Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD.

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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 Gq/11 and/or β-arrestins. FR900359, Sar1Ile4Ile8-angiotensin II (SII), TRV120027 and TRV120055 were used as selective Gq/11 inhibitor and biased agonists to activate noncanonical β-arrestin and canonical Gq/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 Gq/11-dependent signaling pathways of mechanoactivated AT1R but not G protein-independent, noncanonical pathways in smooth muscle cells.

Hypertension is associated with increased production and circulation of vasoconstrictors, resulting in enhanced signalling through their cognate G protein-coupled receptors (GPCR). Prolonged vasoconstrictor GPCR signalling increases arterial contraction and stimulates signalling pathways that promote vascular smooth muscle cell (VSMC) proliferation, contributing to the development of atherosclerotic plaques, re-stenosis lesions and vascular remodelling. GPCR signalling through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) promotes VSMC proliferation. In VSMC, G protein-coupled receptor kinase 2 (GRK2) is known to regulate numerous vasoconstrictor GPCRs and their downstream signalling pathways. As GRK2 is implicated in controlling various aspects of cellular growth, we examined whether GRK2 could affect VSMC proliferation. Using two indices of cell growth, we show that PI3K inhibition and depletion of GRK2 expression produced a similar ablation of pro-proliferative vasoconstrictor-stimulated VSMC growth. Furthermore, GRK2-knockdown ablated the sustained phase of endothelin-1 and angiotensin-II-stimulated Akt phosphorylation, whilst the peak (5 min) phase was unaffected. Conversely, the GRK2 inhibitor compound 101 did not affect vasoconstrictor-driven Akt phosphorylation. Vasoconstrictor-stimulated phosphorylation of the Akt substrates GSK3α and GSK3β was ablated following RNAi-mediated GRK2 depletion, or after PI3K inhibition. Moreover, GRK2 knockdown prevented endothelin-1 and angiotensin-II from increasing cyclin D1 expression. These data suggest GRK2 expression is essential to facilitate vasoconstrictor-driven VSMC proliferation through its ability to promote efficient prolonged PI3K-Akt signalling, and thus relieve the GSK3-mediated block on cell cycling. Considering VSMC GRK2 expression increases early in the development of hypertension, this highlights the potential for GRK2 to promote VSMC growth and exacerbate hypertensive pathophysiological vascular remodelling.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.

The vasomotor role of K2P potassium channels during early postnatal development has never been investigated. We tested the hypothesis that TASK-1 channel (K2P family member) contribution to arterial vascular tone and BP is higher in the early postnatal period than in adulthood.
We studied 10- to 15-day-old (\"young\") and 2- to 3-month-old (\"adult\") male rats performing digital PCR (dPCR) (using endothelium-intact saphenous arteries), isometric myography, sharp microelectrode technique, quantitative PCR (qPCR) and Western blotting (using endothelium-denuded saphenous arteries), and arterial pressure measurements under urethane anaesthesia.
We found mRNA of Kcnk1-Kcnk7, Kcnk12, and Kcnk13 genes to be expressed in rat saphenous artery, and Kcnk3 (TASK-1) and Kcnk6 (TWIK-2) were most abundant in both age groups. The TASK-1 channel blocker AVE1231 (1 μmol·L-1 ) prominently depolarized arterial smooth muscle and increased basal tone level and contractile responses to methoxamine of arteries from young rats but had almost no effect in adult rats. The level of TASK-1 mRNA and protein expression was higher in arteries from young compared with adult rats. Importantly, intravenous administration of AVE1231 (4 mg·kg-1 ) had no effect on mean arterial pressure in adult rats but prominently raised it in young rats.
We showed that TASK-1 channels are important for negative feedback regulation of vasocontraction in young but not adult rats. The influence of TASK-1 channels most likely contributes to low BP level at perinatal age.

PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specific Pkd2 knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca2+ influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca2+ influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2-mediated Na+ currents (INa) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of INa activation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.

Alcohol (ethyl alcohol; ethanol) and caffeine are the two most widely used psychoactive substances in the world. Caffeine and ethanol have both been reported to constrict cerebral arteries in several species, including humans. We have recently shown that application of 10-μM caffeine mixed with 50 mM ethanol to in vitro pressurized cerebral arteries of rats reduced ethanol-induced constriction. This effect was dependent on the presence of nitric oxide (NO•) and could be observed in de-endothelialized arteries supplied with the NO donor sodium nitroprusside (SNP). The molecular target(s) of ethanol-caffeine interaction in cerebral arteries has remained unknown. In the present work, we used rat and mouse middle cerebral arteries (MCA) to identify the extra-endothelial effectors of NO-mediated, caffeine-induced protection against ethanol-evoked arterial constriction. Constriction of intact MCA of rat by either 50 mM ethanol or 10 μM caffeine was ablated in the presence of a selective TRPV1 pharmacological blocker. TRPV1 pharmacological block, but not block of TRPA1, PKG, or BK channels, removed caffeine-induced protection against ethanol-evoked rat MCA constriction, whether evaluated in arteries with intact endothelium or in SNP-supplemented, de-endothelialized arteries. In mouse arteries, caffeine-induced protection against ethanol-induced MCA constriction was significantly amplified, resulting in actual vasodilation, upon pharmacological block of TRPV1, and in TRPV1 knock-out arteries. Despite some species-specific differences, our study unequivocally demonstrates the presence of functional, extra-endothelial TRPV1 that participates in both endothelium-independent MCA constriction by separate exposure to ethanol or caffeine and caffeine-induced protection against ethanol-evoked MCA constriction.

Potassium channels are key regulators of smooth muscle membrane potential and arterial tone. However, the roles of potassium channels in vascular tone regulation in the systemic circulation during early postnatal development are poorly understood. Therefore, this study tested the hypothesis that the negative feedback regulation of vasocontraction by potassium channels changes during maturation.
Experiments were performed on endothelium-denuded saphenous arteries from 10- to 15-day-old and 2- to 3-month-old male rats. Isometric force and membrane potential were recorded using wire myography and the sharp microelectrode technique respectively; mRNA and protein contents were determined by qPCR and Western blotting.
The effects of Kv 1, Kir and Kv 7 channel blockers (DPO-1, BaCl2 , XE991) on methoxamine-induced contraction were larger in arteries of 10- to 15-day-old compared to 2- to 3-month-old animals. In contrast, the BKC a channel blocker iberiotoxin had a stronger influence in 2- to 3- month-old rats. The effects of KATP and Kv 2 channel blockers (glibenclamide, stromatoxin) were not pronounced at both ages. The larger influence of Kv 7 and Kir channel blockade on arterial contraction in 10- to 15-day-old rats was associated with more prominent smooth muscle depolarization. The developmental alterations in potassium channel functioning were generally consistent with their mRNA and protein expression levels in arterial smooth muscle.
The negative feedback regulation of vasocontraction by potassium channels varies during maturation depending on the channel type. A dominating contribution of Kv 7 channels to the regulation of basal tone and agonist-induced contraction was observed in arteries of 10- to 15-day-old animals.

Cholesterol (CLR) conversion into bile acids (BAs) in the liver constitutes the major pathway for CLR elimination from the body. Moreover, these steroids regulate each other\'s metabolism. While the roles of CLR and BAs in regulating metabolism and tissue function are well known, research of the last two decades revealed the existence of specific protein receptors for CLR or BAs in tissues with minor contribution to lipid metabolism, raising the possibility that these lipids serve as signaling molecules throughout the body. Among other lipids, CLR and BAs regulate ionic current mediated by the activity of voltage- and Ca2+-gated, K+ channels of large conductance (BK channels) and, thus, modulate cell physiology and participate in tissue pathophysiology. Initial work attributed modification of BK channel function by CLR or BAs to the capability of these steroids to directly interact with bilayer lipids and thus alter the physicochemical properties of the bilayer with eventual modification of BK channel function. Based on our own work and that of others, we now review evidence that supports direct interactions between CLR or BA and specific BK protein subunits, and the consequence of such interactions on channel activity and organ function, with a particular emphasis on arterial smooth muscle. For each steroid type, we will also briefly discuss several mechanisms that may underlie modification of channel steady-state activity. Finally, we will present novel computational data that provide a chemical basis for differential recognition of CLR vs lithocholic acid by distinct BK channel subunits and recognition sites.