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  • br Materials and methods br Results br Discussion In

    2022-08-04


    Materials and methods
    Results
    Discussion In this study, we demonstrated that zaprinast (a cGMP-PDE inhibitor) induced the intracellular calcium mobilization in the cpi 1 coexpressing GPR35 and Gqi5, Gqo5, or Gα16. Induction of intracellular calcium mobilization by zaprinast in the GPR35-expressing transfectants was due to selective activation of the GPR35-Gα (Gqi5, Gqo5, or Gα16) signaling pathway, rather than inhibition of phosphodiesterases (PDE5, PDE6, PDE9, PDE10, and PDE11), direct activation of Gα proteins, or non-selective stimulation of endogenous GPCR-Gα signaling pathway, because expression of both GPR35 and Gα proteins (Gqi5, Gqo5, or Gα16) was necessary preconditions for this action of zaprinast (Fig. 1, Fig. 3). Thus, these observations strongly suggest that zaprinast acts as an agonist for GPR35. Zaprinast potently induced intracellular calcium mobilization in the transfectant coexpressing rGPR35 and Gqi5 with an EC50 value of 16nM (Fig. 3), while effects of zaprinast on PDEs are moderate or weak (see Section 1). In addition, the selective PDE5/PDE6 inhibitors T-0156 and T-1032 did not have any effect on the transfectants coexpressing GPR35 and Gqi5 (Fig. 4B). Furthermore, 8Bromo-cGMP did not induce intracellular calcium mobilization in our assay (Fig. 4B). These facts also support our conclusion. Several recent reports have suggested that zaprinast may possess pharmacological activities other than PDEs inhibition. For example, Wibberley et al. demonstrated a nitric oxide (NO)-independent role for zaprinast in the regulation of urethral sphincter tone [15]. Yoon et al. reported that intrathecal zaprinast had an antinociceptive effect in the rat formalin test, and that this effect was not related to the NO-cGMP-potassium channel pathway [16]. Because cGMP PDEs are deeply involved in the NO-cGMP signaling pathway (NO activates soluble guanylyl cyclase to increase the intracellular cGMP level, while cGMP PDE terminates NO/cGMP-dependent signals by degrading cGMP) [17], these NO/cGMP-independent effects of zaprinast may be mediated by GPR35 activation. Numerous reports about pharmacological studies of PDEs inhibition by zaprinast have been published [7]. However, it may be necessary to repeat those experiments with different structural classes of selective PDE inhibitors since the GPR35 agonist activity of zaprinast was revealed in this study. It is important to note that zaprinast is a lead compound for sildenafil (Viagra™) [7], indicating that zaprinast has favorable chemical properties for drug design. It may be feasible to identify potent and selective agonists and/or antagonists for hGPR35 without phosphodiesterase inhibitory activity from among compounds related to zaprinast. In fact, we have already identified a number of GPR35 agonists with different potency and species selectivity among zaprinast derivatives (Taniguchi et al, Patent Application WO2005085867(A2)), suggesting that zaprinast may serve as a lead compound to develop drugs that modulate GPR35 activity. During the review process of this manuscript, Wang et al. reported that kynurenic acid was a natural ligand for GPR35 [18]. They independently showed that GPR35 acted as a Gαi/o- and Gα16-coupled receptor using kynurenic acid, which was consistent with our results obtained with zaprinast. However, affinity of kynurenic acid for GPR35 is relatively low (EC50 values of 7.4–39.2μM) [18]. Thus, other chemical classes of natural ligand with higher affinity may be expected. Because GPR35 shares homology with GPCRs belonging to the P2Y family [2] and zaprinast is a xanthine derivative [7], our findings may provide a hint to discover the natural ligand for GPR35.
    Acknowledgments
    Objectives
    Methods
    Results Ischemic/hypoxic injury increased GPR35 expression in cardiomyocytes in a time-depended manner (P≤0.05). GPR35 downregulation and calpain inhibitor protected cardiomyocytes from MI injury with preserved contractile function of heart (P≤0.05), decreased fibrosis area (P≤0.05), decreased mitochondrial fragmentation and ROS overload (P≤0.05). The increased level of Calpain1/2 expression triggered by MI injury had been decreased by GPR35 siRNA (P≤0.05). What’s more, calpain2 upregulation reversed the protective function of GPR35 siRNA(P≤0.05).