Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Gßγ subunit signalling underlies neuropeptide Y-stimulated vasoconstriction in rat mesenteric and coronary arteries.

BACKGROUND AND PURPOSE: Raised serum concentrations of the sympathetic co-transmitter neuropeptide Y (NPY) are linked to cardiovascular diseases. However, the signalling mechanism for vascular smooth muscle (VSM) constriction to NPY is poorly understood. Therefore, the present study investigated the mechanisms of NPY-induced vasoconstriction in rat small mesenteric (RMA) and coronary (RCA) arteries. EXPERIMENTAL APPROACH: Third-order mesenteric or intra-septal arteries from male Wistar rats were assessed in wire myographs for isometric tension, VSM membrane potential and VSM intracellular Ca2+ events. KEY RESULTS: NPY stimulated concentration-dependent vasoconstriction in both RMA and RCA, which was augmented by blocking NO synthase or endothelial denudation in RMA. NPY-mediated vasoconstriction was blocked by the selective Y1 receptor antagonist BIBO 3304 and Y1 receptor protein expression was detected in both the VSM and endothelial cells in RMA and RCA. The selective Gßγ subunit inhibitor gallein and the PLC inhibitor U-73122 attenuated NPY-induced vasoconstriction. Signalling via the Gßγ-PLC pathway stimulated VSM Ca2+ waves and whole-field synchronised Ca2+ flashes in RMA and increased the frequency of Ca2+ flashes in myogenically active RCA. Furthermore, in RMA, the Gßγ pathway linked NPY to VSM depolarization and generation of action potential-like spikes associated with intense vasoconstriction. This depolarization activated L-type voltage-gated Ca2+ channels, as nifedipine abolished NPY-mediated vasoconstriction. CONCLUSIONS AND IMPLICATIONS: These data suggest that the Gßγ subunit, which dissociates upon Y1 receptor activation, initiates VSM membrane depolarization and Ca2+ mobilisation to cause vasoconstriction. This model may help explain the development of microvascular vasospasm during raised sympathetic nerve activity.

Tracking endothelium-dependent NO release in pressurized arteries.

Background: Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Methods: Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), Cu2FL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or en face imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1 µM), and NO donor (SNAP, 10 µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR0. Results: In cell-free chambers using either Cu2FL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both Cu2FL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with Cu2FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR0 following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30 min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP. Conclusion: These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αßγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δßγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Signaling and structures underpinning conducted vasodilation in human and porcine intramyocardial coronary arteries.

Background: Adequate blood flow into coronary micro-arteries is essential for myocardial function. Here we assess the mechanisms responsible for amplifying blood flow into myogenically-contracting human and porcine intramyocardial micro-arteries ex vivo using endothelium-dependent and -independent vasodilators. Methods: Human and porcine atrial and ventricular small intramyocardial coronary arteries (IMCAs) were studied with pressure myography and imaged using confocal microscopy and serial section/3-D reconstruction EM. Results: 3D rendered ultrastructure images of human right atrial (RA-) IMCAs revealed extensive homo-and hetero-cellular contacts, including to longitudinally-arranged smooth muscle cells (l-SMCs) found between the endothelial cells (ECs) and radially-arranged medial SMCs (r-SMCs). Local and conducted vasodilatation followed focal application of bradykinin in both human and porcine RA-IMCAs, and relied on hyperpolarization of SMCs, but not nitric oxide. Bradykinin initiated asynchronous oscillations in endothelial cell Ca2+ in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca2+-activated K+ channels (KCa) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these KCa channels. Extensive electrical coupling was demonstrated between r-SMCs and l-SMCs, providing an additional pathway to facilitate the well-established myoendothelial coupling. Conducted dilation was still evident in a human RA-IMCA with poor myogenic tone, and heterocellular contacts were visible in the 3D reconstructed artery. Hyperpolarization and conducted vasodilation was also observed to adenosine which, in contrast to bradykinin, was sensitive to combined block of ATP-sensitive (KATP) and inwardly rectifying (KIR) K+ channels. Conclusions: These data extend our understanding of the mechanisms that coordinate human coronary microvascular blood flow and the mechanistic overlap with porcine IMCAs. The unusual presence of l-SMCs provides an additional pathway for rapid intercellular signaling between cells of the coronary artery wall. Local and conducted vasodilation follow hyperpolarization of the ECs or SMCs, and contact-coupling between l-SMCs and r-SMCs likely facilitates this vasodilation.

Fine-grained classification based on multi-scale pyramid convolution networks.

The large intra-class variance and small inter-class variance are the key factor affecting fine-grained image classification. Recently, some algorithms have been more accurate and efficient. However, these methods ignore the multi-scale information of the network, resulting in insufficient ability to capture subtle changes. To solve this problem, a weakly supervised fine-grained classification network based on multi-scale pyramid is proposed in this paper. It uses pyramid convolution kernel to replace ordinary convolution kernel in residual network, which can expand the receptive field of the convolution kernel and use complementary information of different scales. Meanwhile, the weakly supervised data augmentation network (WS-DAN) is used to prevent over fitting and improve the performance of the model. In addition, a new attention module, which includes spatial attention and channel attention, is introduced to pay more attention to the object part in the image. The comprehensive experiments are carried out on three public benchmarks. It shows that the proposed method can extract subtle feature and achieve classification effectively.

Acute cigarette smoke or extract exposure rapidly activates TRPA1-mediated calcium influx in primary human airway smooth muscle cells.

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors.

BACKGROUND: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. METHODS: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. RESULTS: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl- currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl- currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. CONCLUSIONS: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl- currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors.

Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells.

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.