hc-067047 and Hypoxia

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic

Topics: Amides; Animals; Chronic Disease; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Eicosanoids; Hypertension, Pulmonary; Hypoxia; Ion Channel Gating; Male; Mice, Inbred C57BL; Morpholines; Pulmonary Artery; Pyrroles; Serotonin; TRPV Cation Channels; Vasoconstriction

The phenomenon of water intake reduction during the 1(st) day of hypobaric hypoxia has been known for a long time. However, the reason for the same is yet unknown. The transient receptor potential vanilloid (TRPV) channels, including TRPV1 and TRPV4, are located in the subfornical organ (SFO). These are calcium permeable cationic channels gated by various stimuli such as cell swelling, low pH, and high temperature, and participate in anti-dipsogenic effects when activated. We aimed to explore the drinking behavior of rats and the mechanism of TRPVs under hypoxia. Chemical TRPV4 inhibitors (HC-067047 and Gadolinium) or TRPV4 knockout, but not TRPV1 inhibitor SB-705498, could restore the water intake under hypoxia. Hypoxia-mediated direct activation of TRPV4 may be the reason of anti-dipsogenic effects because the serum sodium, pH, and intracranial temperature are unaltered. Interestingly, we found that hypoxia immediately increased the intracellular Ca(2+) concentration ([Ca(2+)]i) in HEK293-TRPV4 cells and primary neurons from SFO region, but not in the HEK293-TRPV1 cells. Moreover, hypoxia-induced [Ca(2+)]i increase depended on the indispensable hemeoxygenase-2 (HO-2) and TRPV4. HO-2 and TRPV4 were also confirmed to form a complex in SFO neurons. These results demonstrated that SFO cells sense hypoxia and activate via the HO-2/TRPV4 multiple channels, which are associated with anti-dipsogenic effects.

Topics: Animals; Calcium; Cell Line; Drinking; Gadolinium; HEK293 Cells; Heme Oxygenase (Decyclizing); Humans; Hydrogen-Ion Concentration; Hypoxia; Male; Morpholines; Neurons; Pyrroles; Pyrrolidines; Rats; Rats, Sprague-Dawley; Sodium; Subfornical Organ; Temperature; TRPV Cation Channels; Urea

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone, and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted pulmonary arteries (PAs) of trpv4(-/-) mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 μM HC-067047 significantly reduced the sensitivity of serotonin (5-HT)-induced contraction in wild-type (WT) PAs but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4(-/-) PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca(2+) response was attenuated by HC-067047 in WT PASMCs but not in trpv4(-/-) PASMCs, suggesting TRPV4 is a major Ca(2+) pathway for 5-HT-induced Ca(2+) mobilization. Nifedipine also attenuated 5-HT-induced Ca(2+) response in WT PASMCs but did not cause further reduction in the presence of HC-067047, suggesting interdependence of TRPV4 and voltage-gated Ca(2+) channels in the 5-HT response. Chronic exposure (3-4 wk) of WT mice to 10% O2 caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4(-/-) mice and HC-067047 had no further effect on the 5-HT induced response. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmaco-mechanical coupling and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH.

Topics: Animals; Calcium Signaling; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Familial Primary Pulmonary Hypertension; Hypertension, Pulmonary; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Pulmonary Artery; Pyrroles; Serotonin; Sulfonamides; Time Factors; TRPV Cation Channels; Vasoconstriction; Vasoconstrictor Agents

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

Topics: Animals; Caffeine; Calcium Channel Agonists; Calcium Signaling; Cell Hypoxia; Cells, Cultured; Dantrolene; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Macrocyclic Compounds; Male; Morpholines; Muscle Contraction; Muscle Relaxants, Central; Myocytes, Smooth Muscle; Oxazoles; Patch-Clamp Techniques; Phorbols; Pulmonary Artery; Pyrroles; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; TRPV Cation Channels