Scott Earley

Localized TRPA1 channel Ca2+ signals stimulated by reactive oxygen species promote cerebral artery dilation

Peroxidized lipid metabolites trigger calcium influx through the channel TRPA1 to dilate cerebral arteries. Blood Vessel Dilation with Peroxidized Lipids Cerebral arteries must maintain constant blood flow to the brain even though blood pressure fluctuates constantly. Sullivan et al. characterized a signaling pathway that is specific to the endothelial cells that line cerebral arteries. Reactive oxygen species (ROS) cause lipid peroxidation. In endothelial cells in cerebral arteries, locally produced ROS oxidized lipids, which triggered calcium influx through the ion channel TRPA1. In turn, this calcium influx activated a potassium-permeable channel, resulting in dilation of cerebral arteries. Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca2+ signals representing Ca2+ influx through single TRPA1 channels, which we called “TRPA1 sparklets.” TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca2+ entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca2+ channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca2+-sensitive K+ channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca2+ influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.

A PLCγ1-Dependent, Force-Sensitive Signaling Network in the Myogenic Constriction of Cerebral Arteries

The signaling pathway that links the sensing of increased blood pressure to constriction in cerebral arteries is delineated. Maintaining Blood Flow to the Brain Cerebral arteries continually adjust to changes in blood pressure to ensure constant blood flow to the brain. In response to increased blood pressure, the smooth muscle cells in cerebral arteries contract, resulting in blood vessel constriction. This response requires two cell surface ion channels—TRPC6, a channel that is activated by the stretch caused by increased blood pressure, and TRPM4, a channel that triggers the electrical impulses necessary for blood vessel constriction. Gonzales et al. found that activation of TRPC6 stimulated TRPM4 through calcium-dependent pathways. TRPC6, TRPM4, and the enzyme PLCγ1 were located in close proximity to each other in smooth muscle cells, indicating that a pressure-sensitive signaling network keeps blood flowing in the brain. Maintaining constant blood flow in the face of fluctuations in blood pressure is a critical autoregulatory feature of cerebral arteries. An increase in pressure within the artery lumen causes the vessel to constrict through depolarization and contraction of the encircling smooth muscle cells. This pressure-sensing mechanism involves activation of two types of transient receptor potential (TRP) channels: TRPC6 and TRPM4. We provide evidence that the activation of the γ1 isoform of phospholipase C (PLCγ1) is critical for pressure sensing in cerebral arteries. Inositol 1,4,5-trisphosphate (IP3), generated by PLCγ1 in response to pressure, sensitized IP3 receptors (IP3Rs) to Ca2+ influx mediated by the mechanosensitive TRPC6 channel, synergistically increasing IP3R-mediated Ca2+ release to activate TRPM4 currents, leading to smooth muscle depolarization and constriction of isolated cerebral arteries. Proximity ligation assays demonstrated colocalization of PLCγ1 and TRPC6 with TRPM4, suggesting the presence of a force-sensitive, local signaling network comprising PLCγ1, TRPC6, TRPM4, and IP3Rs. Src tyrosine kinase activity was necessary for stretch-induced TRPM4 activation and myogenic constriction, consistent with the ability of Src to activate PLCγ isoforms. We conclude that contraction of cerebral artery smooth muscle cells requires the integration of pressure-sensing signaling pathways and their convergence on IP3Rs, which mediate localized Ca2+-dependent depolarization through the activation of TRPM4.

CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Rationale: T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results: Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles

Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves - innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

No Static at All: Tuning Into the Complexities of Ca2+ Signaling in the Endothelium

A correlation between increases in the intracellular Ca2+ concentration of vascular endothelial cells and release of endothelium-dependent relaxing factors was first reported decades ago by Luckhoff et al.1 In agreement with these formative observations, subsequent studies demonstrated that Ca2+-sensitive biosynthetic pathways, such as endothelial nitric oxide synthase, as well as small and intermediate conductance Ca2+-activated K+ channels (KCa2.3 and KCa3.1) are primary drivers of endothelium-dependent vasodilation. Because endothelial dysfunction and loss of vasodilatory capacity are a common hallmark of cardiovascular diseases, the underlying Ca2+ signaling mechanisms are of considerable interest. Technological advances, including the development of high-affinity fluorescent Ca2+ indicator dyes, transgenic mice expressing genetically encoded Ca2+ indicator proteins selectively in the endothelium,2 high-speed, high-resolution confocal Ca2+ imaging, and total internal reflection fluorescent microscopy allow the Ca2+ signals controlling endothelium-dependent vasodilation to be probed in ever increasing detail. Application of these methods has revealed that Ca2+ mobilization pathways in the endothelium are unexpectedly complex, dynamic, and diverse. For example, spreading intracellular and intercellular Ca2+ waves are stimulated by the well-characterized endothelium-dependent vasodilator acetylcholine in many vascular beds.2 These propagating Ca2+ events may be critically important for conducted vasodilatory responses.3 Another type of Ca2+ signal was reported by Ledoux et al,4 who demonstrated that in mouse mesenteric arteries, acetylcholine enhances localized release of Ca2+ from the endoplasmic reticulum through inositol trisphosphate receptors (IP3R). These subcellular Ca2+ signals, referred to as Ca2+ pulsars, are localized to membrane domains that project through the internal elastic lamina separating the endothelium from underlying vascular smooth muscle cells. Ca2+ pulsar sites colocalize with areas densely expressing KCa3.1 channels, suggesting that Ca2+ pulsars may …

TRPs in the kidney – location, location, location

A recent study by Chen et al., featured in this issue of Acta Physiologica, may provide the molecular basis for new treatment avenues for acute kidney injury (AKI) by targeting of specific members of the vanilloid (V) transient receptor potential (TRP) subfamily of cation channels (Chen et al., 2014). Maintenance of ideal tissue hemodynamics is vital for organ homeostasis and relies in part on the finely tuned ability of small arteries and arterioles to constrict or dilate in response to changes in perfusion pressure. Pathological states can impair this intrinsic ability, leading to increases in vascular resistance and ischemic injuries. A reduction in blood flow to the kidneys, usually caused by acute hypovolemia, sepsis, surgery and major trauma, can lead to AKI, a disease characterized by rapid loss of kidney function and reduction in glomerular filtration rate (GFR), mainly due to reduction in renal blood flow. AKI is a disease with a high mortality rate, ranging from 40-75%. Impairment of endothelium-dependent dilation of the renal microcirculation, mainly through generation of nitric oxide (NO), is involved in the development of AKI and its progression to chronic kidney disease (Basile and Yoder, 2014). Currently, treatment of AKI consists of pharmacological interventions to mitigate the underlying cause and maintain blood electrolytes values within normal ranges, and dialysis. The findings of Chen and colleagues described in this issue could lead to the development of therapeutics that specifically restore blood flow to the renal pre-glomerular microvasculature during AKI. Heterogeneous tissue distribution of the members of the TRP channel superfamily is well appreciated, but the study by Chen et al. shows that there is differential activity, likely due to preferential expression, of a single TRP channel, TRPV1, within the renal vascular tree. The authors show that activation of Ca2+-permeable TRPV1 channels with capsaicin, a substance derived from hot peppers, dilated vessels in the renal preglomerular microvasculature, characterized by a blunted vascular resistance in isolated perfused kidneys. This effect was inhibited by capsazepine, a selective TRPV1 inhibitor, and was absent from TRPV1−/− mice. Interestingly, large conductance renal arteries only dilated to high concentrations of capsaicin, an effect that was still observed in TRPV1−/− mice, suggesting TRPV1-independent dilatory mechanisms (Chen et al., 2014). In addition, isolated arterioles from the medullary vasa recta were unresponsive to capsaicin stimulation, strengthening the argument that involvement of TRPV1 in endothelium-dependent dilation is indeed restricted to the pre-glomerular renal microcirculation. These novel data compellingly highlight functional heterogeneity of TRP channel activity within the renal vascular tree. Such differential responses may allow the development of tailored therapies aimed at reducing vascular resistance in the renal pre-glomerular microvasculature that will improve renal blood flow and function during AKI. A second intriguing finding of this study is that TRPV4 channels are functional in all segments of the renal circulation, and that TRPV4 vasodilatory activity seems to be coupled to the generation of NO by the enzyme NO synthase (NOS). These data add another dimension to our current understanding of TRPV4 channels in the regulation of vascular tone. Most studies report the importance of TRPV4 in dilations mediated by endothelium-derived hyperpolarization independent of NO production (Sonkusare et al., 2012, Earley et al., 2009). However, TRPV4 has recently been shown to be important for the rescue of NO production in endothelial cells after hypoxic pre-conditioning both in vitro and in vivo (Rath et al., 2012), showing its vital role in the organ protective responses facing ischemic injuries. Thus, TRPV4-mediated NO generation could emerge as a prominent drug target for the treatment of AKI. The present study raises stimulating questions that warrant further investigation. It remains to be determined what genetic factors regulate the selective expression of TRPV1 in the renal pre-glomerular microcirculation, and if this occurs during development or post-development. In addition, it would be interesting to determine if TRPV4 and NOS are located in close proximity, such as that the inflow of Ca2+ caused by TRPV4 activity in the form of TRPV4 sparklets (Sullivan et al., 2012, Sonkusare et al., 2012) generate Ca2+ microdomains with locally elevated Ca2+ concentration that directly activate NOS. It is also possible TRPV4-mediated Ca2+ influx causes a global increase in intracellular Ca2+ by Ca2+-induced Ca2+ release from intracellular stores. It should be noted that TRPV4 is present in membrane caveolae rich in the protein caveolin-1, which is a stereotactic inhibitor of the endothelial NOS (Trane et al., 2014). Unraveling the interplay between TRPV4 and NOS would provide further insight in the intracellular organization of membrane subdomains. Such data would complement recent studies that provide evidence that TRPV4 channels in smooth muscle cells are present in signaling complexes organized by A-kinase anchor protein 150 (AKAP150) allowing activation downstream of G-protein coupled receptors (Mercado et al., 2014). In contrast, the endogenous regulation of TRPV1 channels in the vasculature remains unknown. In summary, the report by Chen et al. elegantly shows through functional studies using isolated arteries and perfusion of whole kidneys differential functional consequences of TRPV1 and TRPV4 activity in different segments of the renal circulation. Pharmacological activation of TRPV1 may emerge as a promising candidate for treatment of AKI to selectively target the pre-glomerular microcirculation, possibly restoring renal blood flow, GFR and, with them, renal function.

Neuroprotective effects of TRPA1 channels in the cerebral endothelium following ischemic stroke

Hypoxia and ischemia are linked to oxidative stress, which can activate the oxidant-sensitive transient receptor potential ankyrin 1 (TRPA1) channel in cerebral artery endothelial cells, leading to vasodilation. We hypothesized that TRPA1 channels in endothelial cells are activated by hypoxia-derived reactive oxygen species, leading to cerebral artery dilation and reduced ischemic damage. Using isolated cerebral arteries expressing a Ca2+ biosensor in endothelial cells, we show that 4-hydroxynonenal and hypoxia increased TRPA1 activity, detected as TRPA1 sparklets. TRPA1 activity during hypoxia was blocked by antioxidants and by TRPA1 antagonism. Hypoxia caused dilation of cerebral arteries, which was disrupted by antioxidants, TRPA1 blockade and by endothelial cell-specific Trpa1 deletion (Trpa1 ecKO mice). Loss of TRPA1 channels in endothelial cells increased cerebral infarcts, whereas TRPA1 activation with cinnamaldehyde reduced infarct in wildtype, but not Trpa1 ecKO, mice. These data suggest that endothelial TRPA1 channels are sensors of hypoxia leading to vasodilation, thereby reducing ischemic damage.

A TRPC3 signalling complex promotes cerebral artery remodelling during hypertension

This editorial refers to ‘TRPC3 channel confers cerebrovascular remodelling during hypertension via transactivation of EGF receptor signalling’ by M. Wang et al. , pp. 34–43. Members of the transient receptor potential (TRP) superfamily of cation channels are key players in critical aspects of vascular function, such as angiogenesis, endothelium-dependent vasodilation, functional hyperaemia, and pressure-induced autoregulation of cerebral artery tone.1 Recent work highlights the importance of TRP channels as components of complex subcellular signalling networks that perform some of these functions. For example, the sole member of the ankyrin TRP subfamily, TRPA1, co-localizes with NADPH-oxidase 2 (NOX2) and intermediate- and small-conductance Ca2+-activated K+ channels (IK and SK, respectively) in the endothelial face of myoendothelial projections. In this complex, NOX2 activity leads to opening of TRPA1 channels, causing a localized influx of Ca2+ that activates nearby IK and SK to hyperpolarize the endothelial cell plasma membrane, leading to dilatation of cerebral pial arteries.2 Vanilloid (V) TRPV4 channels in the renal vasculature are coupled with endothelial nitric oxide synthase and TRPV4 activation causes vasodilation of renal arteries through the generation of nitric …

Science Signaling Podcast: 6 January 2015

Reactive oxygen species create peroxidized lipids that promote dilation of cerebral arteries by activating the ion channel TRPA1. This Podcast features an interview with Scott Earley, author of a Research Article that appears in the 6 January 2015 issue of Science Signaling, about how reactive oxygen species (ROS) trigger dilation of cerebral arteries. ROS can damage cellular proteins and nucleic acids, but they can also act as signaling molecules. In the cerebral circulatory system, ROS trigger vasodilation, an increase in the diameter of blood vessels, which increases blood flow. In peripheral arteries, ROS trigger vasoconstriction, the narrowing of blood vessels, which reduces blood flow. Exactly how ROS elicits opposite effects in different types of blood vessels is not clear. Sullivan et al. found that localized sources of extracellular ROS in cerebral arteries oxidized plasma membrane lipids, which in turn promoted calcium influx through the ion channel TRPA1 in endothelial cells. This influx of calcium triggered opening of potassium channels, resulting in hyperpolarization of both endothelial and vascular smooth muscle cell membranes, thereby causing vasodilation.