New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction

BACKGROUND: Despite increasing evidence for the presence of voltage-gated Na(+) channels (Na(v)) isoforms and measurements of Na(v) channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Na(v) channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Na(v) channels in the control of rat aortic contraction. METHODOLOGY/PRINCIPAL FINDINGS: Na(v) channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Na(v) channels and smooth muscle alpha-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Na(v) transcripts: Na(v)1.2, Na(v)1.3, and Na(v)1.5. Only the Na(v)1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Na(v) channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Na(v) channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2-10 mM), which induced moderate membrane depolarization (e.g., from -55.9+/-1.4 mV to -45.9+/-1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 microM) and blocked by TTX (1 microM). KB-R7943, an inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX. CONCLUSIONS/SIGNIFICANCE: These results define a new role for Na(v) channels in arterial physiology, and suggest that the TTX-sensitive Na(v)1.2 isoform, together with the Na(+)/Ca(2+) exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization

Disrupted endothelial cell heterogeneity and network organization impair vascular function in prediabetic obesity

Background: Obesity is a major risk factor for diabetes and cardiovascular diseases such as hypertension, heart failure, and stroke. Impaired endothelial function occurs in the earliest stages of obesity and underlies vascular alterations that give rise to cardiovascular disease. However, the mechanisms that link weight gain to endothelial dysfunction are ill-defined. Increasing evidence suggests that endothelial cells are not a population of uniform cells but are highly heterogeneous and are organized as a communicating multicellular network that controls vascular function. Purpose: To investigate the hypothesis that disrupted endothelial heterogeneity and network-level organization contribute to impaired vascular reactivity in obesity. Methods and Results: To study obesity-related vascular function without complications associated with diabetes, a state of prediabetic obesity was induced in rats. Small artery diameter recordings confirmed nitric-oxide mediated vasodilator responses were dependent on increases in endothelial calcium levels and were impaired in obese animals. Single-photon imaging revealed a linear relationship between blood vessel relaxation and population-wide calcium responses. Obesity did not alter the slope of this relationship, but impaired calcium responses in the endothelial cell network. The network comprised structural and functional components. The structural architecture, a hexagonal lattice network of connected cells, was unchanged in obesity. The functional network contained sub-populations of clustered specialized agonist-sensing cells from which signals were communicated through the network. In obesity there were fewer but larger clusters of sensory cells and communication path lengths between clusters increased. Communication between neighboring cells was unaltered in obesity. Altered network organization resulted in impaired, population-level calcium signaling and deficient endothelial control of vascular tone. Conclusions: The distribution of cells in the endothelial network is critical in determining overall vascular response. Altered cell heterogeneity and arrangement in obesity decreases endothelial function and provides a novel framework for understanding compromised endothelial function in cardiovascular disease

Calcium mobilization via intracellular ion channels, store organization and mitochondria in smooth muscle

In smooth muscle, Ca2+ release from the internal store into the cytoplasm occurs via inositol trisphosphate (IP3R) and ryanodine receptors (RyR). The internal Ca2+ stores containing IP3R and RyR may be arranged as multiple separate compartments with various IP3R and RyR arrangements, or there may be a single structure containing both receptors. The existence of multiple stores is proposed to explain several physiological responses which include the progression of Ca2+ waves, graded Ca2+ release from the store and various local responses and sensitivities. We suggest that, rather than multiple stores, a single luminally-continuous store exists in which Ca2+ is in free diffusional equilibrium throughout. Regulation of Ca2+ release via IP3R and RyR by the local Ca2+ concentration within the stores explains the apparent existence of multiple stores and physiological processes such as graded Ca2+ release and Ca2+ waves. Close positioning of IP3R on the store with mitochondria or with receptors on the plasma membrane creates ‘IP3 junctions’ to generate local responses on the luminally-continuous store

Endothelial Transient Receptor Potential Channels and Vascular Remodeling: Extracellular Ca2 + Entry for Angiogenesis, Arteriogenesis and Vasculogenesis

Vasculogenesis, angiogenesis and arteriogenesis represent three crucial mechanisms involved in the formation and maintenance of the vascular network in embryonal and post-natal life. It has long been known that endothelial Ca2+ signals are key players in vascular remodeling; indeed, multiple pro-angiogenic factors, including vascular endothelial growth factor, regulate endothelial cell fate through an increase in intracellular Ca2+ concentration. Transient Receptor Potential (TRP) channel consist in a superfamily of non-selective cation channels that are widely expressed within vascular endothelial cells. In addition, TRP channels are present in the two main endothelial progenitor cell (EPC) populations, i.e., myeloid angiogenic cells (MACs) and endothelial colony forming cells (ECFCs). TRP channels are polymodal channels that can assemble in homo- and heteromeric complexes and may be sensitive to both pro-angiogenic cues and subtle changes in local microenvironment. These features render TRP channels the most versatile Ca2+ entry pathway in vascular endothelial cells and in EPCs. Herein, we describe how endothelial TRP channels stimulate vascular remodeling by promoting angiogenesis, arteriogenesis and vasculogenesis through the integration of multiple environmental, e.g., extracellular growth factors and chemokines, and intracellular, e.g., reactive oxygen species, a decrease in Mg2+ levels, or hypercholesterolemia, stimuli. In addition, we illustrate how endothelial TRP channels induce neovascularization in response to synthetic agonists and small molecule drugs. We focus the attention on TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPV1, TRPV4, TRPM2, TRPM4, TRPM7, TRPA1, that were shown to be involved in angiogenesis, arteriogenesis and vasculogenesis. Finally, we discuss the role of endothelial TRP channels in aberrant tumor vascularization by focusing on TRPC1, TRPC3, TRPV2, TRPV4, TRPM8, and TRPA1. These observations suggest that endothelial TRP channels represent potential therapeutic targets in multiple disorders featured by abnormal vascularization, including cancer, ischemic disorders, retinal degeneration and neurodegeneration

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E-rev) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E-rev of nicotine-induced current as a function of extracellular Na+ concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K+/Na+ permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca2+ concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na+, which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine