GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries

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.


Introduction
GPR18 is an orphan G protein-coupled receptor, which, although showing little structural similarity to cannabinoid CB 1 and CB 2 receptors (identity of amino acid sequence of 13 and 8%, respectively; [1]), responds to the natural cannabinoid ∆ 9 -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 CB 1 /CB 2 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].
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).

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 EC 60 ). 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.

Interaction of PSB-CB-27 and O-1918 with the Agonist-Induced Relaxation in Huma Pulmonary Arteries
To investigate the involvement of GPR18 in the effect of the four agonist interaction with the new GPR18 antagonist PSB-CB-27 was studied and compared previously proposed antagonist O-1918. The concentration-response curves for t   Table 3). The potency of NAGly (pEC 50 ) 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. 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.  Table 3 for further details.   Table 3 for further details.

gpr18 mRNA Expression in Human Bronchioles and Pulmonary Arteries
In order to confirm gpr18 mRNA distribution in lung tissue a whether gpr18 presents a differential expression pattern in bronchiole arteries, a real-time quantitative PCR (qPCR) analysis of the gen performed. The results showed that gpr18 mRNA expression was high arteries compared to bronchioles ( Figure 5).

Discussion
Our previous studies on the human pulmonary artery revealed that several cannabinoids including Abn-CBD [25], anandamide (AEA; [26]), cannabidiol [18], 2-arachidonoy lglycerol [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 A 2 analogue U46619. Thromboxane A 2 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 (pEC 50 ) 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 G i protein coupling [4] could not be unambiguously confirmed so far [36]. The reason why the pEC 50 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 (pEC 50 ) 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 pEC 50 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 hCB 1 and hCB 2 receptors, pIC 50 < 5), exhibits a pIC 50 of 6.2 at the hGPR18 in the βarrestin recruitment assay [33]. Its antagonistic potency is identical to the apparent pA 2 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 pA 2 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 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 antinociceptive 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].

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% O 2 and 5% CO 2 (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.

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 EC 60 . 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).

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 -AATCC AGCAGGTCAGCAAAG-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].

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): 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 (pEC 50 = −logEC 50 or pEC 25 = −logEC 25 , where EC 50 or EC 25 is the concentration (M) of the agonist that produced 50 or 25% of the maximal effect (R max )) [47].
Antagonist-or endothelium removal-induced rightward shifts of the CRCs relative to the control curve were calculated on the basis of the EC 25  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.

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.