes enhanced release of several vasoconstrictors, including endothelins and angiotensin, ³ which were inhibited by picotamide in interlobar and coronary arteries in our study. In other studies, picotamide improved intrarenal blood flow and renal function in patients with type II diabetes. ⁵⁰ , ⁵¹ , ⁵² This has been referred to improved renal blood flow resulting from antiaggregatory effects, as no data on renal vascular resistance were available. ⁵⁰ , ⁵¹ , ⁵² Our current study suggests that previously observed improvements of kidney function occured by inhibition of intrarenal vasocontraction. Regarding that picotamide inhibits vasocontraction in at least three vessel types, effects of picotamide on other vessel types, e.g. resistance vessels being critical for arterial blood pressure regulation appear possible as well. However, picotamide does not affect systolic or diastolic blood pressure. ³ , ⁷ , ⁵² Evidence suggesting decreases in mean arterial blood pressure is to the best of our knowledge limited to mice. ⁵³ Finally, inhibition of vasocontraction may contribute to reduced numbers of vascular deaths, which was observed in clinical trials using picotamide and was previously explained by antiaggretory effects. ⁷

5. CONCLUSIONS

Together, our findings suggest that picotamide inhibits contractions induced by a broad panel of vasoconstrictors in porcine interlobar and coronary arteries. The pattern of inhibition differed between vasoconstrictors and vessel types. Our findings from interlobar arteries are in line with previous observations, suggesting decreases of renal vascular resistance and improvements of renal blood flow and renal function in clinical studies.

DISCLOSURE

The authors declare that they have no conflict of interest.

AUTHORS' CONTRIBUTIONS

Bingsheng Li performed methodology and model creation, study design, data analyses, investigation, writing of the original draft and visualization, project administration, and funding acquisition. Ru Huang performed methodology and model creation, study design, data analyses, investigation, and critical revision of the manuscript. Ruixiao Wang performed investigation and critical revision of the manuscript. Yuhan Liu performed investigation and critical revision of the manuscript. Christian G. Stief performed conceptualization, resources, and funding acquisition. Martin Hennenberg performed conceptualization, methodology and model creation, study design, data analyses, investigation, writing of the original draft and visualization, supervision and project administration, and funding acquisition.

ETHICS STATEMENT

This study was performed using arteries obtained from domestic animals, bred and sacrificed for meet production, i.e. from pig kidneys and pig hearts obtained from a local slaughterhouse by a butcher. Consequently, no ethical approval and no approval for animal experiments was required.

DATA AVAILABILITY STATEMENT

The raw data of this study are available from the corresponding author upon reasonable request. All data that support the findings of this study are included in the article.