Search

The arterial myogenic response is an inherent property of resistance arteries. Myogenic tone is crucial for maintaining a relatively constant blood flow in response to changes in intraluminal pressure and protects delicate organs from excessive blood flow. Although this fundamental physiological phenomenon has been extensively studied, the underlying molecular mechanisms are largely unknown. Recent studies identified a crucial role of mechano-activated angiotensin II type 1 receptors (AT1R) in this process. The development of myogenic response is affected by aging. In this review, we summarize recent progress made to understand the role of AT1R and other mechanosensors in the control of arterial myogenic response. We discuss age-related alterations in myogenic response and possible underlying mechanisms and implications for healthy aging.

Background Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) arterial tone. However, many of the molecular determinants of this response are unknown. We previously found that mice with targeted disruption of the gene encoding the angiotensin II type 1a receptor (AT1AR) ( Agtr1a ), the major murine angiotensin II type 1 receptor (AT1R) isoform, showed reduced myogenic tone; however, uncontrolled genetic events (in this case, gene ablation) can lead to phenotypes that are difficult or impossible to interpret. Methods and Results We tested the mechanosensitive function of AT1R using tamoxifen‐inducible smooth muscle‐specific AT1aR knockout (smooth muscle‐ Agtr1a −/− ) mice and studied downstream signaling cascades mediated by G q/11 and/or β‐arrestins. FR900359, Sar1Ile4Ile8‐angiotensin II (SII), TRV120027 and TRV120055 were used as selective G q/11 inhibitor and biased agonists to activate noncanonical β‐arrestin and canonical G q/11 signaling of the AT1R, respectively. Myogenic and Ang II‐induced constrictions were diminished in the perfused renal vasculature, mesenteric and cerebral arteries of smooth muscle‐ Agtr1a −/− mice. Similar effects were observed in arteries of global mutant Agtr1a −/− but not Agtr1b −/− mice. FR900359 decreased myogenic tone and angiotensin II‐induced constrictions whereas selective biased targeting of AT1R‐β‐arrestin signaling pathways had no effects. Conclusions This study demonstrates that myogenic arterial constriction requires G q/11 ‐dependent signaling pathways of mechanoactivated AT1R but not G protein‐independent, noncanonical pathways in smooth muscle cells.

Background Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) arterial tone. However, many of the molecular determinants of this response are unknown. We previously found that mice with targeted disruption of the gene encoding the angiotensin II type 1a receptor (AT1AR) (

Aging is an independent risk factor for hypertension, cardiovascular morbidity, and mortality. However, detailed mechanisms linking aging to cardiovascular disease are unclear. We studied the aging effects on the role of perivascular adipose tissue and downstream vasoconstriction targets, voltage-dependent KV7 channels, and their pharmacological modulators (flupirtine, retigabine, QO58, and QO58-lysine) in a murine model. We assessed vascular function of young and old mesenteric arteries in vitro using wire myography and membrane potential measurements with sharp electrodes. We also performed bulk RNA sequencing and quantitative reverse transcription-polymerase chain reaction tests in mesenteric arteries and perivascular adipose tissue to elucidate molecular underpinnings of age-related phenotypes. Results revealed impaired perivascular adipose tissue-mediated control of vascular tone particularly via KV7.3–5 channels with increased age through metabolic and inflammatory processes and release of perivascular adipose tissue-derived relaxation factors. Moreover, QO58 was identified as novel pharmacological vasodilator to activate XE991-sensitive KCNQ channels in old mesenteric arteries. Our data suggest that targeting inflammation and metabolism in perivascular adipose tissue could represent novel approaches to restore vascular function during aging. Furthermore, KV7.3–5 channels represent a promising target in cardiovascular aging.

Vascular smooth muscle cells (VSMCs) of small peripheral arteries contribute to blood pressure control by adapting their contractile state. These adaptations depend on the VSMC cytosolic Ca2+ concentration, regulated by complex local elementary Ca2+ signaling pathways. Ca2+ sparks represent local, transient, rapid calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial SMCs, Ca2+ sparks activate nearby calcium-dependent potassium channels, cause membrane hyperpolarization and thus decrease the global intracellular [Ca2+] to oppose vasoconstriction. Arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux through RyRs. Cav3.2 T-type channels contribute to a minor extend to Ca2+ spark generation in certain types of arteries. Their localization within cell membrane caveolae is essential. We summarize present data on local elementary calcium signaling (Ca2+ sparks) in arterial SMCs with focus on RyR isoforms, large-conductance calcium-dependent potassium (BKCa) channels, and cell membrane-bound calcium channels (Cav1.2 and Cav3.2), particularly in caveolar microdomains.

Aging is an independent risk factor for hypertension, cardiovascular morbidity, and mortality. However, detailed mechanisms linking aging to cardiovascular disease are unclear. We studied the aging effects on the role of perivascular adipose tissue and downstream vasoconstriction targets, voltage-dependent KV7 channels, and their pharmacological modulators (flupirtine, retigabine, QO58, QO58-lysine) in a murine model. We assessed vascular function of young and old mesenteric arteries in vitro using wire myography. We also performed bulk RNA sequencing and quantitative reverse transcription polymerase chain reaction tests in mesenteric arteries and perivascular adipose tissue to elucidate molecular underpinnings of age-related phenotypes. Results revealed impaired perivascular adipose tissue-mediated control of vascular tone particularly via KV7.3-5 channels with increased age through metabolic and inflammatory processes and release of perivascular adipose tissue-derived relaxation factors. Moreover, QO58 was identified as novel pharmacological vasodilator to activate XE991-sensitive KCNQ channels in old mesenteric arteries. Our data suggest that targeting inflammation and metabolism in perivascular adipose tissue could represent novel approaches to restore vascular function during aging. Furthermore, QO58 represents a novel tool for cardiovascular and hypertension research in aging.

BackgroundThe myogenic response is an inherent vasoconstrictive property of resistance arteries to keep blood flow constant in response to increases in intravascular pressure. Angiotensin II (Ang II) type 1 receptors (AT1R) are broadly distributed, mechanoactivated receptors, which have been proposed to transduce myogenic vasoconstriction. However, the AT1R subtype(s) involved and their downstream G protein- and β-arrestin-mediated signaling pathways are still elusive.ObjectiveTo characterize the function of AT1aR and AT1bR in the regulation of the myogenic response of resistance size arteries and possible downstream signaling cascades mediated by Gq/11and/or β-arrestins.MethodsWe usedAgtr1a-/-,Agtr1b-/-and tamoxifen-inducible smooth muscle-specific AT1aR knockout mice (SM-Agtr1amice). FR900359, [Sar1, Ile4, Ile8] Ang II (SII) and TRV120055 were used as selective Gq/11protein inhibitor and biased agonists to activate non-canonical β-arrestin and canonical Gq/11signaling of the AT1R, respectively.ResultsMyogenic and Ang II-induced vasoconstrictions were diminished in the perfused renal vasculature ofAgtr1a-/-andSM-Agtr1amice. Similar results were observed in isolated pressurized mesenteric and cerebral arteries. Myogenic tone and Ang II-induced vasoconstrictions were normal in arteries fromAgtr1b-/-mice. The Gq/11blocker FR900359 decreased myogenic tone and Ang II vasoconstrictions while selective biased targeting of AT1R β-arrestin signaling pathways had no effects.ConclusionThe present study demonstrates that myogenic arterial constriction requires Gq/11-dependent signaling pathways of mechanoactivated AT1aR but not G protein-independent, noncanonical alternative signaling pathways in the murine mesenteric, cerebral and renal circulation.

Objective— This study examined whether caveolae position Ca V 3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca 2+ influx to trigger Ca 2+ sparks and large-conductance Ca 2+ -activated K + channel feedback. Approach and Results— Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that Ca V 3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-β-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed Ca V 3.2 activity along with large-conductance Ca 2+ -activated K + –mediated spontaneous transient outward currents in cells from C57BL/6 but not Ca V 3.2 −/− mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca 2+ channel activity, including Ca V 3.2. These observations indicate a mistargeting of Ca V 3.2 in caveolin 1 −/− mice, a view supported by a loss of Ni 2+ -sensitive Ca 2+ spark generation and colocalization signal (Ca V 3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the Ca V 3.2-RyR axis functionally impaired the ability of large-conductance Ca 2+ -activated K + to set tone in pressurized caveolin 1 −/− arteries. Conclusions— Caveolae play a critical role in protein targeting and preserving the close structural relationship between Ca V 3.2 and RyR needed to drive negative feedback control in resistance arteries.

Key points In arterial smooth muscle, Ca2+ sparks are elementary Ca2+ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+ -sensitive K+ (BKCa ) channels. This study elucidated the contribution of T-type Cav 3.2 channels in caveolae and their functional interaction with L-type Cav 1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Cav 1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T-type Cav 3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Cav 3.2 channels and RyRs to ignite Ca2+ sparks. Abstract Recent data suggest that T-type Cav 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav 3.2 channels and their functional interaction with Cav 1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Cav 1.2-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+ , Cd2+ and methyl-β-cyclodextrin were used to inhibit Cav 3.2 channels, Cav 1.2 channels and caveolae, respectively. Ni2+ (50 μmol L-1 ) and methyl-β-cyclodextrin (10 mmol L-1 ) decreased Ca2+ spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 μmol L-1 ) suppressed Ca2+ sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl-β-cyclodextrin. Our data demonstrate that Ca2+ influx through CaV 1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.

Caveolae position CaV3.2 (T‐type Ca2+ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large‐conductance Ca2+‐activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Cav3.2 channel inhibition by Ni2+ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Cav3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Cav1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2‐RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 µM), but insensitive to CaV1.2 and CaV3.2 channel blockade. Our data demonstrate that the VSMC CaV3.2‐RyR axis is down‐regulated by aging. This defective CaV3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs., Cav3.2‐RyR axis in smooth muscle cell is down‐regulated by age‐related ultrastructural alterations of caveolae and reduced Cav3.2 expression. This defective Cav3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs.

Background and Purpose: KV 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (VSMC) of diverse arteries, including mesenteric arteries. Based on pharmacological evidence using R-L3 (KV 7.1 channel opener), HMR1556, chromanol 293B (KV 7.1 channel blockers), stimulation of these channels has been suggested to evoke profound relaxation in various vascular beds of rats. However, the specificity of these drugs in vivo is uncertain. Experimental Approach: We used Kcnq1(-/-) mice and pharmacological tools to determine whether KV 7.1 channels play a role in the regulation of arterial tone. Key Results: R-L3 produced similar concentration-dependent relaxations (EC50 ~ 1.4 {mu}M) of arteries from wild-type (Kcnq1(+/+) ) and Kcnq1(-/-) mice, pre-contracted with either phenylephrine or 60 mM KCl. This relaxation was not affected by 10 {mu}M chromanol 293B, 10 {mu}M HMR1556 or 30 {mu}M XE991 (pan-KV 7 channel blocker). The anti-contractile effects of the perivascular adipose tissue (PVAT) were normal in Kcnq1(-/-) arteries. Chromanol 293B and HMR1556 did not affect the anti-contractile effects of (PVAT). Isolated VSMCs from Kcnq1(-/-) mice exhibited normal peak KV currents. The KV 7.2-5 channel opener retigabine caused similar relaxations in Kcnq1(-/-) and wild-type vessels. Conclusion and Implications: We conclude that KV 7.1 channels were apparently not involved in the control of arterial tone by α1 -adrenoceptor agonists and PVAT. In addition, R-L3 is an inappropriate pharmacological tool for studying the function of native vascular KV 7.1 channels in mice.

Objective- This study examined whether caveolae position Ca

AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels.METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels.RESULTS: The TRPV1 agonist capsaicin relaxed mouse mesenteric arteries with an EC50 of 25 nm, but large mouse renal arteries or rat descending vasa recta only at >100-fold higher concentrations. The vasodilatory effect of capsaicin in the low-nanomolar concentration range was endothelium-dependent and absent in vessels of Trpv1 -/- mice. The TRPV4 agonist GSK1016790A relaxed large conducting renal arteries, mesenteric arteries and vasa recta with EC50 of 18, 63 nm and ~10 nm respectively. These effects were endothelium-dependent and inhibited by a TRPV4 antagonist, AB159908 (10 μm). Capsaicin and GSK1016790A produced vascular dilation in isolated mouse perfused kidneys with EC50 of 23 and 3 nm respectively. The capsaicin effects were largely reduced in Trpv1 -/- kidneys, and the effects of GSK1016790A were inhibited in Trpv4 -/- kidneys.CONCLUSION: Our results demonstrate that two TRPV channels have unique sites of vasoregulatory function in the kidney with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.

Rationale : Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein–coupled receptors can elicit a stretch response. Objective : To determine whether angiotensin II type 1 receptors (AT 1 R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. Methods and Results : We used mice deficient in AT 1 R signaling molecules and putative ion channel targets, namely AT 1 R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K + (K v 7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT 1 R subtype a to a G q/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT 1 R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT 1 R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive K v channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K + current and myogenic contractions persist in arteries deficient in these channels. Conclusions : Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT 1 R subtype a. The AT 1 R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance.

KWe used Kcnq1R-L3 produced similar concentration-dependent relaxations (ECWe conclude that K

Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of small mesenteric arteries of mice, with calcium sparklets providing elementary calcium influx through single TRPV4 channels. The role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized that TRPV1 and TRPV4 play an important role in endothelium-dependent vasodilation of renal blood vessels. We performed myography experiments on isolated renal arteries and perfused isolated kidneys of C57BL/6J mice. Isolated mesenteric arteries served as control vessels. The TRPV1 agonist capsaicin relaxed mesenteric arteries with an EC 50 of ~9 nM, but not large renal arteries (EC 50 >10 μM). This relaxation was inhibited by the TRPV1 antagonist capsazepine (10 μM). The capsaicin effect was absent in vessels of TRPV1 knock-out mice. Cytochrome P450 (CYP) 4A- and CYP4F-derived hydroxyeicosatetraenoic acid (20-HETE), which is a proinflammatory mediator of endotoxin-induced acute systemic inflammation, activated TRPV1 channels in patch clamp studies. The TRPV4 agonist GSK1016790A relaxed renal and mesenteric arteries with EC 50 of ~12 nM and 70 nM, respectively. These effects were inhibited by a specific TRPV4 antagonist, AB159908 (10 μM). Capsaicin and GSK1016790A produced vascular dilation in isolated kidneys with EC 50 of ~11 nM and ~2 nM, respectively. The capsaicin effects were absent in kidneys of TRPV1 knock-out mice, whereas the effects of GSK1016790A were also observed in TRPV1 knock-out mice. Our results demonstrate that TRPV4 channels are capable to promote endothelial relaxation in large and small renal arteries, whereas TRPV1 channels play a specific vasodilatory role in small arteries of the kidney. We suggest that specific activation of TRPV1 and TRPV4 may be a novel promising strategy for treatment of sepsis-associated renal vasoconstriction.

Recent preclinical data indicate that activators of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) may improve the outcome of ischaemic acute kidney injury (AKI). The underlying mechanisms are unclear, but may involve TRPV1 channels in dorsal root ganglion neurones that innervate the kidney. Recent data identified TRPV4, together with TRPV1, to serve as major calcium influx channels in endothelial cells. In these cells, gating of individual TRPV4 channels within a four-channel cluster provides elementary calcium influx (calcium sparklets) to open calcium-activated potassium channels and promote vasodilation. The TRPV receptors can also form heteromers that exhibit unique conductance and gating properties, further increasing their spatio-functional diversity. This review summarizes data on electrophysiological properties of TRPV1/4 and their modulation by endogenous channel agonists such as 20-HETE, phospholipase C and phosphatidylinositide 3-kinase (PI3 kinase). We review important roles of TRPV1 and TRPV4 in kidney physiology and renal ischaemia reperfusion injury; further studies are warranted to address renoprotective mechanism of vanilloid receptors in ischaemic AKI including the role of the capsaicin receptor TRPV1 in primary sensory nerves and/or endothelium. Particular attention should be paid to understand the kidneys' ability to respond to ischaemic stimuli after catheter-based renal denervation therapy in man, whereas the discovery of novel pharmacological TRPV modulators may be a successful strategy for better treatment of acute or chronic kidney failure.