TRP Channels in Hypertension

Pulmonary Arterial Hypertension and Essential Hypertension

In all vascular beds, precise regulation of circulatory hemodynamics is predominantly controlled by the cardiac output and vascular resistance. The pulmonary circulation, being a high flow, low-pressure system, has less than one tenth of the flow resistance observed in the high-pressure systemic vasculature.

Essential hypertension (or primary systemic arterial hypertension) is characterized by a sustained blood pressure over 140/90 mmHg. In the early stages of the disease, cardiac output is increased although total peripheral resistance remains constant; cardiac output drops when the disease is sustained and total peripheral resistance is elevated, with changes in peripheral resistance mainly reflecting the degree of arterial tone. Over 90% of all cases of adult systemic arterial hypertension have no clear cause and are therefore referred to as essential or primary hypertension.

Pulmonary arterial hypertension (PAH) develops when the mean pulmonary artery pressure is sustained at an elevated level ≥25 mmHg at rest or 30 mmHg during exercise and is associated with a progressive and sustained increase in pulmonary vascular resistance. Most of the patients with PAH have normal cardiac output, indicating that increased pulmonary vascular resistance is the major cause for the elevated pulmonary artery pressure in these patients. PAH is characterized by progressive pulmonary vasculopathy which, if left untreated, leads to right heart failure with poor prognosis. Increased pulmonary vascular resistance in PAH patients may be caused by sustained pulmonary vasoconstriction and considerable obstruction of the lumen of small arteries caused primarily by excessive proliferation of pulmonary artery smooth muscle cells (PASMC) in the vascular wall.

Based on the most recent classification of pulmonary hypertension, the two predominant forms of PAH are currently classified as idiopathic and familial PAH. Idiopathic PAH is rare, principally affecting women, and its underlying cause remains undetermined. Familial PAH represents hereditary communication of PAH and is thought to be the cause of 6–10 % of PAH cases. Familial PAH is genetically predicted where successive family generations develop PAH. Pulmonary vascular intimal and medial hypertrophy is the characterizing features of idiopathic and familial PAH.

The pulmonary circulation is essential to the maintenance of sufficient circulating O₂ levels. When alveolar O₂ levels are compromised or partial pressure of O₂ is lower than 60 mmHg, it responds uniquely by constricting pulmonary arteries and diverting blood flow to the well-ventilated areas in the lung to ensure maximal oxygenation of the venous blood by adequately matching ventilation with perfusion. The alveolar hypoxia-mediated vasoconstrictive phenomenon is known as hypoxic pulmonary vasoconstriction. Vasoconstriction may also be triggered by a number of receptor-activated mechanisms involving endothelin-1 (ET-1) acting on ET_A and ET_B receptors [1], angiotensin II acting on AT₁ receptors [2], and endoperoxides diffusing from endothelial cells acting on smooth muscle cell (SMC) TP-receptors [3]. Sustained pulmonary vasoconstriction is often accompanied by vascular remodelling, i.e., the muscularization of smaller arteries and arterioles due to SMC proliferation and migration. In severe forms of pulmonary hypertension (such as idiopathic and familial PAH), pulmonary artery remodelling is extensive resulting from intimal fibrosis and medial hypertrophy. Such occlusion of the pulmonary arterioles is associated with the formation of plexiform lesions resultant of the proliferation of endothelial cells, migration and proliferation of SMC, and accumulation of circulating cells (including macrophages and endothelial progenitor cells).

In both pulmonary and systemic circulation, the blood flow and intraluminal pressure are mainly regulated by changes in vessel diameter (or radius). The contractility and proliferation of smooth muscle is reliant upon increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The fundamental systems coordinating changes in [Ca²⁺]_i are: a) Ca2+ entry via voltage-dependent Ca²⁺ channels, receptor-operated cation channels (ROC), and/or store-operated channels (SOC); b) Ca²⁺ release and sequestration from and into the sarcoplasmic (SR) or endoplasmic (ER) reticulum; c) Ca²⁺ extrusion to the extracellular space by Ca²⁺-Mg²⁺ ATPase pumps; d) outward transportation of Ca²⁺ by the forward mode of Na⁺-Ca²⁺ exchangers (NCX) and inward transportation of Ca²⁺ by the reverse mode of NCX; e) mitochondrial Ca²⁺ release and sequestration; and f) release and sequestration from and into other intracellular Ca²⁺ stores (e.g., lysosomes).

The precise molecular entity of ROC and SOC, in particular, remained indefinable until recently. It is now believed that transient receptor potential channels (TRPs) participate in the formation of functional ROC and SOC in the vasculature. TRP channels have emerged at the forefront of research in the physiological and pathophysiological regulation of the vasculature having a wide tissue distribution and diversity of functions. This review examines the current state of knowledge of TRP expression and function in the vasculature, and the potentially pivotal pathophysiological changes in the expression of TRP channels associated with both pulmonary and essential hypertension. Both essential and pulmonary hypertension can lead to cardiac hypertrophy, which itself is also highly dependent on the expression and function of TRP-encoded cation channels. The role of TRPs in cardiac function is not discussed in the review, but readers are invited to peruse related articles for more information [4–8].

Transient Receptor Potential channels (TRP)

TRP channels belong to the superfamily of cation channels formed by tetramers of six transmembrane domains subunits which enclose a pore near the C-terminal end [9] (Fig. 1). Unlike voltage-gated ion (Ca²⁺ and K⁺) channels, TRP subunits do not possess a voltage-sensing moiety, making their activity insensitive to changes in membrane potential. TRP channels therefore function as voltage-independent, non-selective cation channels which are permeable to Na⁺, K⁺, Cs⁺, Li⁺, Ca²⁺, and Mg²⁺ [10]. TRP subunits are split into several subfamilies according to their activation stimuli and the presence of regulatory domains on the cytosolic N- and C-termini (Fig. 1). Canonical TRP (TRPC) is activated by G protein-coupled receptors and receptor tyrosine kinases linked to phosphoinositide hydrolysis via phospholipase C (PLC) activation [11, 12] (Fig. 2). A variety of chemical and physical stimuli including capsaicin, lipids, acid, heat, shear stress, and hypoosmolarity can activate vanilloid receptor related TRP (TRPV). Melastatin related TRP (TRPM) [11] are either constitutively active or activated in response to increased [Ca²⁺]_i, oxidative stress, or exposure to the cold. Less distinctly related families associated with specific genetic disorders, include the polycystins (TRPP), mucolipidins (TRPML), mechanoreceptor potential C (TRPN), and ankyrin (TRPA) [11] TRP subfamilies.

Expression Pattern and Regulation of TRPs in the Vasculature

In vascular smooth muscle cells more than ten TRP isoforms have been detected (Table 1). Their expression patterns in vascular myocytes (both smooth muscle and endothelial) are discussed further below.

TRP Channels in Agonist-mediated Vascular Contraction and Vascular SMC Proliferation

TRP channels play an important functional role in the regulation of vascular tone, being involved in agonist mediated vascular contractility and mitogen-mediated smooth muscle cell proliferation. Phenylephrine-induced contractions in the canine pulmonary artery can be attributed to SOC being inhibited by SK&F 96365 [62]. Interestingly, serotonin-induced pulmonary vasoconstriction is attributed to ROC as it is insensitive to SOC inhibitors and to store depletion by cyclopiazonic acid [63]. TRPC1 and TRPC6 are currently thought underlie ROC responsible for agonist- and hypoxia-induced vasoconstriction of pulmonary arteries [31, 33] as well as in SMC from aorta [64, 65], portal vein [22, 25, 66], and mesenteric arteries [67]. One recent study, however, disputes the involvement of TRPC6-encoded ROC channels in chronic hypoxia-induced pulmonary hypertension [33]; TRPC6^−/− mice exhibited no significant changes in heart mass and pulmonary artery muscularization in response to chronic hypoxia (compared to their wild-type littermates). Conversely, expression of TRPC channels can also promote vasoconstriction via SOC-mediated mechanism in these vascular tissues [16, 18, 42, 49, 68–73]. TRPM4 may function as a mechanosensor to promote pressure-induced vasoconstriction in cerebral arteries [26] and TRPV4 may cause hypotonicity-induced airway contraction [74].

An intimate relationship between [Ca²⁺]_i and cell proliferation exists and TRP channels may indeed be responsible for increased Ca²⁺ influx stimulating proliferation. Indeed, enhanced proliferation is associated with augmented CCE via TRP-mediated SOC channels in PASMC [14, 17, 28, 29, 41, 71].

Functional Interaction of TRPs with the Cytoskeleton and within Caveolae

The cytoskeleton and plasma membrane structures may play an important role not only in TRP localization to the plasma membrane, but also to TRP function itself. Caveolae are cholesterol-rich compartments in the plasma membrane. Caveolae function to centralize cooperative receptors, ion channels, transporters, signaling moieties, and effectors within plasma membrane microdomains [75] (Fig. 3). These regions are important sites for ligand-mediated activation of receptors and Ca²⁺ entry in both smooth muscle and, more prominently, in endothelial cells [76, 77] and are closely associated with the endoplasmic or sarcoplasmic reticulum [78], as shown in Figure 3.

Caveolin-1 (cav-1), the main structural protein in caveolae may play a crucial role in the plasma membrane localization of both TRPC1 and TRPC3. Indeed, ET-1-induced vascular contraction and TRPC1 co-localization with cav-1 are decreased by caveolae disruption [16, 79–81]. TRPC1 and cav-1 have been proposed to physically interact. An N-terminal cav-1 binding motif on the TRPC1 gene is involved in the binding of cav-1 and the regulation of TRPC1 plasma membrane localization and the integrity of lipid rafts [82, 83]. Indeed, functioning of TRPC1 or capacitative Ca²⁺ entry is decreased by cholesterol depletion [16, 80], suggesting that TRPC function is dependent upon the formation of caveolae. In addition, cav-1 may regulate both TRPC1 localization and function via interaction with the caveolin-scaffolding domain, thereby enhancing ER/SR-SOC channel interactions [82, 84].

Other TRPC channels have also been implicated in having functional co-localization with Ca²⁺ signaling pathways. For example, TRPC4 has been shown to interact with a PDZ domain on NHERF (Na⁺-H⁺ exchanger regulatory factor) which makes interaction with PLC possible [85]. Remodeling of the actin cytoskeleton enables coupling between IP₃ type II receptors and TRPC1 channels in platelets [86] and actin stabilization prevents the internalization of TRPC3 channels and loss of CCE [87, 88]. Recently, Cioffi and co-workers demonstrated that the activation of SOC channels in PAEC necessitates the interaction of TRPC4 with cytoskeletal protein 4.1 [48].

Lately, Ca²⁺ entry via the reverse mode of the Na⁺/Ca²⁺ exchanger, which is functionally expressed in cultured human PASMC, is suggested to contribute to store depletion-mediated increases in [Ca²⁺]_i [89] (Figs. 2 and 3). Potentially, blockade of the reverse mode of the NCX could be a novel therapeutic approach for treatment of pulmonary hypertension [89]. Rosker and colleagues had previously shown that TRPC3 interacts with the NCX providing a novel idea in TRP-NCX-mediated Ca²⁺ signalling [90]. Supporting data included a) the extracellular Na⁺ dependence of Ca²⁺ signals generated by TRPC3 over-expression in HEK293 cells and b) potent suppression of TRPC3-mediated Ca²⁺ entry by inhibition of the NCX with KB-R9743 [90]. Cell fractionation and co-immunoprecipitation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions [90]. It is thus possible that, as both TRPC3 and NCX expression are detected, this signalling pathway is localized in caveolae (Fig. 3).

Differential Physiological Functions of TRP Channels in PASMC and PAEC

Interestingly, the expression of TRPs in smooth muscle and endothelial cells may lead to different physiological effects. As described above, TRP channel mediated Ca²⁺ influx in vascular SMC causes vasoconstriction and also stimulates the proliferation of smooth muscle cells and medial hypertrophy. Conversely, Ca²⁺ influx into EC stimulates the production of both endothelium-derived relaxing ( EDRF, e.g., nitric oxide, prostacyclin) and hyperpolarizing factors ( EDHF). By opening Ca²⁺-activated K⁺ channels, increased [Ca²⁺]_i in EC also promotes K⁺ efflux to the intercellular space between EC and SMC, which induces membrane hyperpolarization in SMC by activating inward rectifier K⁺ channels [91]. All the processes ultimately lead to EC-dependent vasodilation.

Several studies have demonstrated an important role for Ca²⁺ influx via SOC in vascular endothelial cells. In TRPC4^−/− mice, a strong correlation has been established between TRPC4, ATP- and acetylcholine-induced Ca²⁺ influx, and relaxation in aortic EC [44]; thrombin-induced Ca²⁺ influx was abolished in PAEC isolated from TRPC4^−/− mice [43, 44]. Overexpression of TRPC3 has been directly associated to increased Ca²⁺ influx in response to ATP and bradykinin in PAEC [92]. Bradykinin activates Ca²⁺ influx in mesenteric arterial EC which express TRPC1 and TRPC3 channel mRNA [93]. TRPC1, especially, is implicated in SOC function in PAEC [16, 48].

Endocannabinoid-activated TRPV1 and TRPV4 channels have been recently implicated in the control of vascular tone. In mesenteric artery, endocannabinoid-mediated vasodilation may also be regulated by these channels as the TRPV1-specific inhibitor, capsazepine, reduced relaxation in response to anandamide [94]. Furthermore, 2-arachidonoyl-glycerol activated TRPV1-mediated Ca²⁺ influx by phosphorylating vasodilator-stimulated phosphoprotein (VASP) which may, in turn, contribute to vascular relaxation by stimulating protein kinases G and/or A [95].

Important functional roles for TRP isoforms in the regulation of vascular permeability have also been proposed. Thrombin-induced elevation of [Ca²⁺]_i in EC activates pathways resulting in endothelial cell contraction and disassembly of vascular endothelial barriers, which subsequently increases vascular permeability [96, 97]. Permeability of the microvasculature of the lung in response to hypoxia correlates with an increased expression of TRPC4 and enhanced CCCE in PAEC [44, 45]. Additional supporting evidence for the role of TRPC in regulating pulmonary vascular permeability includes: a) TRPC4-dependent Ca²⁺ entry in mouse lung vascular EC increases microvascular permeability [44, 98]; b) over-expression of TRPC1 augments, and anti TRPC1 antibody decreases, thrombin- and VEGF-induced increases in vascular permeability [99], and inhibition of TRPC1 by antisense oligonucleotides reduces store-operated Ca²⁺ entry [100]; c) VEGF-mediated increase vascular permeability is mimicked by TRPC6-activating agents OAG and flufenamic acid [101]; and d) activation of TRPV1 by VASP is associated with increased vascular permeability [95].

Upregulated TRP Expression and/or Function in Pulmonary and Essential Hypertension

Arterial pressure is a function of cardiac output and vascular resistance. Hypertension occurs due to increased cardiac output and/or elevated vascular resistance. In both the systemic and pulmonary vasculature, sustained vasoconstriction, obliteration of small arteries due to physical occlusion of the vascular lumen by thromboemboli, and severe vascular wall thickening are the major underlying mechanisms for the increased resistance to blood flow, and subsequently for the increased arterial pressure. What follows is a discussion of how TRP channels contribute to the onset and maintenance of pulmonary and essential (systemic) hypertension.

Genetic Variations in TRP Genes and Their Correlation with Pulmonary and Essential Hypertension

The discovery of specific genetic mutations predisposing to both familial and idiopathic PAH may accelerate the understanding of the mechanisms in the pathogenesis of PAH. In patients with idiopathic PAH, a C-to-G single-nucleotide polymorphism (SNP) has been identified in the promoter region (nt. −254) of TRPC6 gene; the allele frequency of the −254(C→G) SNP is significantly higher in idiopathic PAH patients than in normal subjects and patients with secondary pulmonary hypertension [119]. Genotype analysis demonstrated that 6.3% of idiopathic PAH patients carried homozygous −254G/G, whereas none of the normal subjects did. Moreover, the −254(C→G) SNP creates a binding sequence for the transcription factor nuclear-factor–κB (NF-κB). Functional analyses showed that the −254(C→G) SNP enhanced NF-κB-mediated promoter activity and stimulated TRPC6 expression in PASMC. Inhibition of NF-κB attenuated TRPC6 expression in PASMC of idiopathic PAH patients harbouring the −254G allele [119]. These results suggest that the −254(C→G) SNP may predispose individuals to high-risk of idiopathic PAH by upregulating TRPC6 expression in PASMC and, additionally, by linking abnormal TRPC6 transcription in PASMC to NF-κB, an inflammatory transcription factor [119].

Recently, monogenic human hypertension has been linked to mutations in the gene coding for WNK4, a kinase of the WNK family which regulates the expression of TRPV4. Fu and colleagues have shown that co-expression of WNK4 down-regulates TRPV4 function by decreasing its cell surface expression in HEK-293 cells [120]. Collectively this study demonstrates functional regulation of TRPV4 by WNK4 and speculates that this pathway may impact systemic Ca²⁺ balance [120].

TRP Channels as Potential Therapeutic Targets

Development of novel and effective therapeutic approaches for patients with pulmonary hypertension and essential hypertension is important. The functional correlation of TRP channel expression with changes in blood pressure in both essential and pulmonary hypertension highlights these channels as potential therapeutic targets for the abrogation of vasoconstriction and SMC proliferation characteristic of hypertensive disease states. Indeed, bosentan, an endothelin receptor antagonist which is currently approved by FDA for the treatment of PAH, has been shown to inhibit ET-1- and PDGF-mediated PASMC growth in association with the downregulation of TRPC6 channel protein expression [29]. New evidence also suggests that targeting of other TRP isoforms may also prove beneficial in attenuating essential hypertension in some models [118].

Therapeutic strategies should focus not only on inhibiting TRP channel expression and function, but also on disrupting the functional “partners” and microenvironment for TRP channels. Therefore, agents should be developed which a) inhibit TRP channel trafficking and re-insertion; b) disrupt functional and physical coupling of TRP channels with membrane receptors and transporters, intracellular organelles, and IP₃ receptors; c) downregulate STIM/Orai-l or other messengers and intermediates that promote store depletion-mediated Ca²⁺; and d) disrupt caveolae by inhibiting cholesterol and caveolin production.

Conclusion

This review serves to highlight the importance of TRP channels as integral pathogenic components and as potential new drug targets to combat the detrimental effects of vasoconstriction and vascular remodelling (due to SMC proliferation) in hypertensive disease states (Fig. 5). In the vasculature, several cation-permeable TRP channels have been identified as being involved in both the physiological and pathophysiological regulation of vascular tone and smooth muscle cell proliferation. In the pulmonary vasculature, increased Ca²⁺ influx via store- and receptor-operated Ca²⁺ channels encoded by TRP genes may underlie both the enhanced vascular medial hypertrophy as well as the endothelial permeability which contributes to both pulmonary vascular remodeling and pulmonary edema. In essential hypertension, findings are not as clear, especially as it relates to non-TRPC channel isoforms. What is apparent is that TRPC channel upregulation is a strong promoter of vascular SMC proliferation in both types of hypertension. The potential protective role of TRPV and TRPM channels vis à vis blood pressure regulation in essential hypertension provides us with a novel therapeutic target in the treatment of hypertension.