Next Article in Journal
Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics
Next Article in Special Issue
Sex Differences in Cardiovascular Diseases: A Matter of Estrogens, Ceramides, and Sphingosine 1-Phosphate
Previous Article in Journal
Clinical Breakpoint of Apramycin to Swine Salmonella and Its Effect on Ileum Flora
Previous Article in Special Issue
Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 3
10.3390/ijms23031427
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries

by
Hanna Kozłowska
1,*,
Barbara Malinowska
1,
Marta Baranowska-Kuczko
1,2,
Magdalena Kusaczuk
3,
Miłosz Nesterowicz
4,
Mirosław Kozłowski
4,
Christa E. Müller
5,6,
Katarzyna Kieć-Kononowicz
7 and
Eberhard Schlicker
8
1
Department of Experimental Physiology and Pathophysiology, Medical University of Białystok, ul. Mickiewicza 2A, 15-222 Białystok, Poland
2
Department of Clinical Pharmacy, Medical University of Białystok, ul. Mickiewicza 2A, 15-222 Białystok, Poland
3
Department of Pharmaceutical Biochemistry, Medical University of Białystok, ul. Mickiewicza 2A, 15-222 Białystok, Poland
4
Department of Thoracic Surgery, Medical University of Białystok, ul. M.C. Skłodowska 4A, 15-276 Białystok, Poland
5
Department of Pharmaceutical & Medicinal Chemistry, Pharmaceutical Institute, PharmaCenter Bonn, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
6
Research Training Group 1873, University of Bonn, Venusberg-Campus 1, 53127 Bonn, Germany
7
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University, Medical College, ul. Medyczna 9, 30-688 Kraków, Poland
8
Department of Pharmacology and Toxicology, University of Bonn, Venusberg-Campus 1, 53127 Bonn, Germany
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(3), 1427; https://doi.org/10.3390/ijms23031427
Submission received: 18 November 2021 / Revised: 21 January 2022 / Accepted: 24 January 2022 / Published: 26 January 2022
(This article belongs to the Special Issue G Protein-Coupled Receptor and Their Kinases in Cell Biology and Disease 2.0)
Browse Figures
Versions Notes

Abstract

:
GPR18 receptor protein was detected in the heart and vasculature and appears to play a functional role in the cardiovascular system. We investigated the effects of the new GPR18 agonists PSB-MZ-1415 and PSB-MZ-1440 and the new GPR18 antagonist PSB-CB-27 on isolated human pulmonary arteries (hPAs) and compared their effects with the previously proposed, but unconfirmed, GPR18 ligands NAGly, Abn-CBD (agonists) and O-1918 (antagonist). GPR18 expression in hPAs was shown at the mRNA level. PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD fully relaxed endothelium-intact hPAs precontracted with the thromboxane A2 analog U46619. PSB-CB-27 shifted the concentration-response curves (CRCs) of PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD to the right; O-1918 caused rightward shifts of the CRCs of PSB-MZ-1415 and NAGly. Endothelium removal diminished the potency and the maximum effect of PSB-MZ-1415. The potency of PSB-MZ-1415 or NAGly was reduced in male patients, smokers and patients with hypercholesterolemia. In conclusion, the novel GPR18 agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs and the effect is inhibited by the new GPR18 antagonist PSB-CB-27. GPR18, which appears to exhibit lower activity in hPAs from male, smoking or hypercholesterolemic patients, may become a new target for the treatment of pulmonary arterial hypertension.

Graphical Abstract

1. Introduction

GPR18 is an orphan G protein-coupled receptor, which, although showing little structural similarity to cannabinoid CB1 and CB2 receptors (identity of amino acid sequence of 13 and 8%, respectively; [1]), responds to the natural cannabinoid Δ9-tetrahydrocannabinol (THC). In the mouse, GPR18 is located in the central nervous system, the gastrointestinal tract, in the lymphoid system [2,3], in peripheral blood leukocytes, several hematopoietic cell lines [4,5] and in the eye [6,7]. GPR18 expression has recently been verified in skeletal muscle from obese rats [8]. In humans, this receptor is expressed in the brain including the hypothalamus [9], in cells of the immune system [4,5], and in the colon [10]. GPR18 is also expressed in cultured cells, including human lymphoid cells [4], HEC-1B endometrial cells [11], BV2 murine microglial cells [12], and metastatic melanoma [13]. Although the (patho)physiological role of GPR18 is controversial and unclear, it is believed that it may be involved in immunological processes, inhibition of inflammatory reactions including intestinal inflammation [10,14] and in lowering intraocular pressure in mice [7,15].
GPR18 receptor protein was also detected in the cardiovascular system, including the heart [16], isolated vessels and endothelial cells of mesenteric arteries [17], retinal vessels of the rat [6], pulmonary arteries [18], and placental vascular smooth muscle of humans [19] as well as various vascular endothelial cell lines [6,20,21]. It was also found to be expressed in the rat’s rostral ventrolateral medulla (RVLM), a brain region involved in the regulation of cardiovascular function [9].
Some functional studies indeed suggest that GPR18 plays a role in the cardiovascular system. Thus, chronic administration of an agonist to the RVLM was reported to lower mean blood pressure, suppress the cardiac sympathetic dominance and improve left ventricular function in conscious rats [16], and to display similar cardioprotective effects in a rat model of streptozotocin-induced diabetes [22]. Moreover, mesenteric vasodilation and hypotension in mice lacking CB1/CB2 receptors has been shown [23], and an endothelium-dependent vasorelaxation has been described in small mesenteric arteries of the rat [17,24] and in human pulmonary arteries (hPAs; [25,26]). All of these data are based on cannabinoids such as THC and abnormal cannabidiol (Abn-CBD), which were proposed to act as agonists, and O-1918, proposed to act as an antagonist, both in HEK293-GPR18 transfected cells [11] and in various vascular preparations. The receptor involved in the vasodilation of mesenteric arteries of rodents was described earlier than GPR18 and labeled “endothelial cannabinoid receptor” [27]. Some authors suggested that the latter receptor, which is not defined in terms of molecular biology, may be GPR18 [12].
There are, however, difficulties in the unambiguous assessment of GPR18 since the agonistic or antagonistic effects of the cannabinoids mentioned above (except for THC) were not confirmed [28,29,30]. This also holds true for N-arachidonyl-glycine (NAGly; [31]), which was proposed as the endogenous ligand of GPR18 [4]). Interesting enough, NAGly activates (1) large calcium-dependent potassium channels (BKCa; [17,32], (2) small calcium-dependent potassium channels (SKCa; [6], and (3) the Na+/Ca2+ exchanger (NCX) leading to NO production [24]. Each of the latter three mechanisms would lead to vascular relaxation.
Recently, novel ligands for GPR18 have become available, including the agonists PSB-MZ-1415 and PSB-MZ-1440 and the antagonist PSB-CB-27 [29,33]. Taking into account the fact that GPR18 protein is expressed in the human pulmonary artery [18], the aim of the present study was to investigate the effects of the three novel GPR18 ligands on isolated hPAs and to compare their effects with the previously proposed, but unconfirmed, GPR18 ligands NAGly, Abn-CBD, and O-1918. In addition, we searched for gpr18 mRNA, using reverse transcriptase polymerase chain reaction (RT-PCR).

2. Results

2.1. General

The vasorelaxant effects of agonists on hPAs were studied in vascular rings precontracted with U46619 (0.01 μM, a concentration approximately equivalent to its EC60). In all experimental groups, the U46619-induced increase in tone was comparable (Table 1) and the U46619-induced contractions were not affected by 30 min incubation periods with the antagonists or their vehicles.

2.2. Influence of Agonists on U46619-Precontracted Human Pulmonary Arteries

The previously proposed GPR18 agonists NAGly and Abn-CBD, and the novel GPR18 agonists PSB-MZ-1415 and PSB-MZ-1440, were studied in a concentration range of 0.01–30 μM. PSB-MZ-1415 produced a full concentration-dependent relaxation of endothelium-intact isolated hPAs precontracted with U46619; endothelial removal reduced both its potency and its maximum effect (Figure 1 and Figure 2A,C, Table 1). PSB-MZ-1440, NAGly and Abn-CBD also fully relaxed endothelial-dependent and U46619-precontracted hPAs (Figure 2B and Figure 3A,B; for original traces see Figure 2D and Figure 3C,D). The rank order of potencies (according to their pEC50 values) was Abn-CBD ≥ PSB-MZ-1415 ≥ NAGly ≥ PSB-MZ-1440 (for pEC50, pEC25, and Rmax, see Table 1). Repeated administration of the vehicles DMSO and ethanol had a very slight vasorelaxant effect only (Figure 2 and Figure 3, Table 1).

2.3. Interaction of PSB-CB-27 and O-1918 with the Agonist-Induced Relaxation in Human Pulmonary Arteries

To investigate the involvement of GPR18 in the effect of the four agonists, their interaction with the new GPR18 antagonist PSB-CB-27 was studied and compared to the previously proposed antagonist O-1918. The concentration-response curves for the PSB compounds are shown in Figure 2, and those of NAGly and Abn-CBD are depicted in Figure 3; original traces are presented in the bottom part of the two figures. pEC25 values in the absence and presence of the antagonists and their apparent pA2 values are given in Table 1 and Table 2, respectively.
PSB-CB-27 was studied against each of the four GPR18 agonists at 10 µM and, in the case of NAGly, also at 1 µM. The GPR18 antagonist PSB-CB-27 (1 and 10 µM) shifted the concentration-response curve (CRC) for NAGly to the right by a factor of 4 and 9.5, respectively. In the case of Abn-CBD, PSB-MZ-1415, and PSB-MZ-1440, PSB-CB-27 (10 µM) elicited rightward shifts of 25-, 16- and 25-fold, respectively. The antagonist O-1918 (10 μM) was studied against NAGly and PSB-MZ-1415 only and caused a 12.5- and 9.5-fold rightward shift of their CRCs, respectively.

2.4. Post hoc Analysis of the Influence of Co-Morbidities on PSB-MZ-1415- and NAGly-Mediated Vasorelaxation in Human Pulmonary Arteries

Post hoc analysis was performed to establish selected relationships between PSB-MZ-1415 and NAGly responses and patient characteristics (Figure 4, Table 3). The potency of NAGly (pEC50) was reduced in smokers (4.9 ± 0.1) compared to non-smokers (5.5 ± 0.2, p < 0.05; Figure 4F, Table 3), and in patients with hypercholesterolemia (4.6 ± 0.1) compared to normocholesterolemic patients (5.2 ± 0.1, p < 0.01; Figure 4H, Table 3), whereas the potency of PSB-MZ-1415 was reduced in males (5.0 ± 0.1) compared to females (5.5 ± 0.1, p < 0.01; Figure 4A) and showed a tendency towards a decrease in smokers and in patients with hypercholesterolemia (Figure 4B,D; Table 3). Heart diseases had no influence on the potency of these drugs (Figure 4C,G). We could not perform an analysis for all diseases and for PSB-MZ-1440 and Abn-CBD, as patient numbers were too small for adequate statistical analysis.

2.5. gpr18 mRNA Expression in Human Bronchioles and Pulmonary Arteries

In order to confirm gpr18 mRNA distribution in lung tissue and to investigate whether gpr18 presents a differential expression pattern in bronchioles and pulmonary arteries, a real-time quantitative PCR (qPCR) analysis of the gene transcript was performed. The results showed that gpr18 mRNA expression was higher in pulmonary arteries compared to bronchioles (Figure 5).

3. Discussion

Our previous studies on the human pulmonary artery revealed that several cannabinoids including Abn-CBD [25], anandamide (AEA; [26]), cannabidiol [18], 2-arachidonoylglycerol [34], and virodhamine [35] elicit an endothelium-dependent vasorelaxation. The effect of Abn-CBD, AEA, and virodhamine is antagonized by O-1918 [25,26,35], suggesting the involvement of the so-called endothelial cannabinoid receptor. There is some evidence that the latter receptor (which is not defined in terms of molecular biology) is identical to the orphan receptor GPR18 [12]. Using an immunohistochemical technique, we detected GPR18 protein in the endothelial and muscular layer of the human pulmonary artery [18]. In the present study, we examined whether also GPR18 mRNA is detectable in the human pulmonary artery and compared the effects of NAGly, a previously proposed, but unconfirmed, endogenous agonist of GPR18 [4], of two newly developed GPR18 agonists, and of a newly developed GPR18 antagonist [33] to the effects of Abn-CBD and O-1918.
Using the RT-PCR technique, we were indeed able to detect gpr18 mRNA in hPAs; gpr18 mRNA expression in pulmonary arteries is more abundant than in bronchioles. These differences in regional mRNA expression may not link to the same changes in protein. Following the detection of gpr18 mRNA and protein [18], further experiments dedicated to the third level of GPR18, i.e., to its function, were carried out on isolated hPAs, which were contracted by the thromboxane A2 analogue U46619. Thromboxane A2 receptors are present in large amounts in pulmonary vessels [34], and U46619 has been used to study cannabinoid ligands in our previous work [18,34].
The new agonists PSB-MZ-1415 and PSB-MZ-1440 were optimized based on an initial lead [36]. Their potency (pEC50) at GPR18 was studied in β-arrestin recruitment assays in CHO cells stably expressing the hGPR18 and amounted to 7.7 and 7.2, respectively (publication in preparation). However, the coupling of GPR18 is currently unknown. Early reports on Gi protein coupling [4] could not be unambiguously confirmed so far [36]. The reason why the pEC50 values of the newly developed GPR18 agonists in a cellular functional assay (7.2–7.7) were relatively high when compared to relaxation of hPAs (4.9–5.2) is unclear but not surprising, given the more complex system.
For the sake of comparison, we also studied NAGly and Abn-CBD, the agonistic effect of which was shown in HEK293-GPR18 transfected cells [12], but later disputed by other groups [28,29,30,31]. The novel and older compounds resembled each other in terms of maximum effect and potency. All investigated agonists induced a concentration-dependent full relaxation. The concentration range (0.1–30 μM) was the same for each of the four compounds. The potencies (pEC50) of the four drugs in the hPAs were Abn-CBD (5.5) ≥ PSB-MZ-1415 (5.2) ≥ NAGly (5.1) ≥ PSB-MZ-1440 (4.9); differences were not statistically significant. NAGly and Abn-CBD had been previously studied also in precontracted rat small mesenteric arteries [17,27], which they almost fully relaxed, exhibiting pEC50 values of ~ 5.7.
In order to further clarify whether GPR18 is involved in the relaxation of hPAs, studies were carried out with the new GPR18 antagonist PSB-CB-27 [33]. This compound (inactive at hCB1 and hCB2 receptors, pIC50 < 5), exhibits a pIC50 of 6.2 at the hGPR18 in the β-arrestin recruitment assay [33]. Its antagonistic potency is identical to the apparent pA2 value (6.2–6.3) against the two novel GPR18 agonists obtained in hPAs. This suggests that the receptor under consideration is a GPR18 since, in the case of antagonists, only the interaction between agonist and antagonist at the receptor level is measured and values obtained in different experimental models should be very similar. Interesting enough, the apparent pA2 of PSB-CB-27 against the two other agonists Abn-CBD and NAGly was also 6.2–6.3. These results are compatible with the view, but do not prove, that in our experimental model also the latter two agonists activate GPR18.
The antagonist at the endothelial cannabinoid receptor, O-1918, had a pA2 of 6.0 (mean of the values against PSB-MZ-1415 and NAGly), which is in the middle of the range of pA2 values obtained for the vasorelaxant effect in various vascular preparations including hPAs ([25]—5.1, [35]—6.3, [26]—5.6), rat pulmonary arteries ([37]—5.7) and mesenteric arteries ([17]—6.7, [27]—5.8). Moreover, we found that vasorelaxation to PSB-MZ-1415 was strongly reduced in endothelium-denuded hPAs, indicating that PSB-MZ-1415 can act endothelium-dependently and, to a lesser extent, endothelium-independently. Our results are similar to those obtained by Kotańska et al. 2021 [38], who found that another GPR18 receptor agonist, PSB-KD-107, caused endothelium-dependent vasorelaxation of the rat aorta.
While GPR18 mRNA, protein expression, and function have been shown in hPAs, the question arises whether this receptor has practical relevance. Post hoc analysis revealed that the potency of the agonists for GPR18-related relaxation was reduced in males, by smoking, and hypercholesterolemia compared to respective controls. This is in line with the fact that smoking and hypercholesterolemia are well-known important risk factors for endothelial dysfunction [39]. Pulmonary arterial hypertension can be elicited by a multitude of pathophysiological alterations [40], and GPR18 agonists may become an additional strategy for its treatment, thereby adding to the armamentarium of drugs, including prostacyclin analogues, endothelin receptor antagonists, phosphodiesterase 5 inhibitors, and soluble guanylate cyclase stimulators [41]. Interestingly, anti-inflammatory and anti-nociceptive activities of PSB-MZ-1415 have been shown in animal models of intestinal inflammation and inflammatory pain [10], and beneficial effects of a GPR18 agonist (PSB-MZ-1415) and of GPR18 antagonists (PSB-CB-5 and PSB-CB-27) on food intake and body weight gain in rats have also been reported [42].

4. Materials and Methods

4.1. Tissue Preparation

Experimental protocols were approved by the Human Ethics Committee of the Medical University of Białystok, Poland (R-I-002/59/2017). The tissue donors provided written informed consent for the use of their blood vessels.
Lung tissue was received from 28 patients (19 men and 9 women; patient characteristics are shown in Table 3) undergoing lobectomy or pneumonectomy during the resection of lung carcinoma or tuberculoma with a surgical margin of healthy tissue. Before the operation, they received cephalosporins and heparin as anti-infection and antithrombotic prophylaxis, respectively. Anesthesia was induced intravenously by etomidate and maintained with inhaled sevoflurane. Pancuronium and sufentanil were applied for muscle relaxation and analgesia, respectively. Isolation of the human pulmonary arteries has been described previously [18]. Arterial rings (3–5 mm in length and 2–4 mm in outer diameter) were mounted in 10 mL organ baths containing Tyrode’s solution (for composition, see [18]) gassed with 95% O2 and 5% CO2 (37 °C, pH 7.4). The hPAs were allowed to equilibrate for 90 min at a resting tension of ~20–25 mN. After the equilibration period, all rings were exposed to high KCl (60 mM) to establish tissue viability. Then, rings were exposed to phenylephrine (1 µM) followed by the induction of at least 80% relaxation in response to acetylcholine (1 µM) to verify if the endothelium was intact. For some experiments, the endothelium was removed by gently rubbing off the intima. Successful endothelial denudation was confirmed by the absence of acetylcholine-induced vasorelaxation.
Muscle tension was recorded by a force displacement transducer (BIO-SYS-TECH, Białystok, Poland) [18].

4.2. Vasorelaxation Study

In each individual preparation, only one CRC was determined. All experiments were performed in paired vessels, i.e., the effect of a drug was studied in one vessel and another vessel from the same patient served as a control.
Human pulmonary arteries were sub-maximally contracted with U46619 (a prostanoid TP receptor agonist) at 0.01 µM, which is approximately equivalent to its EC60. After a stable level of tone was maintained, CRCs were generated by the cumulative application of NAGly, Abn-CBD, PSB-MZ-1415, PSB-MZ-1440, or their vehicle. When necessary, tissues were pre-treated for 30 min with one of the following antagonists: PSB-CB-27 (1 or 10 μM; a novel GPR18 receptor antagonist) or O-1918 (10 μM; a previously proposed antagonist).
In vehicle-treated tissues, identical volumes of the respective vehicles of the agonists or antagonists were administered (dimethyl sulfoxide (DMSO) or ethanol, see below).

4.3. RNA Isolation and Gene Expression Analysis

Specimens of lung and bronchial tissues from 6 patients (from the above-mentioned group of 25 patients) were immediately flash-frozen in liquid nitrogen and stored at −80 °C. Approximately 5–10 mg of each tissue were taken to perform RNA purification. Samples were finely ground to a powder with a chilled stainless-steel mortar and pestle. Total RNA was isolated according to the procedure described previously [43]. Briefly, the ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA) was used for the purification procedure, and traces of genomic DNA were excluded by DNase I treatment following the manufacturer’s instructions. Spectrophotometric measurements (A260/A280) were carried out to assess the quantity and quality of the extracted RNA (NanoPhotometer, Implen, München, Germany). Synthesis of the cDNA was performed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Briefly, 0.5 µg of purified total RNA were used in a 20 µL reaction mixture containing random octamers, oligo dT-16 primers, dNTPs, and MuLV reverse transcriptase (RT). Two microliters of cDNA served as a template for real-time qPCR reactions. Amplification of the product was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). A pre-designed set of primers for human Grp18 gene was purchased from Bio-Rad (GPR18 PrimePCR™ PreAmp for SYBR® Green Assay (accession no. NC_000013.10)). As an internal control, two reference genes, Rpl13A and Hprt1, were tested, and Rpl13A was chosen for further analysis. The sequences of the housekeeping genes were as previously described: Rpl13A sense 5′-CTATGACCAATAGGAAGAGCAACC-3′, antisense 5′-GCAGAGTATATGACCAGGT-GGAA-3′ [44], and Hprt1 sense 5′-TGCTCGAGATGTGATGAAGC-3′, antisense 5′-AATCCAGCAGGTCAGCAAAG-3′ [45]. Primer accuracy was checked by using the Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast, accessed on 24 January 2022). The following reaction parameters were applied: initial denaturation at 98 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 40 s. Melt curve analysis was performed from 65 to 95 °C in 0.5 °C steps, 10 s for the first step and 5 s for each step thereafter. The CFX Connect Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) was used to perform real-time qPCR assays. Reactions were run in triplicate and expression levels were analyzed using the relative quantification method modified by [46].

4.4. Drugs

In this study, three selective ligands of human GPR18 were used, including two agonists with undisclosed structure (PSB-MZ-1415, EC50 0.019 µM and PSB-MZ-1440, EC50 0.061 µM; chemical details will be shortly published elsewhere) and the antagonist (Z)-2-(3-(6-(4-chlorophenoxy)hexyloxy)benzylidene)-6,7-dihydro-2H-imidazo[2,1-b][1,3]thiazin-3(5H)-one (PSB-CB-27, IC50 0.65 µM). The three ligands were synthetized at the Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University, Kraków, Poland, and identified and characterized in the Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, Germany ([33] and unpublished results).
U46619 ((5Z)-7-{(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxyoct-1-en-1-yl]-2-oxabicyclo[2.2.1]-heptan-5-yl}hept-5-enoic acid), O-1918 (1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]benzene), NAGly, and Abn-CBD were purchased from Tocris Bioscience (Bristol, UK). Stock solutions of these substances were made to 10 mM in ethanol; their final concentrations were prepared by dilutions with deionized water, which led to final concentrations of ethanol of <0.7% v/v. Stock solutions of PSB-MZ-1415, PSB-MZ-1440, and PSB-CB-27 were made only to 10 mM in dimethyl sulfoxide (DMSO). The final concentration of DMSO was <1.0% v/v.

4.5. Calculations and Statistical Analysis

Results are given as the mean ± SEM of n (number of patients). The relaxation elicited by the GPR18 agonists (NAGly, Abn-CBD, PSB-MZ-1415, PSB-MZ-1440) or the appropriate solvent was expressed as a percentage of the precontraction induced by U46619.
GraphPad Prism 5.0 software (San Diego, CA, USA) was used to plot the mean data as sigmoidal concentration-response curves (CRCs):
Y = B o t t o m + ( T o p − B o t t o m ) / ( 1 + 10 ( L o g E C 50 − X ) )
where X is the logarithm of the molar concentration of the agonist and Y is the percent response. The curves were used to determine potency (pEC50 = −logEC50 or pEC25 = −logEC25, where EC50 or EC25 is the concentration (M) of the agonist that produced 50 or 25% of the maximal effect (Rmax)) [47].
Antagonist- or endothelium removal-induced rightward shifts of the CRCs relative to the control curve were calculated on the basis of the EC25 values. The antagonistic potency (apparent pA2) of PSB-CB-27 or O-1918 against GPR18 agonists was calculated from the equation: apparent pA2 = log (CR − 1) − log[B], where [B] is the molar concentration of the antagonist and CR is the concentration ratio of the EC25 values of GPR18 agonists in the presence and absence of an antagonist.
When two or more treatment groups were compared to the same control, a one-way analysis of variance (ANOVA) followed by Dunnett’s test was used. Post hoc tests were run only if F achieved the necessary level of statistical significance and there was no significant variance inhomogeneity. Student’s t-test for unpaired data was used when two groups (one treatment and one control group) were compared. Data were analyzed using GraphPad Prism version 5.00 for Windows, GraphPad Software (La Jolla, CA, USA). Differences were considered to be statistically significant at p < 0.05.

5. Conclusions

In conclusion, the following facts lend further support to the role of GPR18 in isolated human pulmonary arteries: (i) Two new agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs, (ii) the vasodilatory effect of these agonists is inhibited by the new GPR18 antagonist PSB-CB-27, and (iii) expression of gpr18 mRNA is shown. GPR18, the activity of which is reduced by endothelial denudation and is lower in hPAs from male, hypercholesterolemic, or smoking patients, may become a new target for the treatment of pulmonary arterial hypertension.

Author Contributions

Conceptualization, H.K. and B.M.; methodology, H.K., M.B.-K., M.K. (Magdalena Kusaczuk), M.N., M.K. (Mirosław Kozłowski): software, H.K., M.B.-K.; validation, H.K., M.B.-K., M.K. (Magdalena Kusaczuk); formal analysis, H.K., M.B.-K.; investigation, H.K.; synthesis of the three GPR18 ligands, K.K.-K.; identification and characterization of the three GPR18 ligands; C.E.M.; data curation, M.N., M.K. (Mirosław Kozłowski).; writing—original draft preparation, H.K.; writing—review and editing, E.S., H.K., B.M., C.E.M., K.K.-K.; visualization, H.K.; supervision, E.S., B.M., C.E.M., K.K.-K.; project administration, H.K. and B.M.; funding acquisition, H.K. and B.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Medical University of Białystok (Poland; grant No. SUB/2/DN/21/003/2213).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

Acknowledgments

The authors would like to thank Marzanna Cechowska-Pasko from the Department of Pharmaceutical Biochemistry (Medical University of Białystok, Poland) for the possibility of RT-PCR analysis.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

Abn-CBDabnormal cannabidiol
AEAanandamide
CRCconcentration-response curve
DMSOdimethyl sulfoxide
hPAshuman pulmonary arteries
NAGlyN-arachidonyl-glycine
PSB-MZ-1415GPR18 agonist
PSB-MZ-1440GPR18 agonist
PSB-CB-27GPR18 antagonist
THCΔ9-tetrahydrocannabinol
RVLMrostral ventrolateral medulla

References

  1. Rajaraman, G.; Simcocks, A.; Hryciw, D.H.; Hutchinson, D.S.; McAinch, A.J. G protein coupled receptor 18: A potential role for endocannabinoid signaling in metabolic dysfunction. Mol. Nutr. Food Res. 2016, 60, 92–102. [Google Scholar] [CrossRef]
  2. Gantz, I.; Muraoka, A.; Yang, Y.K.; Samuelson, L.C.; Zimmerman, E.M.; Cook, H.; Yamada, T. Cloning and chromosomal localization of a gene (GPR18) encoding a novel seven transmembrane receptor highly expressed in spleen and testis. Genomics 1997, 42, 462–466. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, X.; Sumida, H.; Cyster, J.G. GPR18 is required for a normal CD8αα intestinal intraepithelial lymphocyte compartment. J. Exp. Med. 2014, 211, 2351–2359. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Kohno, M.; Hasegawa, H.; Inoue, A.; Muraoka, M.; Miyazaki, T.; Oka, K.; Yasukawa, M. Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18. Biochem. Biophys. Res. Commun. 2006, 347, 827–832. [Google Scholar] [CrossRef] [PubMed]
  5. Takenouchi, R.; Inoue, K.; Kambe, Y.; Miyata, A. N-arachidonoyl glycine induces macrophage apoptosis via GPR18. Biochem. Biophys. Res. Commun. 2012, 418, 366–371. [Google Scholar] [CrossRef] [PubMed]
  6. MacIntyre, J.; Dong, A.; Straiker, A.; Zhu, J.; Howlett, S.E.; Bagher, A.; Denovan-Wright, E.; Yu, D.Y.; Kelly, M.E. Cannabinoid and lipid-mediated vasorelaxation in retinal micro vasculature. Eur. J. Pharmacol. 2014, 735, 105–114. [Google Scholar] [CrossRef]
  7. Miller, S.; Leishman, E.; Oehler, O.; Daily, L.; Murataeva, N.; Wager-Miller, J.; Bradshaw, H.; Straiker, A. Evidence for a GPR18 role in diurnal regulation of intraocular pressure. Investig. Ophthalmol. Vis. Sci. 2016, 57, 6419–6426. [Google Scholar] [CrossRef] [Green Version]
  8. Simcocks, A.C.; O’Keefe, L.; Jenkin, K.A.; Cornall, L.M.; Grinfeld, E.; Mathai, M.L.; Hryciw, D.H.; McAinch, A.J. The role of atypical cannabinoid ligands O-1602 and O-1918 on skeletal muscle homeostasis with a focus on obesity. Int. J. Mol. Sci. 2020, 21, 5922. [Google Scholar] [CrossRef]
  9. Penumarti, A.; Abdel-Rahman, A.A. The novel endocannabinoid receptor GPR18 is expressed in the rostral ventrolateral medulla and exerts tonic restraining influence on blood pressure. J. Pharmacol. Exp. Ther. 2014, 349, 29–38. [Google Scholar] [CrossRef] [Green Version]
  10. Fabisiak, A.; Fabisiak, N.; Mokrowiecka, A.; Małecka-Panas, E.; Jacenik, D.; Kordek, R.; Zielińska, M.; Kieć-Kononowicz, K.; Fichna, J. Novel selective agonist of GPR18, PSB-KK-1415 exerts potent anti-inflammatory and anti-nociceptive activities in animal models of intestinal inflammation and inflammatory pain. Neurogastroenterol. Motil. 2020, 33, e14003. [Google Scholar] [CrossRef]
  11. McHugh, D. GPR18 in microglia: Implications for the CNS and endocannabinoid system signalling. Br. J. Pharmacol. 2012, 167, 1575–1582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. McHugh, D.; Hu, S.S.; Rimmerman, N.; Juknat, A.; Vogel, Z.; Walker, J.M.; Bradshaw, H.B. N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci. 2010, 11, 44. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Qin, Y.; Verdegaal, E.M.; Siderius, M.; Bebelman, J.P.; Smit, M.J.; Leurs, R.; Willemze, R.; Tensen, C.P.; Osanto, S. Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: The consti tutively active orphan GPCR GPR18 as novel drug target. Pigment Cell Melanoma Res. 2011, 24, 207–218. [Google Scholar] [CrossRef] [PubMed]
  14. Burstein, S.H. N-Acyl amino acids (elmiric acids): Endogenous signaling molecules with therapeutic potential. Mol. Pharmacol. 2018, 93, 228–238. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Caldwell, M.D.; Hu, S.S.; Viswanathan, S.; Bradshaw, H.; Kelly, M.E.; Straiker, A.A. GPR18-based signalling system regulates IOP in murine eye. Br. J. Pharmacol. 2013, 169, 834–843. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Matouk, A.I.; Taye, A.; El-Moselhy, M.A.; Heeba, G.H.; Abdel-Rahman, A.A. The effect of chronic activation of the novel endocannabinoid receptor GPR18 on myocardial func tion and blood pressure in conscious rats. J. Cardiovasc. Pharmacol. 2017, 69, 23–33. [Google Scholar] [CrossRef] [Green Version]
  17. Parmar, N.; Ho, W.S. N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels. Br. J. Pharmacol. 2010, 160, 594–603. [Google Scholar] [CrossRef] [Green Version]
  18. Baranowska-Kuczko, M.; Kozłowska, H.; Kloza, M.; Sadowska, O.; Kozłowski, M.; Kusaczuk, M.; Kasacka, I.; Malinowska, B. Vasodilatory effects of cannabidiol in human pulmonary and rat small mesenteric arteries: Modification by hypertension and the potential pharmacological opportunities. J. Hypertens. 2020, 38, 896–911. [Google Scholar] [CrossRef]
  19. Ulu, A.; Sahoo, P.K.; Yuil-Valdes, A.G.; Mukherjee, M.; Van Ormer, M.; Muthuraj, P.G.; Thompson, M.; Anderson Berry, A.; Hanson, C.K.; Natarajan, S.K.; et al. Omega-3 fatty acid-derived resolvin D2 regulates human placental vascular smooth muscle and extravillous trophoblast activities. Int. J. Mol. Sci. 2019, 20, 4402. [Google Scholar] [CrossRef] [Green Version]
  20. Console-Bram, L.; Brailoiu, E.; Brailoiu, G.C.; Sharir, H.; Abood, M.E. Activation of GPR18 by cannabinoid compounds: A tale of biased agonism. Br. J. Pharmacol. 2014, 171, 3908. [Google Scholar] [CrossRef] [Green Version]
  21. Wilhelmsen, K.; Khakpour, S.; Tran, A.; Sheehan, K.; Schumacher, M.; Xu, F.; Hellman, J. The endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) and synthetic cannabinoid WIN55,212-2 abate the inflammatory activation of human endothelial cells. J. Biol. Chem. 2014, 289, 13079–13100. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Matouk, A.I.; Taye, A.; El-Moselhy, M.A.; Heeba, G.H.; Abdel-Rahman, A.A. Abnormal can nabidiol confers cardioprotection in diabetic rats independent of glycemic control. Eur. J. Pharmacol. 2018, 820, 256–264. [Google Scholar] [CrossRef] [PubMed]
  23. Járai, Z.; Wagner, J.A.; Varga, K.; Lake, K.D.; Compton, D.R.; Martin, B.R.; Zimmer, A.M.; Bonner, T.I.; Buckley, N.E.; Mezey, E.; et al. Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc. Natl. Acad. Sci. USA 1999, 96, 14136–14141. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Al Suleimani, Y.M.; Al Mahruqi, A.S. The endogenous lipid N-arachidonoyl glycine is hypotensive and nitric oxide-cGMP-dependent vasorelaxant. Eur. J. Pharmacol. 2017, 794, 209–215. [Google Scholar] [CrossRef] [PubMed]
  25. Kozlowska, H.; Baranowska, M.; Schlicker, E.; Kozlowski, M.; Laudanski, J.; Malinowska, B. Identification of the vasodilatory endothelial cannabinoid receptor in the human pulmonary artery. J. Hypertens. 2007, 25, 2240–2248. [Google Scholar] [CrossRef] [PubMed]
  26. Baranowska-Kuczko, M.; Kozlowska, H.; Kozlowski, M.; Schlicker, E.; Kloza, M.; Surazynski, A.; Grzeda, E.; Malinowska, B. Mechanisms of endothelium-dependent relaxation evoked by anandamide in isolated human pulmonary arteries. Naunyn. Schmiedebergs Arch. Pharmacol. 2014, 387, 477–486. [Google Scholar] [CrossRef] [Green Version]
  27. Offertáler, L.; Mo, F.-M.; Bátkai, S.; Liu, J.; Begg, M.; Razdan, R.K.; Martin, B.R.; Bukoski, R.D.; Kunos, G. Selective ligands and cellular effectors of a G protein-coupled endothelial cannabinoid receptor. Mol. Pharmacol. 2003, 63, 699–705. [Google Scholar] [CrossRef] [Green Version]
  28. Finlay, D.B.; Joseph, W.R.; Grimsey, N.L.; Glass, M. GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine. PeerJ 2016, 4, e1835. [Google Scholar] [CrossRef] [Green Version]
  29. Neumann, A.; Engel, V.; Mahardhika, A.B.; Schoeder, C.T.; Namasivayam, V.; Kieć-Kononowicz, K.; Müller, C.E. Computational investigations on the binding mode of ligands for the cannabinoid-activated G protein-coupled receptor GPR18. Biomolecules 2020, 10, 686. [Google Scholar] [CrossRef]
  30. Alexander, S.P.; Christopoulos, A.; Davenport, A.P.; Kelly, E.; Mathie, A.; Peters, J.A.; Veale, E.L.; Armstrong, J.F.; Faccenda, E.; Harding, S.D.; et al. The Concise Guide to Pharmacology 2021/22: G protein-coupled receptors. Br. J. Pharmacol. 2021, 178 (Suppl. S1), S27–S156. [Google Scholar] [CrossRef]
  31. Yin, H.; Chu, A.; Li, W.; Wang, B.; Shelton, F.; Otero, F.; Nguyen, D.G.; Caldwell, J.S.; Chen, Y.A. Lipid G protein-coupled receptor ligand identification using beta-arrestin PathHunter assay. J. Biol. Chem. 2009, 284, 12328–12338. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Bondarenko, A.I.; Drachuk, K.; Panasiuk, O.; Sagach, V.; Deak, A.T.; Malli, R.; Graier, W.F. N-Arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs. Br. J. Pharmacol. 2013, 169, 933–948. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Schoeder, C.T.; Kaleta, M.; Mahardhika, A.B.; Olejarz-Maciej, A.; Łażewska, D.; Kieć-Kononowicz, K.; Müller, C.E. Structure-activity relationships of imidazothiazinones and analogs as antagonists of the cannabinoid-activated orphan G protein-coupled receptor GPR18. Eur. J. Med. Chem. 2018, 155, 381–397. [Google Scholar] [CrossRef] [PubMed]
  34. Karpińska, O.; Baranowska-Kuczko, M.; Kloza, M.; Ambrozewicz, E.; Kozłowski, T.; Kasacka, I.; Malinowska, B.; Kozłowska, H. Activation of CB1 receptors by 2-arachidonoylglycerol attenuates vasoconstriction induced by U46619 and angiotensin II in human and rat pulmonary arteries. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2017, 312, R883–R893. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Kozlowska, H.; Baranowska, M.; Schlicker, E.; Kozlowski, M.; Laudanski, J.; Malinowska, B. Virodhamine relaxes the human pulmonary through the endothelial cannabinoid receptor and indirectly through a COX product. Br. J. Pharmacol. 2008, 155, 1034–1042. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Schoeder, C.T.; Mahardhika, A.B.; Drabczyńska, A.; Kieć-Kononowicz, K.; Müller, C.E. Discovery of tricyclic xanthines as agonists of the cannabinoid-activated orphan G-protein-coupled receptor GPR18. ACS Med. Chem. Lett. 2020, 11, 2024–2031. [Google Scholar] [CrossRef]
  37. Baranowska-Kuczko, M.; MacLean, M.R.; Kozlowska, H.; Malinowska, B. Endothelium-dependent mechanisms of the vasodilatory effect of the endocannabinoid, anandamide, in the rat pulmonary artery. Pharmacol. Res. 2012, 66, 251–259. [Google Scholar] [CrossRef]
  38. Kotańska, M.; Kubacka, M.; Bednarski, M.; Nicosia, N.; Szafarz, M.; Jawień, W.; Müller, C.E.; Kieć-Kononowicz, K. The GPR18 agonist PSB-KD-107 exerts endothelium-dependent vasorelaxant effects. Pharmaceuticals 2021, 14, 799. [Google Scholar] [CrossRef]
  39. Su, J.B. Vascular endothelial dysfunction and pharmacological treatment. World J. Cardiol. 2015, 7, 719–741. [Google Scholar] [CrossRef]
  40. Galie, N.; Hoeper, M.M.; Humbert, M.; Torbicki, A.; Vachiery, J.L.; Barbera, J.A.; Beghetti, M.; Corris, P.; Gaine, S.; Gibbs, J.S.; et al. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur. Heart J. 2016, 37, 67–119. [Google Scholar] [CrossRef]
  41. Sommer, N.; Ghofrani, H.A.; Pak, O.; Bonnet, S.; Provencher, S.; Sitbon, O.; Rosenkranz, S.; Hoeper, M.M.; Kiely, D.G. Current and future treatments of pulmonary arterial hypertension. Br. J. Pharmacol. 2021, 178, 6–30. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Kotańska, M.; Mika, K.; Szafarz, M.; Kubacka, M.; Müller, C.E.; Sapa, J.; Kieć-Kononowicz, K. Effects of GPR18 ligands on body weight and metabolic parameters in a female rat model of excessive eating. Pharmaceuticals 2021, 14, 270. [Google Scholar] [CrossRef] [PubMed]
  43. Karpińska, O.; Baranowska-Kuczko, M.; Malinowska, B.; Kloza, M.; Kusaczuk, M.; Gęgotek, A.; Golec, P.; Kasacka, I.; Koz łowska, H. Mechanisms of l-alpha-lysophosphatidylinositol-induced relaxation in human pulmonary arteries. Life Sci. 2018, 192, 38–45. [Google Scholar] [CrossRef] [PubMed]
  44. Kusaczuk, M.; Krętowski, R.; Stypułkowska, A.; Cechowska-Pasko, M. Molecular and cellular effects of a novel hydroxa mate-based HDAC inhibitor-belinostat-in glioblastoma cell lines: A preliminary report. Investig. New Drugs 2016, 34, 552–564. [Google Scholar] [CrossRef] [Green Version]
  45. Yoo, S.A.; You, S.; Yoon, H.J.; Kim, D.H.; Kim, H.S.; Lee, K.; Ahn, J.H.; Hwang, D.; Lee, A.S.; Kim, K.J.; et al. A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis. J. Exp. Med. 2012, 209, 871–886. [Google Scholar] [CrossRef]
  46. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001, 29, e45. [Google Scholar] [CrossRef]
  47. Kenakin, T.P. A Pharmacology Primer: Techniques for More Effective and Strategic Drug Discovery, 5th ed.; Academic Press: San Diego, CA, USA, 2019. [Google Scholar]
Ijms 23 01427 g001 550
Figure 1. Influence of endothelium removal on the vasorelaxation induced by PSB-MZ-1415 in isolated human pulmonary arteries. Results are expressed as the percentage relaxation of the isometric contraction induced by U46619 (n = 3 per group) in comparison to the respective control.
Figure 1. Influence of endothelium removal on the vasorelaxation induced by PSB-MZ-1415 in isolated human pulmonary arteries. Results are expressed as the percentage relaxation of the isometric contraction induced by U46619 (n = 3 per group) in comparison to the respective control.
Ijms 23 01427 g001
Ijms 23 01427 g002 550
Figure 2. Influence of PSBCB–27 (A–D) and O–1918 (A,C) on the vasorelaxation induced by PSB–MZ–1415 (A,C) and PSB–MZ–1440 (B,D) in endothelium-intact isolated human pulmonary arteries. Concentration-response curves (mean ± SEM of 5–7 tissue samples) and representative original traces of the PSB–MZ–1415–(A,C) or PSB–MZ–1440–(B,D) induced relaxation (also in the presence of PSB–CB–27 or O–1918) are shown in the upper and lower panels, respectively. In (A,B), the results are expressed as the percentage relaxation of the isometric contraction induced by U46619. See Table 1 and Table 2 for the statistical analysis and for potencies. CONTROL refers to the effects of the agonists in the absence of the particular antagonist. The effects of increasing concentrations of DMSO (max. < 1%), given repeatedly, are shown as well (B,D). Arrows show the moment of application of a particular concentration of PSB–MZ–1415 or PSB–MZ–1440.
Figure 2. Influence of PSBCB–27 (A–D) and O–1918 (A,C) on the vasorelaxation induced by PSB–MZ–1415 (A,C) and PSB–MZ–1440 (B,D) in endothelium-intact isolated human pulmonary arteries. Concentration-response curves (mean ± SEM of 5–7 tissue samples) and representative original traces of the PSB–MZ–1415–(A,C) or PSB–MZ–1440–(B,D) induced relaxation (also in the presence of PSB–CB–27 or O–1918) are shown in the upper and lower panels, respectively. In (A,B), the results are expressed as the percentage relaxation of the isometric contraction induced by U46619. See Table 1 and Table 2 for the statistical analysis and for potencies. CONTROL refers to the effects of the agonists in the absence of the particular antagonist. The effects of increasing concentrations of DMSO (max. < 1%), given repeatedly, are shown as well (B,D). Arrows show the moment of application of a particular concentration of PSB–MZ–1415 or PSB–MZ–1440.
Ijms 23 01427 g002
Ijms 23 01427 g003 550
Figure 3. Influence of PSB–CB–27 (A–D) and O–1918 (A,C) on the vasorelaxation induced by NAGly (A,C) and Abn–CBD (B,D) in endothelium-intact isolated human pulmonary arteries. Concentration-response curves (mean ± SEM of 7–13 tissue samples) and representative original traces of the NAGly–(A,C) or Abn–CBD–(B,D) induced relaxation (also in the presence of PSB–CB–27 or O–1918) are shown in the upper and lower panels, respectively. In (A,B), the results are expressed as the percentage relaxation of the isometric contraction induced by U46619. See Table 1 and Table 2 for the statistical analysis and for potencies. CONTROL refers to the effects of the agonists in the absence of the particular antagonist. The effects of increasing concentrations of ethanol (max. < 0.6%), given repeatedly, are shown as well (B,D). Arrows show the moment of application of a particular concentration of NAGly or Abn–CBD.
Figure 3. Influence of PSB–CB–27 (A–D) and O–1918 (A,C) on the vasorelaxation induced by NAGly (A,C) and Abn–CBD (B,D) in endothelium-intact isolated human pulmonary arteries. Concentration-response curves (mean ± SEM of 7–13 tissue samples) and representative original traces of the NAGly–(A,C) or Abn–CBD–(B,D) induced relaxation (also in the presence of PSB–CB–27 or O–1918) are shown in the upper and lower panels, respectively. In (A,B), the results are expressed as the percentage relaxation of the isometric contraction induced by U46619. See Table 1 and Table 2 for the statistical analysis and for potencies. CONTROL refers to the effects of the agonists in the absence of the particular antagonist. The effects of increasing concentrations of ethanol (max. < 0.6%), given repeatedly, are shown as well (B,D). Arrows show the moment of application of a particular concentration of NAGly or Abn–CBD.
Ijms 23 01427 g003
Ijms 23 01427 g004 550
Figure 4. Post hoc analysis of the effect of sex and co-morbidities on PSB–MZ–1415–(A–D) and NAGly–(E–H) mediated vasorelaxation in endothelium-intact isolated human pulmonary arteries. The results are presented as the mean ± SEM of n (given in parentheses) tissue samples for each curve. Student’s t-test for unpaired data was used to compare the curves (pEC50 values). (A) p = 0.0096; (B) p = 0.2133; (C) p = 1.0; (D) p = 0.2524; (E) p = 0.2197; (F) p= 0.0174; (G) p = 1.0; (H) p = 0.0022. See Table 3 for further details.
Figure 4. Post hoc analysis of the effect of sex and co-morbidities on PSB–MZ–1415–(A–D) and NAGly–(E–H) mediated vasorelaxation in endothelium-intact isolated human pulmonary arteries. The results are presented as the mean ± SEM of n (given in parentheses) tissue samples for each curve. Student’s t-test for unpaired data was used to compare the curves (pEC50 values). (A) p = 0.0096; (B) p = 0.2133; (C) p = 1.0; (D) p = 0.2524; (E) p = 0.2197; (F) p= 0.0174; (G) p = 1.0; (H) p = 0.0022. See Table 3 for further details.
Ijms 23 01427 g004
Ijms 23 01427 g005 550
Figure 5. Relative mRNA expression level of gpr18 evaluated by real-time qPCR assay in isolated human bronchioles and pulmonary arteries. The RNA isolated from bronchioles was used as the control, and its expression was set 1.0. Results are shown as a relative fold change in mRNA expression in comparison to control. The results are presented as the mean ± SEM of 6 tissue samples. * p < 0.05.
Figure 5. Relative mRNA expression level of gpr18 evaluated by real-time qPCR assay in isolated human bronchioles and pulmonary arteries. The RNA isolated from bronchioles was used as the control, and its expression was set 1.0. Results are shown as a relative fold change in mRNA expression in comparison to control. The results are presented as the mean ± SEM of 6 tissue samples. * p < 0.05.
Ijms 23 01427 g005
Table
Table 1. Vasodilatory effects of NAGly, Abn-CBD, PSB-MZ-1415, and PSB-MZ-1440 on U46619 (0.01 µM)-precontracted isolated human pulmonary arteries (pEC25, pEC50), interaction with PSB-CB-27 and O-1918, and effect of endothelial removal.
Table 1. Vasodilatory effects of NAGly, Abn-CBD, PSB-MZ-1415, and PSB-MZ-1440 on U46619 (0.01 µM)-precontracted isolated human pulmonary arteries (pEC25, pEC50), interaction with PSB-CB-27 and O-1918, and effect of endothelial removal.
GroupnContraction (mN)pEC25pEC50Rmax (%)
NAGly1310.8 ± 2.55.8 ± 0.15.1 ± 0.192.5 ± 5.6
+PSB-CB-27 (1 μM)79.9 ± 1.35.2 ± 0.1 ***aN.D.N.D.
+PSB-CB-27 (10 μM)710.5 ± 1.84.9 ± 0.1 ***aN.D.N.D.
+O-1918 (10 μM) 79.8 ± 2.14.7 ± 0.1 ***aN.D.N.D.
Abn-CBD510.6 ± 2.56.2 ± 0.25.5 ± 0.197.4 ± 4.1
+PSB-CB-27 (10 μM)79.7 ± 1.94.8 ± 0.1 ***bN.D.N.D.
Ethanol 78.9 ± 2.6N.D.N.D.4.8 ± 4.1 ***b
PSB-MZ-1415109.9 ± 1.25.8 ± 0.25.2 ± 0.2100.3 ± 5.9
+PSB-CB-27 (10 μM)610.2 ± 1.34.6 ± 0.1 ***aN.D.N.D.
+O-1918 (10 μM)69.8 ± 1.84.9 ± 0.2 **aN.D.N.D.
+Endothelium310.3 ± 1.15.9 ± 0.15.0 ± 0.1N.D.
−Endothelium311.3 ± 1.24.8 ± 0.1 **bN.D.N.D.
PSB-MZ-1440810.3 ± 2.06.2 ± 0.24.9 ± 0.1106.5 ± 5.3
+PSB-CB-27 (10 μM)69.5 ± 1.24.8 ± 0.1 ***bN.D.N.D.
DMSO1010.5 ± 1.8N.D.N.D.4.9 ± 4.0 ***b
Data are expressed as the mean ± SEM and represent contractile responses to U46619 (0.01 µM). n, number of patients; ** p < 0.01, *** p < 0.001; compared to the respective control as determined by a one-way ANOVA followed by Dunnett’s post hoc test or b Student’s t-test. N.D., not determined.
Table
Table 2. Apparent pA2 values of PSB-CB-27 and O-1918 for their interaction with NAGly, Abn-CBD, PSB-MZ-1415, and PSB-MZ-1440.
Table 2. Apparent pA2 values of PSB-CB-27 and O-1918 for their interaction with NAGly, Abn-CBD, PSB-MZ-1415, and PSB-MZ-1440.
NAGlyAbn-CBDPSB-MZ-1415PSB-MZ-1440
PSB-CB-276.26.36.26.3
O-19186.1N.D.5.8N.D.
Values were calculated from the concentration-response curves shown in Figure 2 and Figure 3 on the basis of the pEC25 values obtained in the presence and absence of the antagonists. The concentration of the antagonists was 10 µM. PSB–CB–27 was studied against NAGly also at 1 µM, and the pA2 given in the table represents the sum of both single pA2 values. N.D., not determined.
Table
Table 3. Patient characteristics, diagnosis, types of operations, medications and post hoc analysis of the effect of sex and co-morbidities on PSB-MZ-1415- and NAGly-mediated vasorelaxation in isolated human pulmonary arteries.
Table 3. Patient characteristics, diagnosis, types of operations, medications and post hoc analysis of the effect of sex and co-morbidities on PSB-MZ-1415- and NAGly-mediated vasorelaxation in isolated human pulmonary arteries.
Characteristics Vasodilatory Effect of:
PSB-MZ-1415NAGly
NRangeMean ± SEMnpEC50Rmax (%)npEC50Rmax (%)
Ethnicity (Polish white)28
Male19 65.0 ± 0.1103.3 ± 5.495.2 ± 0.198.4 ± 5.2
Female9 45.5 ± 0.1 **95.9 ± 10.455.0 ± 0.195.7 ± 10.6
Age (years)2851–7965.2 ± 1.9
BMI (kg/m2)2818.2–34.125.1 ± 0.8
Non-smokers7 45.3 ± 0.1107.3 ± 4.235.5 ± 0.2105.0 ± 8.1
Smokers21 65.1 ± 0.195.9 ± 9.4114.9 ± 0.1 *89.9 ± 6.0
0–10 cigarettes per day5
10–20 cigarettes per day16
Systolic blood pressure (mmHg)28109–152128.5 ± 2.8
Diastolic blood pressure (mmHg)2864–9573.1 ± 1.8
No heart diseases14 55.2 ± 0.197.9 ± 10.665.0 ± 0.299.2 ± 7.0
Heart diseases14 55.2 ± 0.1102.8 ± 6.585.0 ± 0.188.6 ± 5.9
Normocholesterolemia19 35.2 ± 0.195.3 ± 8.395.2 ± 0.199.4 ± 6.2
Hypercholesterolemia9 65.0 ± 0.1101.6 ± 9.354.6 ± 0.1 **81.9 ± 7.5
Arterial hypertension9
Diabetes mellitus4
Reason for surgery
Operation
Tumor staging
Medications		Cancer (27 patients), tuberculoma (1)
Right upper lobectomy (10), right middle lobectomy (1), right lower lobectomy (7), left upper lobectomy (3), left lower lobectomy (5), left pneumonectomy (1)
IA (7), IB (2), IIA (4), IIB (10), IIIA (4)
α1-Adrenoceptor antagonist (3), ACE inhibitors (8), β blockers (7), statins (9), calcium channel blocker (4), potassium channel blocker (1), diuretics (5), hypoglycemic medication (4), anticoagulants (4), protein pump inhibitors (1)
Values of pEC50 and Rmax for vasodilatory effects of PSB-MZ-1415 and NAGly are based on the concentration-response curves in Figure 4 and represent mean percentage relaxations of the isometric contraction induced by U46619 and with the standard error of the mean (SEM) fit to non-linear regression (curve fit). N, number of all patients; n, the number of patients selected for the respective studies with PSB-MZ-1415 and NAGly. * p < 0.05; ** p < 0.01; compared to male, non-smokers and normocholesterolemic patients, respectively, as determined by Student’s t-test for unpaired data. ACE, angiotensin-converting enzyme; BMI, body mass index; pTNM classification (VIIIth edition).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Kozłowska, H.; Malinowska, B.; Baranowska-Kuczko, M.; Kusaczuk, M.; Nesterowicz, M.; Kozłowski, M.; Müller, C.E.; Kieć-Kononowicz, K.; Schlicker, E. GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries. Int. J. Mol. Sci. 2022, 23, 1427. https://doi.org/10.3390/ijms23031427

AMA Style

Kozłowska H, Malinowska B, Baranowska-Kuczko M, Kusaczuk M, Nesterowicz M, Kozłowski M, Müller CE, Kieć-Kononowicz K, Schlicker E. GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries. International Journal of Molecular Sciences. 2022; 23(3):1427. https://doi.org/10.3390/ijms23031427

Chicago/Turabian Style

Kozłowska, Hanna, Barbara Malinowska, Marta Baranowska-Kuczko, Magdalena Kusaczuk, Miłosz Nesterowicz, Mirosław Kozłowski, Christa E. Müller, Katarzyna Kieć-Kononowicz, and Eberhard Schlicker. 2022. "GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries" International Journal of Molecular Sciences 23, no. 3: 1427. https://doi.org/10.3390/ijms23031427

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop