Role of TRP ion channels in cerebral circulation and neurovascular communication

Abstract

The dynamic regulation of blood flow is essential for meeting the high metabolic demands of the brain and maintaining brain function. Cerebral blood flow is regulated primarily by 1) the intrinsic mechanisms that determine vascular contractility and 2) signals from neurons and astrocytes that alter vascular contractility. Stimuli from neurons and astrocytes can also initiate a signaling cascade in the brain capillary endothelium to increase regional blood flow. Recent studies provide evidence that TRP channels in endothelial cells, smooth muscle cells, neurons, astrocytes, and perivascular nerves control cerebrovascular contractility and cerebral blood flow. TRP channels exert their functional effects either through cell membrane depolarization or by serving as a Ca²⁺ influx pathway. Endothelial cells and astrocytes also maintain the integrity of the blood-brain barrier. Both endothelial cells and astrocytes express TRP channels, and an increase in endothelial TRP channel activity has been linked with a disrupted endothelial barrier function. Therefore, TRP channels can play a potentially important role in regulating blood-brain barrier integrity. Here, we review the regulation of cerebrovascular contractility by TRP channels under healthy and disease conditions and their potential roles in maintaining blood-brain barrier function.

Introduction

The brain has high metabolic and energy needs. Since neurons cannot store oxygen and glucose, they rely heavily on a continuous blood supply to fulfill their metabolic requirements. The metabolic requirements are not uniform throughout the brain but instead increased in the regions of high neuronal activity. Therefore, a cellular cross-talk between neurons and the vasculature is of utmost importance in supporting the areas with high neuronal activity. Interruption of this “neurovascular” communication can impair regional blood flow within the brain and have detrimental effects on neuronal structure and function [59]. Contractility of the cerebral vasculature is a crucial determinant of cerebral blood flow. Vascular contractility, in turn, is determined by mechanisms intrinsic to blood vessels and mediators released by neighboring neurons and astrocytes [4, 13]. Intraluminal pressure is one of the most critical regulators of cerebrovascular contractility. Cerebral arteries and arterioles constrict in response to increased intraluminal pressure. This phenomenon, called myogenic constriction, regulates capillary hydrostatic pressure and cerebral blood flow. Myogenic constriction involves depolarization of smooth muscle cell (SMC) membrane and subsequent Ca²⁺ influx that results in SMC contraction [9, 59]. Localized changes in the levels of O₂, H⁺, K⁺, Ca²⁺, glucose, adenosine, and adenosine triphosphate (ATP) can also alter vascular contractility and cerebral blood flow. Additionally, mediators released by nearby astrocytes and neurons can act on endothelial cells (ECs) and SMCs to alter vascular contractility and regional blood flow in the brain.

The cerebral circulation exhibits unique characteristics that minimize injury by maintaining optimal perfusion in response to physiological and pathological stimuli. The brain represents only 2% of the bodyweight but receives approximately 15% of the cardiac output. Blood supply to the brain occurs via two pairs of arteries: right and left common carotid arteries and right and left vertebral arteries. Each common carotid artery divides into the external carotid artery and internal carotid artery, the cerebrum’s principal blood supplier. The basilar artery and two internal carotid arteries form an anastomotic ring at the brain’s base (circle of Willis). The anterior, middle, and posterior cerebral arteries that branch to smaller arteries and arterioles run along the surface and then penetrate the brain parenchyma. The vascular tree branches further into arterioles and capillaries, dramatically increasing the overall vascular area, slowing the blood flow, and allowing diffusion of nutrients and metabolites [22, 23, 105].

The cerebrovascular wall consists of multiple cell types, including ECs, SMCs, pericytes, astrocytes, microglia, perivascular macrophages (PVMs), and perivascular fibroblast-like cells [121]. Vascular contraction and dilation are differentially regulated in the different segments of the cerebrovascular tree. Large arteries (carotid artery and middle cerebral artery) have multiple layers of contractile SMCs, which encircle the endothelial basement membrane. The number of SMCs decreases as the arteries become smaller (pial artery), eventually diminishing to a single cell layer in arterioles (penetrating parenchymal arterioles). The perivascular sensory nerves are limited to the pial arteries. A continuous endothelial monolayer forms the inner layer of the vascular walls. Adjacent ECs are securely connected to one another by tight junctions. On the luminal side, ECs are covered by glycocalyx glycoproteins, proteoglycans, and glycosaminoglycans [99]. Pericytes are embedded into the basement membrane of capillaries and pre-capillary arterioles and play a crucial role in regulating CBF and blood-brain barrier integrity (Fig. 1–3).

Figure 1: TRP channels regulate pial arterial contractility.

Endothelial Cells: Ca²⁺ influx through TRPV3, TRPV4, and TRPA1 channels activates nearby intermediate (IK) and small (SK) conductance Ca²⁺-activated potassium channels, causing vasodilation. TRPM2 channels are another Ca²⁺ entry pathway in cerebral endothelial cells. Smooth muscle cells: Ca²⁺ influx through plasma membrane TRPV4 channels or Ca²⁺ release through late endo-lysosome (LEL) TRPML1 channel activates ryanodine receptors (RyR), resulting in Ca²⁺ release (Ca²⁺ Sparks) from the sarcoplasmic reticulum (SR). Ca²⁺ sparks, in turn, stimulate large-conductance Ca²⁺-activated potassium (BK) channels, leading to SMC relaxation and vasodilation. Ca²⁺ entry through TRPC6, TRPC3, and TRPP1 channels triggers Ca²⁺ release from the SR via IP3 receptors (IP3R). IP3R Ca²⁺ signals activate sodium influx through TRPM4 channels, leading to SMC depolarization and vasoconstriction. Perivascular Sensory Nerves: Ca²⁺ entry via TRPA1 and TRPV1 channels induces the release of CGRP (calcitonin gene-ralated peptide) and vasodilation.

Figure 3: TRP channel regulation of neurovascular communication at the level of capillaries.

Ca²⁺ influx through TRPV4 channels is promoted by Gq protein-coupled receptor (GqPCR) signaling in the capillary endothelium. Activation of phospholipase C by Gq proteins lowers phosphatidylinositol 4,5-bisphosphate (PIP2) levels. PIP2 inhibits TRPV4 channels and activates inwardly rectifying potassium (Kir) channels. Thus, GqPCR signaling disinhibits TRPV4 channels and lowers the activity of Kir channels. Ca²⁺ influx signals through TRPA1 channels stimulate nearby Pannexin 1 channels, promoting ATP release. ATP activates Ca²⁺-permeable purinergic P2X receptor ion channels. The capillary Ca²⁺ events propagate upstream to the parenchymal arteriolar endothelium resulting in vasodilation. The role of TRP channels in pericytes is largely unknown.

The term “neurovascular communication (NVC)” represents the interactions of neurons or astrocytes with cerebrovascular ECs and SMCs [23, 47]. Vascular cells (ECs, SMCs, pericytes), astrocytes, and neurons work in tandem to regulate blood flow in the brain. Astrocytes are the regulatory hubs that receive signals from the active neurons and relay them to blood vessels, facilitating increased blood flow to the active neurons [5]. The network of astrocytes projects from neurons to parenchymal arterioles/capillaries, where astrocytic “endfeet” encase the parenchymal arterioles/capillaries. ECs and astrocytes also maintain the integrity of the blood-brain barrier [14]. The blood-brain barrier is absolutely essential for excluding neurotoxic plasma-derived components, red blood cells, leukocytes, and pathogens from the central nervous system (CNS) [4]. Blood-brain barrier integrity is maintained by specialized tight junctions that limit the hydraulic conductivity and transcellular movement. Cellular signaling mechanisms in the neurovascular unit control cerebral blood flow and blood-brain barrier integrity. While NVC implies passage of information from neurons to the vasculature, communication in the opposite direction (vasculo-neuronal coupling or VNC) has also been proposed. Together, NVC and VNC balance the energy supply with demands while preventing brain hyperperfusion or hypoperfusion. It is thought that the NVC overrides VNC during high neuronal activity [93].

Transient receptor potential (TRP) ion channels have emerged as essential regulators of cerebrovascular function and neurovascular communication. TRP channels are members of the superfamily of cation-conducting membrane proteins widely expressed in both excitable and non-excitable cells. TRP channels are activated by diverse stimuli, including temperature, light, pressure, changes in osmolarity, and G protein-coupled receptor (GPCR) signaling. Several TRP channels are expressed in the cerebral vasculature and neurovascular unit. TRP channels, based on their sequence homology, are classified into six subfamilies: TRPC (Canonical), TRPV (Vanilloid), TRPA (Ankyrin), TRPM (Melastatin), TRPP (Polycystic), and TRPML (Mucolipin). Broadly speaking, TRP channels act as cellular sensors that alter cellular function primarily by changing membrane potential and/or increasing cellular Ca²⁺. The molecular structure of TRP channels is characterized by four subunits, each presenting six hydrophobic transmembrane segments (S1-S6) and cytosolic N-and C- terminal tails [2, 8]. A short hydrophobic conserved domain among TRPV, TRPM, and TRPC subfamilies, referred to as the “TRP box,” is located after S6. TRP box is implicated in fine-tuning TRPV, TRPM, and TRPC channel activity.

TRP channels play crucial roles in various biological processes in the brain, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, pressure regulation, and Ca²⁺ and Mg²⁺ homeostasis. Particularly in blood vessels, GPCR signaling and other endogenous stimuli can activate TRP channels and enable their physiological roles. Accumulating evidence suggests that sex hormones can influence TRP channels expression and function, supporting potentially important roles for TRP channels in determining the sex differences in NVC and VNC [62]. TRP channels in cerebrovascular endothelium have primarily been associated with vasodilation. In contrast, TRP channels in SMCs can cause vasoconstriction or vasodilation depending on the channel involved and its downstream signaling targets. Here, we provide an overview of the role of cerebrovascular TRP channels in neurovascular communication and cerebral blood flow regulation.

TRPM channels

TRPM channels in both the SMC and EC layers play an essential role in regulating cerebrovascular function. The TRPM subfamily contains eight members: TRPM1-TRPM8. Most TRPM subfamily members are non-selective, Ca²⁺-permeable cation channels, except for TRPM4 and TRPM5, which are impermeable to Ca²⁺ [50].

TRPM4 channel has emerged as a critical player in pressure-induced depolarization of cerebrovascular SMCs. The unitary conductance of the TRPM4 channel is approximately 24 pS. TRPM4 channel is present in SMCs from the cerebral pial and parenchymal arterioles [67] (Fig. 1, Fig. 2). It is a Ca²⁺-activated, Ca²⁺-impermeable non-selective cation channel that contains binding sites for calmodulin (CaM) and ATP, and protein kinase C (PKC) phosphorylation sites [81]. Earley and colleagues presented the first evidence that SMC TRPM4 channels contribute to myogenic constriction of rat cerebral arteries [32, 96]. Further studies showed that TRPM4 channel-induced SMC depolarization and contraction are enhanced by angiotensin II receptor 1 (AT1R) stimulation [31] and PKC activation [37, 67] in male Sprague-Dawley rats. On the contrary, PKC inhibition impaired myogenic constriction of cerebral arteries [31]. Suppression of TRPM4 channel expression using antisense oligonucleotide caused a 70% to 85% decrease in myogenic constriction, further supporting an essential role of TRPM4 channels in the development of myogenic constriction of cerebral arteries. TRPM4 channels in cerebral arteries were also critical for purinergic P2Y receptor activation-induced vasoconstriction [67] and acted as a crucial link between P2Y receptors and myogenic constriction. Notably, TRPM4 channels were upregulated in a rat model of subarachnoid hemorrhage, increasing myogenic constriction of cerebral arteries and lowering cerebral blood flow [40]. Gonzales and colleagues observed that co-localization of TRPM4 channels with TRPC6 channels in cerebral artery myocytes facilitates stretch-induced membrane depolarization and myogenic constriction [41] in male Sprague-Dawley rats. The researchers explained that stretch activates TRPC6 channels directly or through AT1R-phospholipase Cγ1 (PLCγ1) signaling. The resulting Ca²⁺ influx activates inositol 1,4,5-trisphosphate (IP₃) receptors on the sarcoplasmic reticular (SR) membrane. IP₃ receptor-mediated Ca²⁺ release promotes the activity of TRPM4 channels on the SMC membrane and leads to SMC membrane depolarization.

Figure 2: TRP channel regulation of parenchymal arteriolar contractility.

Endothelial Cells: Ca²⁺ influx through TRPV3, TRPV4, and TRPA1 channels activates nearby intermediate (IK) and small (SK) conductance Ca²⁺-activated potassium channels, causing vasodilation. Smooth muscle cells: Ca²⁺ entry through TRPC6 channels stimulates Ca²⁺ release from the sarcoplasmic reticulum (SR) via inositol triphosphate receptors (IP3R). IP3R-mediated Ca²⁺ release activates sodium currents through TRPM4 channels, depolarizing the SMC membrane and causing vasoconstriction. Ca²⁺ influx through TRPV4 channels stimulates Ca²⁺ release from the SR through ryanodine receptors (RyR) and activates large-conductance Ca²⁺-activated potassium channels (BK), leading to SMC hyperpolarization and vasodilation. Astrocytic Endfeet: Ca²⁺ signals through TRPV4 channels are amplified by Ca²⁺-induced Ca²⁺ release via IP3Rs. This mechanism increases boosts neurovascular coupling and dilates parenchymal arterioles. TRPC6 channel is another essential regulator of astrocytic Ca²⁺ homeostasis.

TRPM2 channel is a Ca²⁺-permeable non-selective cation channel highly expressed in the CNS [124], cerebral ECs [85], microglia [76], and pericytes [54]. TRPM2 channel is equally permeable to divalent and monovalent cations [101]. The channel is localized in the plasma membrane [120] and/or lysosomal compartments [64, 133]. TRPM2 channel embodies both ion channel functionality and enzymatic ADP-ribose (ADPR) hydrolase activity [119]. Several extracellular stimuli, including reactive oxygen species (ROS), H₂O₂, amyloid β peptide (Aβ), and Tumor necrosis factor- α (TNFα) were shown to induce TRPM2 channel activity via metabolic production of intracellular poly-ADPR [61, 107]. TRPM2 channel has also been proposed as an essential regulator of endothelial Ca²⁺ homeostasis. In dementia of Alzheimer’s disease (AD), the extracellular accumulation of Aβ triggered oxidative-nitrosative stress that activated the DNA repair enzyme poly-ADPR polymerase. The increase in poly-ADPR potentiated TRPM2 channel activity in ECs, leading to endothelial Ca²⁺ overload, endothelial dysfunction, and neurovascular impairment [85]. In the transient middle cerebral artery occlusion (tMCAO) model of stroke in male Sprague Dawley rats, microglial TRPM2 channel was upregulated through microglial activation and cellular infiltration [36]. It was proposed that under ischemia-reperfusion (I/R) conditions, persistent activation of TRPM2 channel results in prolonged membrane depolarization and elevation of [Ca²⁺]_i and [Na⁺]_I, resulting in microglial death. Therefore, targeting TRPM2 channels could be a therapeutic avenue to counteract the deleterious cerebrovascular effects of AD and I/R. Jia et al. [53] reported an important role for TRPM2 channels in sex differences during cerebral ischemia-induced oxidative stress. Inhibition or knockdown of TRPM2 channel significantly protected ischemia-induced neuronal death and reduced the infarct volume in male mice but not in female mice. Recent studies also show that TRPM7 channels contribute to ageing-related dementia [134], whereas TRPM8 channels inhibit TRPV1 channel-associated hypothermia [35]. Thus, the roles of different TRPM channels in the neurovascular system and sex-differences in neurovascular function is an emerging area of research.

TRPA1 channels

Recent developments establish that endothelial TRPA1 channel is a crucial Ca²⁺ entry pathway in neurovascular coupling and regulation of cerebral blood flow. TRPA1, the sole member of the mammalian ankyrin TRP subfamily, is a large-conductance (~100 pS) [79], Ca²⁺-permeable cation channel. TRPA1 channel is permeable to both monovalent and divalent cations, but its relative permeability is higher for Ca²⁺ than for K⁺ or Na⁺ [11]. Moreover, the activity of TRPA1 channels is regulated by both intracellular and extracellular Ca²⁺. TRPA1 channels are activated by several pungent natural compounds in food, including allicin (garlic), allyl isothiocyanate (mustard), and cinnamon (cinnamaldehyde) [6]. Importantly, TRPA1 channels are critical sensors of cellular redox signaling, a hallmark of cerebrovascular disease [90].

Endothelial TRPA1 channels in pial arteries (Fig. 1), parenchymal arterioles (Fig. 2), and capillaries (Fig. 3) play crucial roles in regulating cerebral blood flow. Pires et al. found that hypoxia and cerebral ischemia dilate cerebral pial arteries and parenchymal arterioles via activation of endothelial TRPA1 channels [90]. Acute hypoxia increased the frequency of TRPA1 sparklets (optically detected elementary Ca²⁺ influx signals [108, 113]) and dilated mouse cerebral arteries and penetrating parenchymal arterioles. Further, increased endothelial TRPA1 channel activity reduced cerebral infarct size. Hypoxia-induced dilation of cerebral arteries was disrupted by antioxidants, TRPA1 antagonist A967079, and EC-specific TRPA1 deletion in mice. In a recent study, Thakore et al. showed that capillary endothelial TRPA1 channels initiate a dilatory signal that is transmitted to upstream parenchymal arterioles. Ca²⁺ signals from capillary TRPA1 channels propagate upstream through the capillary network via endothelial Pannexin1 (Panx1, ATP release channel on the cell membrane), extracellular ATP release, and subsequent activation of Ca²⁺-permeable purinergic P2X receptor ion channels [116]. ATP derived from astrocytes, neurons, and capillary ECs can activate purinergic P2X receptors on adjacent ECs. Ca²⁺ signals initiated in the capillary endothelium propagate upstream to the arteriolar endothelium, where they activate Ca²⁺-sensitive SK and IK (small and intermediate conductance K⁺ channels, respectively) channels. SK and IK channel activation results in EC membrane hyperpolarization, which is transmitted to arteriolar SMCs via gap junctions, thereby dilating the parenchymal arterioles. Together, these studies demonstrated a central role for endothelial TRPA1 channels in neurovascular coupling [116].

TRPA1 channel is also found in sensory neurons [7, 110]. Neuronal activation leads to the release of calcitonin gene-related peptide (CGRP), a potent vasodilator. TRPA1 channels co-localize with CGRP in primary sensory neurons [110]. Activation of neuronal TRPA1 channels by mustard oil increases CGRP release and dilates cerebral vasculature [63], causing headaches. This effect is blocked by a CGRP antagonist or TRPA1 channel inhibitor. Similarly, in diet-induced obese rats, higher TRPA1 channel activity in trigeminal ganglia increased the CGRP release and elevated meningeal blood flow [71]. Moreover, activation of TRPA1 channels in perivascular sensory neurons triggered the release of CGRP, decreased vascular resistance, and increased cerebral blood flow [87]. A recent study also showed that TRPA1 channels may play a crucial role in ageing-related memory decline [12]. Thus, activation of endothelial TRPA1 channels has neuroprotective effects, whereas activation of neuronal TRPA1 channels can have detrimental consequences in the brain.

TRPP channels

TRPP1 and TRPP2 are Ca²⁺-permeable, non-selective cation channels with a large unitary conductance (~100 pS) [69]. Recent studies have revealed vital roles for TRPP1 channels in regulating myogenic constriction in different vascular beds [80, 106]. TRPP1 channels are expressed in cerebrovascular SMCs [80] and ECs [10], located on the endoplasmic reticular membrane [104] and plasma membrane [69]. In human and rat cerebral artery SMCs, TRPP1 channels are predominantly expressed at the plasma membrane [80] and contribute to myogenic constriction. RNAi-mediated knockdown of TRPP1 reduced the swelling-activated cation currents in isolated cerebrovascular SMCs and suppressed the development of myogenic constriction in cerebral arteries [80]. Moreover, TRPP1 haploinsufficient mice displayed impaired SMC Ca²⁺ homeostasis and elevated blood pressure [95]. Abdi and colleagues showed that TRPP2 channels interact with ryanodine receptors (RyR) and IP₃ receptors in mouse cerebral artery SMCs [1]. Moreover, TRPP2 haploinsufficiency resulted in arterial remodeling of mouse middle cerebral arteries. Thus, recent evidence supports crucial roles for TRPP channels in regulating SMC Ca²⁺ homeostasis, although further studies are needed to understand these channels’ regulatory mechanisms and physiological roles in the vasculature. The development of specific pharmacological activators or inhibitors of TRPP channels will significantly improve our understanding of their properties and functional roles.

TRPML channels

The TRPML subfamily consists of Ca²⁺-permeable, non-selective cation channels (TRPML1, TRPML2, and TRPML3). TRPML1 is ubiquitously expressed, TRPML2 is expressed in lymphoid and myeloid tissues, and TRPML3 is expressed in the cochlea and the melanocytes [19]. Unlike other TRP channels present on the plasma membrane, TRPML1 is predominantly localized at the late endosomal and lysosomal (LELs) membranes. TRPML1 channels are permeable to Ca²⁺, Fe²⁺, Zn²⁺, Na⁺, and K⁺. The unitary conductance of the TRPML1 channel is 45 pS [125]. Recent studies indicate that TRPML1 channels are expressed in the SMCs from cerebral arteries [118], where they form a stable complex with RyR2s and are functionally coupled with RyR2s. The TRPML1-RyR2 signaling subsequently activates Ca²⁺-sensitive, large-conductance K⁺ (BK) channels in cerebrovascular SMCs, resulting in vasodilation. Global deficiency of the TRPML1 channel results in vascular hypercontractility and hypertension [118]. Therefore, SMC TRPML1 channel appears to be a vital regulator of cerebrovascular resistance and systemic blood pressure.

TRPC channels

TRPC subfamily is grouped into three major categories based on the amino acid sequence homology and functional similarities; TRPC1/4/5, TRPC2, and TRPC3/6/7 [23, 75]. The TRPC channel family consists of Ca²⁺-permeable, non-selective cation channels (TRPC1–7). TRPC1 functions as a receptor-operated, diacylglycerol (DAG)- and IP₃ receptor-activated cation channel. TRPC2 is different from other TRPC subfamily members in that it encodes a non-functional truncated protein (pseudogene) in humans [122]. TRPC4 and TRPC5 function as non-selective cation channels activated by G_q/11 family GPCRs and receptor tyrosine kinases [103]. TRPC3, TRPC6, and TRPC7 show non-selective and double-rectifying cation currents when activated by G_q/11-coupled receptors or DAG as either homomeric or heteromeric channels [48].

TRPC3 channels are expressed in cerebral ECs [100], neurons [17], and SMCs [128]. The unitary conductance for the TRPC3 channel is approximately 68 pS [92]. TRPC3 channel is a key player in the receptor-mediated contraction of cerebral artery SMCs. Reading and colleagues showed that suppression of SMC TRPC3 channel expression with antisense oligonucleotide reduced uridine triphosphate (UTP)-induced constriction of rat cerebral arteries, but not pressure-induced constriction [97]. Further, IP₃ receptor activation was shown to induce constriction through IP₃ receptor–TRPC3 channel signaling in cerebral arteries [128]. In hyperperfusion disorders such as epilepsy and seizures, TRPC3 and TRPC6 channels are upregulated in hippocampal neurons [89, 131]. The upregulation of neuronal TRPC3 and TRPC6 channels correlates with impaired cerebral blood flow and a high degree of moment-to-moment variability in neuronal activity [24]. Deleting TRPC3 channels from SMCs rescued cerebral blood flow and neuronal activity in mice [24], implying a harmful effect of SMC TRPC3 channel activity on neurovascular communication.

TRPC6 channels are an integral part of the force-sensing complex in cerebral artery SMCs and a key regulator of myogenic constriction [41] (Fig. 1). Gonzales et al. showed that Ca²⁺ influx through TRPC6 channels in rat cerebral artery SMCs is activated by a direct mechanical stimulation or through the PLCγ–DAG pathway. Ca²⁺ influx through TRPC6 channels can increase IP₃ receptor-mediated Ca²⁺ release from the SR, which, in turn, stimulates nearby TRPM4 channels [41]. In another study, Welsh and colleagues found that antisense oligonucleotides targeting TRPC6 channel mRNA attenuated pressure-induced membrane depolarization of rat cerebral artery SMCs and reduced myogenic constriction [127]. Notably, global TRPC6^−/− mice showed increased myogenic constriction of cerebral arteries and elevated blood pressure. This surprising finding was attributed to a compensatory upregulation of TRPC3 channels in SMCs [28]. Thus, SMC TRPC3 and TRPC6 channels appear to be crucial controllers of receptor-mediated and myogenic constriction of cerebral arteries, respectively.

In forebrain ischemia, Ca²⁺ influx through SMC TRPC1 and TRPC6 channels and endothelin-1-induced constriction of middle cerebral arteries are increased [55]. Ischemia/reperfusion (I/R) and oxygen/glucose deprivation also upregulated TRPC6 channels in primary cortical neurons [18], contributing to Na⁺ entry, NMDA receptor activation, and Ca²⁺ overload that ultimately resulted in neuronal death. Deletion of TRPC6 channel protected the neurons against I/R-induced damage and excitatory neurotoxins such as glutamate and NMDA. On the contrary, the SMC TRPC6 channel was downregulated in the middle cerebral artery occlusion (MCAO) mouse model of I/R [68]. The brain infarct lesion, apoptosis, and interleukin-6 (IL-6) and IL-1β levels were increased, and the astrocyte population was reduced in this mouse model. Overexpression of astrocytic TRPC6 channel or HYP9, a selective TRPC6 agonist, attenuated the damage to astrocytes in this model [68]. Remarkably, TRPC3/6/7 triple knockout mouse ameliorated brain damage through decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression and increased AKT phosphorylation, resulting in reduced astrocyte apoptosis [20, 94]. Overall, TRPC channels may serve as a crucial link between the nervous system and the vasculature in health and disease.

TRPC channel activity has also been linked with the maintenance of blood-brain barrier function. The expression of TRPC3 channel was upregulated in neurons and ECs within the brain’s piriform cortex in status epilepticus [100]. Upregulation of endothelial TRPC3 channels lowered the levels of SMI-71, a rat endothelial blood-brain barrier antigen, a finding that correlated with blood-brain barrier disruption and neuronal damage. On the contrary, Chlorpyrifos, an insecticide, decreased the expression of endothelial TRPC4 channels and disrupted the blood-brain barrier in Sprague–Dawley rats [66]. An in-depth assessment of the role of TRPC channels in maintaining blood-brain barrier integrity is awaited.

TRPV channels

TRPV channels are expressed on the cell membranes of SMCs, ECs, and perivascular nerves, where they serve as signal transducers activated by endogenous ligands, heat, mechanical stimuli, and osmotic stress. High unitary conductance and permeability for Ca²⁺ are characteristic properties of TRPV channels.

The first TRPV subfamily member, TRPV1, has a unitary conductance of 35 to 70 pS [15]. TRPV1 channels are essential for thermal and chemical nociception in sensory neurons and are activated by capsaicin, changes in pH, and temperature in the noxious range (>42 °C) [8]. TRPV1 channel activation in perivascular sensory nerves stimulates the release of CGRP and leads to vasodilation [43, 123]. TRPV1 reporter mice clearly showed the expression of TRPV1 channels in perivascular sensory neurons [16] and SMCs from cerebral arteries [88]. In contrast to the dilatory effect of neuronal TRPV1 channel activity, SMC TRPV1 channels induce vasoconstriction [16, 88]. Therefore, the activation of TRPV1 channels on SMCs could counteract nerve-related changes in vascular reactivity in response to endogenous TRPV1 agonists.

Pegorini et al. demonstrated that in vivo pretreatment with capsaicin, a TRPV1 channel agonist, was neuroprotective in a rodent model of global cerebral ischemia [86]. In a later study, Khatibi and colleagues confirmed these findings in a different model of brain ischemia [57]. Pre-ischemia injection of capsaicin reduced cerebral infarct volume and normalized vasoreactivity [57]. The authors speculated that capsaicin-induced desensitization of TRPV1 channels prevents Ca²⁺ overload and neurotoxicity following ischemia. Furthermore, genetic deletion of TRPV1 was protective against ischemia-induced neurological and motor deficits [77]. In a more recent study, Hakimizadeh and colleagues showed that inhibiting TRPV1 channels post-ischemia limits neuronal damage by lowering the expression of toll-like receptor 2 (TLR2) and TLR4 [42]. Indeed, TLR2 and TLR4 are upregulated following cerebral ischemia [42, 65] and play an essential role in sustaining neuronal inflammation and death [115]. Based on these reports, TRPV1 channels could be a novel therapeutic target against stroke-induced neuronal damage, and their roles in the pathogenesis of cerebral ischemia need to be further investigated.

Another TRPV subfamily member, TRPV4, is permeable to Ca²⁺ and Na⁺ and is widely distributed in the neurovascular system, found in ECs [45], SMCs [30], astrocytes [29], and neurons [73]. The single-channel conductance of TRPV4 channel is in the range of 90–100 pS for outward currents and 50–60 pS for inward currents [33]. In cerebral pial arteries, endogenous agonist 11,12 epoxyeicosatrienoic acid (11,12-EET) activated SMC TRPV4 channel currents. Ca²⁺ influx through TRPV4 channels signaled through RyR–BK channel to induce SMC hyperpolarization and vasodilation [30]. Elementary Ca²⁺ release signals (Ca²⁺ sparks) through RyRs located on the SR membrane activate BK channels [126]. Reducing the expression of TRPV4 channels with antisense oligonucleotides prevented the 11,12-EET-induced SMC hyperpolarization and dilation of cerebral pial arteries from male Sprague–Dawley rats. In SMCs from penetrating parenchymal arterioles, PKC anchoring by A-kinase-anchoring protein 150 (AKAP150) facilitated Ang II-activation of SMC TRPV4 channels. Furthermore, the AKAP150-TRPV4 channel interactions in SMCs were highly dynamic, with activation of Ang II signaling increasing the proximity between AKAP150 and TRPV4 channels [74]. Thus, TRPV4 channels in cerebrovascular SMCs have been associated with SMC relaxation and vasodilation (Fig. 1, Fig. 2). In this regard, important sex differnces were observed in the activity of SMC TRPV4 channels from cerebral arteries of male and female mice, with the overall activity being lower in female mice [114].

Endothelial TRPV4 channel activity promotes endothelium-dependent dilation in the cerebral arteries and is impaired in cerebrovascular disorders. Chang and colleagues showed that endothelial TRPV4 channels dilate pial arteries. This group further demonstrated that TRPV4 channel-dependent dilation of pial arteries is impaired in a mouse model of Alzheimer’s disease [132]. Using TRPV4 knockout rats, a recent study by Dorrance and colleagues reported that endothelial TRPV4 channel signaling is essential for parenchymal arteriolar dilation and cognitive function [26]. Earlier, Dorrance and colleagues showed that mineralocorticoid receptor antagonism increases TRPV4 channel expression in parenchymal arterioles and improves cognitive function [27] in hypertension, providing further evidence that endothelial TRPV4 channels have a protective effect on cognitive function.

Cerebral capillary ECs also show a robust expression and function of TRPV4 channels (Fig. 3). In the arterial ECs, coupling of TRPV4 channels with IK/SK channels or eNOS [72, 82, 108] results in SMC relaxation and vasodilation. Of note, the TRPV4-eNOS or TRPV4-IK/SK channel coupling is known to occur at endothelial projections to SMCs [84]. In the capillaries, however, there are no SMCs surrounding the ECs. Further, IK/SK channel currents were not observed in capillary ECs [70], suggesting a distinct TRPV4 channel-dependent signaling in capillary ECs compared to arterial ECs. Capillary ECs have been shown to express eNOS [75]. Therefore, it is plausible that TRPV4 channels in capillary ECs activate eNOS, although this possibility has not been verified. Harraz et al. showed that TRPV4 channels in capillary ECs are tonically inhibited by basal levels of phosphatidylinositol 4,5-bisphosphate (PIP2) [45]. PIP2 is a plasma membrane lipid hydrolyzed by G_q protein-coupled receptor (GqPCR) signaling into IP₃ and DAG. In capillary ECs, activation of G_qPCR reduced PIP2 levels and disinhibited TRPV4 channels, simultaneously reducing inwardly rectifying potassium (Kir2.1) channel activity [44]. Thus, PIP2 acts as a functional switch for TRPV4 and Kir2.1 channels (Fig. 3), both significant players in cerebral blood flow regulation.

The expression of TRPV4 channels at the astrocytic endfeet [9, 10] suggests that the channels may play a vital role in neurovascular communication. Astrocytic endfeet are critical microdomains for coupling neuronal activity with changes in cerebral blood flow [32]. In this regard, Straub et al. optically recorded the Ca²⁺ signals occurring at the astrocytic endfeet [111]. Neuronal activity induced a rapid increase in endfeet Ca²⁺, which preceded the dilation of adjacent arterioles. The authors concluded that spatially restricted IP3 receptor Ca²⁺ signals at the endfeet are sufficient to induce vasodilation [111]. Later, Dunn et al. provided the first evidence that Ca²⁺ signals through TRPV4 channels increased the strength of neurovascular coupling by augmenting neuronal activation-induced endfoot Ca²⁺ and arteriolar dilation. The authors reported that the amplification of TRPV4 Ca²⁺ signals by Ca²⁺-induced Ca²⁺ release via IP₃ receptors [29] further boosts neurovascular coupling. Interestingly, Kim et al. demonstrated that astrocytic TRPV4 channels mediate pressure-induced constriction of parenchymal arterioles, thus modulating vascular contractility to match the neuronal activity [58]. Taken together, TRPV4 channel activity appears to be central to the physiological function of multiple cell types involved in neurovascular communication.

TRPV3 channels are large unitary conductance (~150–200 pS), Ca²⁺ and Na⁺ permeable cation channels. They are expressed in the endothelium of cerebral pial arteries and parenchymal arterioles [91]. Activation of TRPV3 channels causes endothelium-dependent hyperpolarization of SMCs in pial arteries and parenchymal arterioles. Ca²⁺ influx through TRPV3 channels activates SK and IK channel currents, resulting in endothelium-dependent vasodilation of pial arteries and parenchymal arterioles [91]. Moreover, the Ca²⁺ flux through a single TRPV3 channel was found to be greater than other TRP channels [91]; approximately three times greater than TRPV4 channels [112] and 1.5 times greater than TRPA1 channels [113]. Further studies are needed to understand the physiological activators and regulatory mechanisms for endothelial TRPV3 channels.

Conclusion and Perspectives

TRP channels in vascular ECs and SMCs, astrocytes, perivascular nerves, and neurons play a critical role in ensuring resource delivery for neural processing and neural homeostasis. TRP channels accomplish this by 1) directly regulating cerebrovascular contractility; and 2) indirectly influencing cerebrovascular contractility via signaling from neurons, astrocytes, and perivascular nerves. TRP channels control cerebrovascular contractility by acting as a Ca²⁺ influx pathway or depolarizing the membrane potential. Current evidence suggests that TRPA1 and TRPV channels are the main Ca²⁺ influx pathways in cerebrovascular ECs. TRPM4, TRPC3, TRPC6, TRPP, and TRPV are the main TRP channels in cerebrovascular SMCs. The diverse biophysical properties of TRP channels and their distinct downstream signaling targets enable their involvement in multiple signaling pathways that control cerebral blood flow. The involvement of TRP channels in cerebral blood flow autoregulation could also be determined by the local stimuli that activate a specific TRP channel, thereby ensuring functional specificity. Cerebral arteries and arterioles are hypercontractile in vascular disorders [51]. Moreover, neurovascular communication is impaired in vascular disorders such as hypertension and stroke [51, 52]. Further research is needed to determine the contribution of TRP channels in impaired neurovascular communication in vascular disorders.

Pericytes line capillaries in the brain. Recent studies show that brain pericytes have a substantial effect on cerebral blood flow [46]. TRP channel-dependent signaling mechanisms in brain capillary pericytes remain unexplored and may be an exciting direction for future investigations. Further, many TRP channels in ECs and SMCs require scaffolding/anchoring proteins for normal function [74, 83]. AKAP150 is an example of a scaffolding protein that facilitates post-translational modifications of TRP channels by anchoring kinases such as Protein Kinase A (PKA) and PKC [60]. Indeed, AKAP150 is expressed in multiple cell types in the brain. In a recent study, Tajada and collegues demonstrated the importance of AKAP150-PKC complexes in enhancing TRPV4 channel activity in myocytes isolated from mouse pial and parenchymal arteries [114]. Interestignly, Pagán and collegues identified a novel neuroprotective role for AKAP150 in astrocytes. AKAP150 was upregulated in astrocytes under ischemic conditions to facilitate astrocytic K⁺ clearance [98]. These studies provide initial evidence that AKAP150 may be a significant player in the regulation of neurovascular comunication.

Caveolin-1 is another important structural protein found within caveolae. Chow and colleagues showed an essential role for endothelial caveolae in neurovascular coupling [21]. Endothelial caveolin-1 anchors eNOS [39], and TRP channel activation has also been linked with eNOS activation [25]. Moreover, caveolin-1 has emerged as a vital scaffolding protein for TRP channels [25]. Therefore, it will be interesting to investigate whether caveolin-1–TRP channel interaction determines eNOS activity, neurovascular coupling, and cerebral blood flow. In systemic arteries, endothelial TRP channels play a central role in myoendothelial feedback, which involves SMC to EC signaling that limits SMC contraction. Endothelial TRPV4 channels were essential for negatively regulating SMC α1 adrenergic receptor-mediated constriction of systemic arteries [49]. Myoendothelial feedback mechanisms have not been studied in the cerebral vasculature and may play potentially important roles in regulating cerebral blood flow. IK/SK channels are not present in the brain capillary endothelium. However, Kir2.1 channels are functional and are crucial for conducted hyperpolarization. TRPV4 channel-induced vasodilation occurred through downstream activation of endothelial Kir2.1 channels in the systemic arteries [109]. Kir2.1 channels were also involved in flow-mediated dilation of systemic arteries [3]. However, a functional interaction between TRPV4 and Kir2.1 channels and its physiological roles in the brain capillary endothelium remain unknown.

Perivascular macrophages (PVM) were recently shown to impair neurovascular coupling through NADPH 2 Oxidase (NOX2) activation and production of superoxide radicals. This effect seems to be mediated by angiotensin II, which activates angiotensin receptor 1 expressed on PVM [34, 56, 129]. Other studies suggest that TRP channels can influence ATR1 activity [130]. Considering the expression of TRP channels in macrophages [102], future studies of PVM TRP channels may provide interesting insigths into the impairment of neurovascular communication.

Finally, the role of TRP channels in controlling the integrity of the blood-brain barrier remains largely unexplored. Endothelial TRP channels have been associated with increased capillary permeability [38, 117]. In this regard, caveolin-1 is an essential component of the transcellular barrier. An increased expression of caveolin-1 was linked with blood-brain barrier leakage following ischemic brain insult [78]. Therefore, the interaction of TRP channels with caveolin-1 or other scaffolding proteins in endothelial cells and astrocytes will be an exciting area for future research on the blood-brain barrier.

Highlights.

• Overview of neurovascular communication and blood brain barrier function

• Biophysical properties and roles of individual TRP channels involved in neurovascular communication

• Conclusions and Future Research Directions