Physiological Functions of Transient Receptor Potential Channels in Pulmonary Arterial Smooth Muscle Cells

Abstract

The transient receptor potential (TRP) gene superfamily, which consists of 7 subfamilies with at least 28 mammalian homologues, is known to encode a wide variety of cation channels with diverse biophysical properties, activation mechanisms, and physiological functions. Recent studies have identified multiple TRP channel subtypes, belonging to the canonical (TRPC), melastatin-related (TRPM), and vanilloid-related (TRPV) subfamilies, in pulmonary arterial smooth muscle cells (PASMCs). They operate as specific Ca²⁺ pathways responsive to stimuli, including Ca²⁺ store depletion, receptor activation, reactive oxygen species, growth factors, and mechanical stress. Increasing evidence suggests that these channels play crucial roles in agonist-induced pulmonary vasoconstriction, hypoxic pulmonary vasoconstriction, smooth muscle cell proliferation, vascular remodeling, and pulmonary arterial hypertension. This chapter highlighted and discussed these putative physiological functions of TRP channels in pulmonary vasculatures. Since Ca²⁺ ions regulate many cellular processes via specific Ca²⁺ signals, future investigations of these novel channels will likely uncover more important regulatory mechanisms of pulmonary vascular functions in health and in disease states.

1 Introduction

In vascular smooth muscle cells (VSMCs), the Ca²⁺ ion serves as a multifunctional messenger responsible for numerous cellular functions, ranging from muscle contraction to gene expression. Depending on the type of agonist and physiological stimulation, [Ca²⁺]_i can be elevated by Ca²⁺ influx through numerous Ca²⁺ pathways on the plasma membrane, including voltage-gated Ca²⁺ channels, receptor- and store-operated Ca²⁺ channels, nonselective cation channels (NSCCs), and Na⁺-Ca²⁺ exchangers, as well as by Ca²⁺ release from the inositol 1,4,5-trisphosphate (IP₃) receptor, ryanodine receptor, and nicotinic acid adenine dinucleotide phosphate (NAADP)-gated intracellular Ca²⁺ stores. Voltage-dependent Ca²⁺ pathways in VSMCs have been well characterized, but the molecular identities and physiological properties of various voltage-independent Ca²⁺ pathways have long been enigmatic. Recent evidence suggests that the mammalian homologues of the Drosophila transient receptor potential (TRP) protein,1 which encode a large repertoire of cation channels, are responsible for many voltage-independent Ca²⁺ pathways in VSMCs.

The TRP superfamily is divided into two groups of a total of seven subfamilies. Group 1 consists of the classical/canonical (TRPC), melastatin-related (TRPM), vanilloid-related (TRPV), ankyrin-related (TRPA), and no mechanoreceptor potential C NOMPC (TRPN) subfamilies; Group 2 includes the polycystin-related (TRPP) and mucolipin-related (TRPML) subfamilies.2 All TRP proteins share the common features of having six transmembrane domains, a pore-forming loop between the fifth and sixth transmembrane segments, and the highly conserved TRP domains. But, they display remarkable diversity in physiological functions, such as cation selectivity and activation mechanisms. They play critical roles in the response to most major external stimuli, including light, sound, chemicals, temperature, and touch. To date, 28 mammalian TRP homologues have been found in a wide variety of cells and tissues, and at least 10 TRPs have been identified as functional channels in VSMCs (Table 7.1). They are implicated in many different vascular functions, such as myogenic response, agonist-induced vasoconstriction, Ca²⁺ and Mg²⁺ homeostasis, VSMC proliferation, and vascular remodeling.3

Table 7.1 Functional TRP channels identified in vascular smooth muscles

2 Expression of TRP Channels in Pulmonary Artery Smooth Muscle Cells

The classical/canonical TRPC subfamily, which is comprised of seven voltage-independent NSCCs, is the best-studied TRP family in pulmonary artery smooth muscle cells (PASMCs). Multiple TRPC messenger RNA (mRNA) transcripts and proteins have been identified. A survey of the literature showed that TRPC1 and TRPC6 are the two major TRPC channels expressed in rat, mouse, and human pulmonary arteries (PAs) and PASMCs (Table 7.2). TRPC3 and TRPC4 also were frequently detected, but their expressions varied depending on species and cell preparation (freshly isolated or cultured), whereas TRPC5 and TRPC7 were generally absent. In contrast, expression of TRPC channels is different in canine PAs. Reverse-transcription polymerase chain reaction (RT-PCR) detected TRPC4, TRPC6, and TRPC7 mRNAs in canine main PAs, with TRPC4 the major expressed isoform.4 A study showed that TRPC1 and TRPC6 levels are higher in distal than in proximal PAs of rats,5 suggesting heterogeneity of TRPC expression exists in different locations along the pulmonary vascular tree.

Table 7.2 Expression of TRP channels in PASMCs

Compared to TRPC channels, information on other TRP subfamilies in the pulmonary vasculature is scant. We previously performed a survey on the expression of TRPM and TRPV channels in deendothelialized rat intralobar PAs and aorta.6 The mRNA of TRPM2, TRPM3, TRPM4, TRPM7, and TRPM8 of the TRPM family and TRPV1, TRPV2, TRPV3, and TRPV4 of the TRPV family were detected in both PAs and aorta. The ranks of relative expression evaluated by quantitative real-time RT-PCR were TRPV4 > TRPV2 > TRPV1 >> TRPV3, and TRPM8 > TRPM4 > TRPM7 > TRPM3 > TRPM2 > TRPM5. Expression of TRPM2, TRPM8, TRPV1, and TRPV4 proteins in PAs was also verified by Western blot. Moreover, the TRPM8 agonist menthol or the TRPV4 agonist 4α-phorbol 12,13-didecanoate (4α-PDD) evoked a Ca²⁺ response in PASMCs. These responses could be abolished by the removal of Ca²⁺ or application of Ni²⁺ but were unaffected by nifedipine. These results indicate that multiple TRPM and TRPV channels are expressed, and at least TRPM8 and TRPV4 channels are functional Ca²⁺ influx pathways in PASMCs. Expression of other TRP subfamilies (TRPA, TRPP, and TRML) has not been reported in pulmonary vasculature.

3 Physiological Functions of TRP Channels in PASMCs

3.1 Store-Operated and Receptor-Operated Ca²⁺ Entry

Store-operated Ca ²⁺ entry (SOCE) is defined as the capacitative Ca²⁺ entry activated by the depletion of intracellular Ca²⁺ stores.7 Several mechanisms, including the diffusible calcium influx factor (CIF), exocytosis, and conformational coupling, have been proposed. Studies have established that the stromal interacting molecule 1 (STIM1), a transmembrane protein with an N-terminal EF-hand Ca²⁺-binding domain, acts as a Ca²⁺ sensor of endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca²⁺ content.8 Depletion of ER/SR Ca²⁺ results in rearrangement of STIM1 in the form of punctae underneath the plasma membrane and subsequent activation of Store-operated cation channel (SOCC). Moreover, the homologue STIM2 may operate as another Ca²⁺ sensor capable of detecting a small decrease in ER/SR [Ca²⁺] and triggering SOCE for feedback regulation of basal cytosolic and ER Ca²⁺ levels.9 In vascular smooth muscle, SOCE is mostly as nonselective cation pathways, in contrast to the highly Ca²⁺-selective Ca²⁺ release-activated current (I _CRAC) recorded in T lymphocytes.10 Receptor-operated Ca²⁺ entry (ROCE) is ­usually defined loosely as voltage-independent Ca²⁺ entry that requires ligand binding to membrane receptors for activation. In fact, the definitions of SOCE and ROCE are not mutually exclusive, and the distinction between the two pathways sometimes is rather murky. For example, an agonist binds to a G_q protein-coupled receptor and activates phospholipase Cβ (PLC-β) to generate IP₃ and diacylglycerol (DAG), leading to dual activation of SOCE and ROCE.

Since their discovery, TRPC channels have been implicated as SOCC and Receptor-operated cation channels (ROCC) because they are nonselective Ca²⁺-permeable channels and are activated by a PLC-dependent mechanism.11 It is now clear that TRPC1, TRPC4, and TRPC5 represent a subgroup of TRPC channels activated by Ca²⁺ store depletion caused by inhibition of SR Ca²⁺-ATPase (adenosine triphosphatase) using cyclopiazonic acid (CPA) or thapsigargin. TRPC3, TRPC6, and TRPC7 form another subgroup of channels participating in ROCE activated directly by DAG independent of protein kinase C (PKC). In a heterologous expression system, members of the TRPC1/4/5 or TRPC3/6/7 subgroup can coassemble directly to form heteromeric channels, but cross association does not occur between members of the two subgroups.2,3,12 However, some studies showed that TRPC3, TRPC4, and TRPC5 can operate as both SOCC and ROCC.13 Studies found that STIM1 not only can directly interact with TRPC1, TRPC4, and TRPC5 to activate SOCE14 but also can mediate heteromerization of TRPC3 with TRPC1 and TRPC6 with TRPC4.15 These new findings raise an important question of whether SOCE and ROCE are separate Ca²⁺ pathways or the same pathway due to heteromerization of various TRPC channels in native cells.

Our previous study provided evidence that SOCE and ROCE are independent Ca²⁺ pathways in rat intralobar PASMCs, where the putative store-operated TRPC1 and receptor-operated TRPC6 are predominantly expressed.16 Direct activation of SOCE with thapsigargin and ROCE with the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) elicited distinctive cation entries that exhibited a 1,000-fold difference in their sensitivity to La³⁺ (IC₅₀ of ∼0.3 μM for SOCE and ∼300 μM for ROCE). Small interfering RNA (siRNA) knockdown of TRPC1 and TRPC6 inhibited the thapsigargin- and OAG-activated cation entry, respectively. Furthermore, TRPC1 siRNA had no effect on OAG-induced cation entry, and TRPC6 siRNA did not alter the thapsigargin-induced response. These results indicate that thapsigargin-induced SOCE and OAG-mediated ROCE are mutually independent pathways, with TRPC1 and TRPC6 the major determinants of SOCE and ROCE, respectively, in rat intralobar PASMCs.

The notion that TRPC1 is critical for SOCE is consistent with other observations that TRPC1-specific antisense oligonucleotides inhibited the TRPC1 expression and blocked SOCE in cultured human PASMCs,17 and overexpression of TRPC1 in rat PAs enhanced SOCE-induced vasoconstriction.18 Moreover, the importance of TRPC6 in ROCE is supported by the observation that OAG failed to activate ROCC in PASMCs of trpc6 ^-/- mice.19 However, a study in cultured rat main PASMCs showed that PDGF (platelet-derived growth factor) upregulated TRPC6 and enhanced SOCE during cell proliferation. Inhibition of TRPC6 with antisense oligonucleotides reduced the amplitude of SOCE and attenuated mitogen-mediated PASMC proliferation. Hence, TRPC6 might exhibit different properties in proliferating PASMCs, perhaps due to heteromerization with other TRPC subtypes through interactions with STIM1 or Orai1.15,20 In addition, TRPC3 and TRPC4 have been reported to mediate SOCE in cultured human PASMCs,21,22 but the TRPC subtypes responsible for ROCE have not been examined in human pulmonary myocytes.

3.2 Agonist-Induced Pulmonary Vasoconstriction

As mentioned, many vasoactive agonists and growth factors can exert their effects by binding to receptors coupled to G_q protein or receptor tyrosine kinases to activate PLC-β or PLC-γ, respectively, to generate IP₃ and DAG. IP₃ triggers Ca²⁺ release from IP₃-receptor-gated Ca²⁺ stores, leading to Ca²⁺ influx through SOCC, and DAG directly activates ROCC and modulates other effectors through PKC. In addition to the PLC-IP₃/DAG pathways, agonists and growth factors may activate receptor-operated TRP channels by direct tyrosine phosphorylation through Src family protein tyrosine kinases, including Src and Fyn.23,24

Vasoactive agonists, including endothelin I, angiotensin II, norepinephrine, PDGF, EGF (epidermal growth factor), and ATP (adenosine triphosphate) have been shown to activate SOCE or ROCE in VSMCs. It is generally assumed that TRPC channels, by mediating SOCE and ROCE, play significant roles in agonist-induced pulmonary vasoconstriction. However, despite evidence that SOCE is able to elicit pulmonary vasoconstriction18,25 and phenylephrine-induced contractions of PAs could be inhibited partially by the nonselectively cation channel blockers SKF-96365 and Ni²⁺,25,26 it is unclear which subtypes of TRPC channels are participating and what contributions TRPC channels have in a particular agonist-induced pulmonary vasoconstriction. In fact, our preliminary observations suggested that agonist-induced vasoconstriction was unabated in PAs of trpc1 ^-/- and trpc6 ^-/- mice (Ref. 27 and unpublished data), similar to a previous report on phenylephrine-induced vasoconstriction in aorta and mesenteric arteries of trpc6 ^-/- mice.28 It is likely that agonists activate vasoconstriction through redundant mechanisms; hence, deletion of one TRP channel subtype could be effectively compensated by other Ca²⁺ pathways. Moreover, 5-hydroxytryptamine or serotonin (5-HT)-induced constriction of small, pressurized rat PAs is mediated through an arachidonic acid-sensitive Ca²⁺ influx,29 which has properties similar to the TRPV4 channels.30 These results highlight the fact that some agonists may induce ROCE and pulmonary vasoconstriction by activating NSCCs other than TRPC channels through PLC-independent signaling pathways.

3.3 Hypoxic Pulmonary Vasoconstriction

Acute reduction in alveolar O₂ tension causes reversible constriction of small resistant PAs. It serves as an adaptive mechanism for diverting blood flow from poorly ventilated to better ventilated regions of the lung to improve ventilation-perfusion matching. It has been proposed that hypoxia alters the redox state of PASMCs, leading to the inhibition of voltage-gated K⁺ channels, membrane depolarization, activation of voltage-gated Ca²⁺ channels, [Ca²⁺]_i increase, and vasoconstriction.31 Increasing evidence suggests that voltage-independent Ca²⁺ entry also plays a critical role in hypoxic pulmonary vasoconstriction (HPV). It was shown first in canine PAs that hypoxia activated a nisoldipine- and ryanodine-insensitive Ca²⁺ influx in the presence of thapsigargin or CPA32 and later in small rat PAs that the initial phase (phase I) of HPV was carried by capacitative Ca²⁺ influx related to a thapsigargin-sensitive store. Phase II contraction was supported by Ca²⁺ influx through a separate voltage-independent pathway.33 In isolated canine and rat PASMCs, hypoxia activated a dihydropyridine-insensitive Ca²⁺ influx, which had a pharmacological profile similar to SOCE.34,35 Antagonists of SOCC/NSCC, including SKF-96365, Ni²⁺, and La³⁺, inhibited HPV in isolated perfused rat lungs at concentrations that inhibit vasoconstrictions induced by SOCE but not by KCl.36

A study showed that the expression levels of STIM1, TRPC1, TRPC4, and TRPC6 were higher in distal than proximal PAs of rats. Enhanced expression of these proteins was associated with a higher magnitude of CPA-induced SOCE and hypoxia-induced Ca²⁺ response in distal PASMCs.5 Moreover, knockdown of the SR Ca²⁺ sensor STIM1 with siRNA in rat PASMCs abolished SOCE and hypoxia-induced Ca²⁺ response.37 Collectively speaking, evidence at the organ, tissue, and cell levels suggests unequivocally that SOCCs/NSCCs participate in HPV, even though the specific TRP channel has not been determined. These observations together with other studies lead to an alternative hypothesis that HPV activates Ca²⁺ release from ryanodine receptor-gated Ca²⁺ stores, causing activation of SOCE.

Direct evidence of a critical role for TRP channels in HPV emerged from the study of trpc6 ^-/- mice.19 The evidence includes the following: The acute phase of HPV was completely absent in isolated perfused lungs of trpc6 ^-/- mice; the hypoxia-induced increase in [Ca²⁺]_i, nonselective cation influx, and membrane currents were missing in Endothelin-1 (ET-1)-primed trpc6- ^/- microvascular PASMCs; and hypoxia-induced Ca²⁺ response was rescued by expressing TRPC6 in trpc6 ^-/- PASMCs.It is intriguing that the hypoxia-induced activation of TRPC6 channels required priming of PASMCs with ET-1 and was mediated by DAG accumulation. These observations are congruent with the widely accepted knowledge that priming of isolated perfused lung, PAs, or PASMCs with an agonist facilitates robust hypoxic responses, but the DAG-mediated activation of TRPC6 indicates a crucial contribution of ROCE instead of the previously suggested SOCE in HPV. It is unclear whether the discrepancy depends on animal species or the presence of priming. It will be important to evaluate the DAG/TRPC6 mechanism in agonist-primed PASMCs of rats and other species for a better understanding of ROCE involvement in HPV.

The TRPC channels may contribute to HPV by providing Ca²⁺ for direct activation of calmodulin/myosin light chain kinase/actin-myosin interactions and by causing membrane depolarization to activate Ca²⁺ influx through L-type Ca²⁺ channels. This is consistent with the fact that both antagonists of SOCE/NSCC and L-type Ca²⁺ channels attenuated independently the hypoxia-induced Ca²⁺ response in PASMCs and HPV in isolated perfused lungs.34,36 Moreover, hypoxia-induced activation of TRPC channels may lead to local accumulation of Na⁺ ions, facilitating Ca²⁺ influx through reverse Na⁺-Ca²⁺ exchange. However, this possibility is still controversial and requires further investigation.

3.4 PASMC Growth and Proliferation

Mitogen-induced proliferation of PASMCs requires elevation of [Ca²⁺]_i to activate Ca²⁺-dependent signaling pathways and Ca²⁺-sensitive transcription factors. Based on a series of articles published by Yuan and associates, it is now established that PASMC proliferation is associated with augmented SOCE mediated by TRPC channel upregulation.38 Depending on species, mitogens, and physiological states, most of the TRPC subtypes expressed in PASMCs, including TRPC1, TRPC3, TRPC4, and TRPC6, have been implicated as a mediator for cell proliferation. In human PASMCs, cell proliferation induced by serum and growth factors was associated with increased resting [Ca²⁺]_i, enhanced capacitative Ca²⁺ entry, and upregulation of TRPC1.17,39 Downregulation of TRPC1 using antisense oligonucleotide, removal of extracellular Ca²⁺, and application of Ni²⁺ all inhibited the enhancement of cell proliferation induced by serum and growth factors, suggesting Ca²⁺ influx via the upregulated TRPC1 mediates PASMC growth. In another study, incubation of human PASMCs with ATP increased phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), TRPC4 expression, SOCE, and cell proliferation. Transfection of a CREB mutant abolished ATP-induced TRPC4 upregulation, and introduction of a siRNA against TRPC4 attenuated SOCE and cell proliferation activated by ATP. These data suggested that ATP exerts its mitogenic effect via CREB-dependent upregulation of TRPC4 channels. Furthermore, PDGF stimulates Signal transducer and activator of transcription 3 (STAT3) phosphorylation, leading to the upregulation of c-Jun, which activates the transcription of TRPC6, resulting in enhanced SOCE and PASMC proliferation.40,41

It is interesting to note that the mentioned studies involved different mitogens, signaling pathways, and TRPC channels; yet they all found enhancement in SOCE and cell proliferation. The simplest explanation is that an enhanced SOCE, irrespective of the TRPC subtype involved, supports the mitogen-induced PASMC proliferation in a nondiscriminatory manner. However, Ca²⁺ signals carried by distinctive Ca²⁺ channels/pathways are known to preferentially regulate specific Ca²⁺-sensitive transcription factors for different physiological processes, according to the signal amplitude and frequency as well as the spatial association with their effectors.42 Indeed, PGDF-induced PASMC proliferation was unaffected by the L-type Ca²⁺ channel inhibitor nifedipine but was inhibited by the SOCE/NSCC inhibitor SKF-96365, suggesting a specific contribution of SOCE-dependent signaling pathways.41 Hence, it is tempting to speculate that various mitogens might regulate different TRPC subtypes to modulate specific Ca²⁺-dependent processes at various stages in the cell cycle.

3.5 Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH), both idiopathic (IPAH) and secondary PAH, involves numerous interacting factors that lead to massive vascular remodeling, increase in vasomotor tone, and alterations in vascular reactivity. Vascular remodeling is characterized by pronounced medial and adventitial thickening due to PASMC proliferation and migration, recruitment and differentiation of progenitor cells, and synthesis of extracellular matrix. Increase in vasomotor tone is evident by the acute reduction in pulmonary arterial pressure (Ppa) in response to vasodilators, such as prostacyclin, nitric oxide, phosphodiesterase V inhibitors, K⁺ channel opener, and Rho kinase inhibitors. Alterations in vascular reactivity are manifested by the enhanced responsiveness to agonists, such as endothelin 1, serotonin, and angiotensin II, and the reduced relaxation to various vasodilators.

3.5.1 Chronic Hypoxia-Induced Pulmonary Hypertension

All the salient characteristics of PAH can be either directly or indirectly related to alterations of Ca²⁺ homeostasis in PASMCs. It has been shown in the chronic hypoxic rat model that major changes in ionic balance occur in PASMCs, including membrane depolarization, reduction in K _V currents, and increase in resting [Ca²⁺]_i. The increase in resting [Ca²⁺]_i in PASMCs is mainly due to Ca²⁺ influx through voltage-independent Ca²⁺ channels because removal of extracellular Ca²⁺ reduces resting [Ca²⁺]_i to the level of normoxic PASMCs, but inhibition of the voltage-gated Ca²⁺ channel with nifedipine has little effect.16,43 In the search of alternative Ca²⁺ entry pathways for the disturbance of resting [Ca²⁺]_i, we found that the expression of TRPC channels in PAs is greatly altered in rats exposed to 10% O₂ for 4 weeks.16 TRPC1 and TRPC6 mRNA and protein levels were more than doubled, while TRPC3 was unchanged in hypoxic PAs. Ca²⁺ measurements and Mn²⁺-quenching experiments showed that both thapsigargin-induced SOCE and OAG-induced ROCE were enhanced proportionally in hypoxic PASMCs. More importantly, inhibition of SOCE with a low concentration of La³⁺ (10 μM) caused a reduction in basal [Ca²⁺]_i similar to the removal of extracellular Ca²⁺, but increased La³⁺ concentration to inhibit ROCE failed to cause additional decline in [Ca²⁺]_i. Similar results were obtained in parallel experiments using PA rings to evaluate basal vascular tone. Since TRPC1 is responsible for SOCE in rat PASMCs,16 these results suggested that the upregulation of TRPC1 and SOCE contribute to the elevated [Ca²⁺]_i in PASMCs and the increase in basal vascular tone of PAs of chronic hypoxic rats.

A subsequent study further extended our findings showing that TRPC1 and TRPC6 upregulation also occurred in cultured PASMCs incubated under 4% O₂ for 60 h, and in nonhypoxic PASMCs overexpressing hypoxia-­inducible factor 1α (HIF-1α).44 The increase of TRPC expression was absent when mice of partial HIF-1α deficiency were exposed to hypoxia. These results clearly indicate that the upregulation of TRPC channels is a direct effect of hypoxia on PASMCs mediated by HIF-1α.

However, several important questions remain to be answered. Are TRPC1 and TRPC6 essential for the development of hypoxic PAH? Are they required specifically for vascular remodeling, increased Ppa, and enhanced vasoconstriction to agonists in PAH? It had been reported that the elevation of right ventricular systolic pressure (RVSP), right heart hypertrophy, and pulmonary vascular remodeling were unaltered in chronic hypoxic trpc6 ^-/- mice.19 This argued against an indispensable role for TRPC6 in chronic hypoxia-induced PAH. In contrast, our preliminary study found that hypoxia-induced PAH was significantly attenuated in trpc1 ^-/- mice.27 This further supports the idea that the store-operated TRPC1 channels participate in the development of hypoxic PAH.

We recently have extended our investigation on the roles of TRPM and TRPV channels in chronic hypoxic PAH.45 Preliminary experiments showed that among the six subtypes of TRPV and eight subtypes of TRPM, the mechanosensitive TRPV4 was the only channel that was upregulated in PAs of rats exposed to hypoxia. Upregulation of TRPV4 was associated with enhanced Ca²⁺ response induced by the TRPV4 agonist 4α-PDD and hypotonicity in hypoxic PASMCs. Significant myogenic tone, sensitive to the TRPV blocker ruthenium red, was also observed in pressurized pulmonary microvessels of chronic hypoxic but not ­normoxic rats. Moreover, the severity of PAH was significantly attenuated in trpv4 ^-/- mice exposed to hypoxia. These results suggest that the mechanosensitive TRPV4 channels are upregulated in PASMCs during chronic hypoxia and play significant roles in the development of myogenic tone and PAH. A schematic is presented in Fig. 7.1 to depict our present understandings on the participation of TRPC and TRPV channels in chronic hypoxia-induced PAH.

Fig. 7.1

Schematic diagram depicting the involvement of TRP channels in chronic hypoxic PAH. Hypoxia activates release of ROS (reactive oxygen species), agonists, and growth factors, leading to SOCE, ROCE, and Non-selective cation entry (NSCE) through TRPC1, TRPC6, and TRPV4 channels, respectively. Increase in intracellular [Ca²⁺]_i enhances basal vascular tone and agonist-induced responses to cause pulmonary vasoconstriction. Increase in Ppa activates mechanosensitive TRPV4 channels to elicit myogenic response. Furthermore, HIF and other Ca²⁺-dependent transcriptional factors mediate gene transcription to upregulate TRPC1, TRPC6, and TRPV4 expression and promote cell proliferation and vascular remodeling

3.5.2 Idiopathic Pulmonary Arterial Hypertension

In addition to chronic hypoxic PAH, a link has also been established between TRPC and IPAH. Elevated levels of TRPC6 and TRPC3 mRNA and protein were found in cultured PASMCs isolated from patients with IPAH but not in PASMCs of patients with secondary PAH or those who were not hypertensive.46 The enhanced TRPC expression appears to be responsible for the higher activity of SOCE and cell proliferation in IPAH PASMCs21,41,46 because downregulation of TRPC6 by siRNA blocked the accelerated proliferation of these cells.46 The underlying cause for the enhanced TRPC expression in IPAH PASMCs is unclear. It is unrelated to mitogenic or autocrine factors released during PAH because elevated TRPC expression was observed in cells after multiple passages. However, it could be a specific phenotype of a population of PASMCs or myofibroblasts that are selectively expanded or recruited during the development of IPAH. It is important to note that the ET receptor antagonist bosentan suppressed TRPC6 expression and proliferation of IPAH PASMCs in the absence of agonist41; and PASMCs from normal subjects and patients with IPAH showed divergence in response to cAMP-dependent regulation on TRPC3 expression and SOCE.21 These observations suggest that aberrations in the constitutive activities of endogenous receptors and transcriptional pathways may contribute to the enhanced TRPC expression in IPAH PASMCs.

4 Conclusion

Ever since the discovery of the first TRP channel 20 years ago,1 TRP channels have continuously amazed the scientific community by their incredibly diverse complexity in structural interactions, modes of activation, molecular regulations, and physiological functions. Novel discoveries about these channels have been reported in a daily basis (>400 papers in 2008). In comparison, research on TRP channels in pulmonary vasculature is still in its infancy. The functions and regulations of most TRP channels of PASMCs, especially members of TRPV, TRPM, TRPA, TRPP, and TRPML subfamilies, have not been explored. Since Ca²⁺ ions regulate multiple cellular processes via specific Ca²⁺ signals from diverse pathways, future investigations of these unexplored TRP channels will likely uncover important regulatory pathways for pulmonary vascular functions in health and in disease states.