Capillary oxygen regulates demand-supply coupling by triggering connexin40-mediated conduction: Rethinking the metabolic hypothesis.

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+.

Advanced glycated end-products inhibit dilation through constitutive endothelial RAGE and Nox1/4 in rat isolated skeletal muscle arteries.

OBJECTIVE: This study investigated the actions of advanced glycated end-products (AGE), their receptors (RAGE), and NAD(P)H oxidase (Nox) subtypes 1, 2, and 4 on mechanisms of endothelium-dependent dilation of the rat cremaster muscle artery (CMA). METHODS: Immunofluorescence studies were used to examine expression of RAGE in rat arteries. ROS accumulation was measured using luminescence and fluorescence assays. Functional studies were performed using pressure myography. RESULTS: High levels of RAGE expression were shown in the endothelial cells of the CMA, compared with low endothelial expression in middle cerebral and mesenteric arteries and the aorta. Exogenous AGE (in vitro glycated bovine serum albumin) stimulated H2O2 accumulation in CMA, which was prevented by the RAGE antagonist FPS-ZM1, the NAD(P)H oxidase (Nox) inhibitor apocynin and inhibited by the Nox1/4 inhibitor setanaxib, but not the Nox2 inhibitor GSK2795039. In functional studies, AGE inhibited vasodilation of CMA stimulated by acetylcholine, sodium nitroprusside, and the BKCa activator NS1619, but not adenosine-induced dilation. FPS-ZM1, apocynin, and setanaxib prevented the inhibitory effects of AGE on responses to acetylcholine and NS-1619. CONCLUSION: These observations suggest RAGE are constitutively expressed in the endothelium of the rat CMA and may be activated by AGE to stimulate Nox1/4 and ROS formation with resulting inhibition of NO and BKCa-mediated endothelium-dependent dilation.

Neurovascular Uncoupling Is Linked to Microcirculatory Dysfunction in Regions Outside the Ischemic Core Following Ischemic Stroke.

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor ß. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P<0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P=0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+ -activated potassium channels.

BACKGROUND: Within the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+ -activated K+ (IKCa ) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. OBJECTIVES: We investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. METHODS: Platelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb /ß3 activation, granule release, and procoagulant platelet formation. RESULTS: Platelet IKCa channel activation with SKA-31 inhibited aggregation in a concentration-dependent manner, an effect reversed by the selective IKCa channel blocker TRAM-34. The QCM model along with confocal microscopy demonstrated that SKA-31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA-31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA-31 also inhibited dense and alpha-granule secretion and integrin αIIb /ß3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. CONCLUSIONS: Platelet IKCa channel activation inhibits aggregation by reducing calcium-signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

Effect of ß1 /ß2 -adrenoceptor blockade on ß3 -adrenoceptor activity in the rat cremaster muscle artery.

BACKGROUND AND PURPOSE: The physiological role of vascular ß3 -adrenoceptors is not fully understood. Recent evidence suggests cardiac ß3 -adrenoceptors are functionally effective after down-regulation of ß1 /ß2 -adrenoceptors. The functional interaction between the ß3 -adrenoceptor and other ß-adrenoceptor subtypes in rat striated muscle arteries was investigated. EXPERIMENTAL APPROACH: Studies were performed in cremaster muscle arteries isolated from male Sprague-Dawley rats. ß-adrenoceptor expression was assessed through RT-PCR and immunofluorescence. Functional effects of ß3 -adrenoceptor agonists and antagonists and other ß-adrenoceptor ligands were measured using pressure myography. KEY RESULTS: All three ß-adrenoceptor subtypes were present in the endothelium of the cremaster muscle artery. The ß3 -adrenoceptor agonists mirabegron and CL 316,243 had no effect on the diameter of pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the ß3 -adrenoceptor agonist SR 58611A and the nonselective ß-adrenoceptor agonist isoprenaline caused concentration-dependent dilation. In the presence of ß1/2 -adrenoceptor antagonists nadolol (10 µM), atenolol (1 µM) and ICI 118,551 (0.1 µM) both mirabegron and CL 316,243 were effective in causing vasodilation and the potency of SR 58611A was enhanced, while responses to isoprenaline were inhibited. The ß3 -adrenoceptor antagonist L 748,337 (1 µM) inhibited vasodilation caused by ß3 -adrenoceptor agonists (in the presence of ß1/2 -adrenoceptor blockade), but L 748,337 had no effect on isoprenaline-induced vasodilation. CONCLUSION AND IMPLICATIONS: All three ß-adrenoceptor subtypes were present in the endothelium of the rat cremaster muscle artery, but ß3 -adrenoceptor mediated vasodilation was only evident after blockade of ß1/2 -adrenoceptors. This suggests constitutive ß1/2 -adrenoceptor activity inhibits ß3 -adrenoceptor function in the endothelium of skeletal muscle resistance arteries.

KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells.

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded kV7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

AIMS: Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain. METHODS AND RESULTS: Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found. CONCLUSION: This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

Intercellular Conduction Optimizes Arterial Network Function and Conserves Blood Flow Homeostasis During Cerebrovascular Challenges.

OBJECTIVE: Cerebral arterial networks match blood flow delivery with neural activity. Neurovascular response begins with a stimulus and a focal change in vessel diameter, which by themselves is inconsequential to blood flow magnitude, until they spread and alter the contractile status of neighboring arterial segments. We sought to define the mechanisms underlying integrated vascular behavior and considered the role of intercellular electrical signaling in this phenomenon. Approach and Results: Electron microscopic and histochemical analysis revealed the structural coupling of cerebrovascular cells and the expression of gap junctional subunits at the cell interfaces, enabling intercellular signaling among vascular cells. Indeed, robust vasomotor conduction was detected in human and mice cerebral arteries after focal vessel stimulation: a response attributed to endothelial gap junctional communication, as its genetic alteration attenuated this behavior. Conducted responses were observed to ascend from the penetrating arterioles, influencing the contractile status of cortical surface vessels, in a simulated model of cerebral arterial network. Ascending responses recognized in vivo after whisker stimulation were significantly attenuated in mice with altered endothelial gap junctional signaling confirming that gap junctional communication drives integrated vessel responses. The diminishment in vascular communication also impaired the critical ability of the cerebral vasculature to maintain blood flow homeostasis and hence tissue viability after stroke. CONCLUSIONS: Our findings highlight the integral role of intercellular electrical signaling in transcribing focal stimuli into coordinated changes in cerebrovascular contractile activity and expose, a hitherto unknown mechanism for flow regulation after stroke.

Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries.

Objective- Inward rectifying K+ (KIR) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba2+-sensitive KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed KIR2.1/KIR2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of KIR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial KIR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle KIR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of KIR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized KIR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

Agonist-evoked endothelial Ca2+ signalling microdomains.

Localized, oscillating Ca2+ signals have been identified in discrete microdomains of vascular endothelial cells. At myoendothelial contacts (between endothelial and smooth muscle cells), both endothelial Ca2+ pulsars (IP3-mediated release of intracellular Ca2+) and Ca2+ sparklets (extracellular Ca2+ entry via TRP channels) contribute to endothelium-dependent hyperpolarization of smooth muscle, vasodilation, and feedback control of vasoconstriction. Ca2+ sparklets occurring at close-contact domains between endothelial cells are possibly involved in conducted hyperpolarization and spreading vasodilation in arterial networks. This review summarizes these Ca2+ signalling phenomena, examines the proposed mechanisms leading to their generation by G-protein-coupled receptor agonists, and explores the proposed physiological roles of these localized and specialized Ca2+ signals.

Smooth muscle Ca2+ sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2+/-G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/-G301R and WT showed similar contractile responses. The Atp1a2+/-G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+ change was smaller than in WT. The enhanced constriction of Atp1a2+/-G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/-G301R mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Blockade of Pannexin-1 Channels and Purinergic P2X7 Receptors Shows Protective Effects Against Cytokines-Induced Colitis of Human Colonic Mucosa.

Introduction: The pannexin-1 (Panx1) channels are found in many cell types, and ATP released from these channels can act on nearby cells activating purinergic P2X7 receptors (P2X7R) which lead to inflammation. Although Panx1 and P2X7R are implicated in the process of inflammation and cell death, few studies have looked at the role they play in inflammatory bowel disease in human. Hence, the aim of the present study was to investigate the function of Panx1 and P2X7R in an ex vivo colitis model developed from human colonic mucosal explants. Materials and Methods: Healthy human colonic mucosal strips (4 × 10 mm) were incubated in carbogenated culture medium at 37°C for 16 h. Proinflammatory cytokines TNFα and IL-1ß (each 10 ng/mL) were used to induce colitis in mucosal strips, and the effects of Panx1 and P2X7R on cytokines-induced tissue damage were determined in the presence of the Panx1 channel blocker 10Panx1 (100 µM) and P2X7R antagonist A438079 (100 µM). The effects of 10Panx1 and A438079 on cytokines-enhanced epithelial permeability were also studied using Caco-2 cells. Results: Histological staining showed that the mucosal strips had severe structural damage in the cytokines-only group but not in the incubation-control group (P < 0.01). Compared to the cytokines-only group, crypt damage was significantly decreased in groups receiving cytokines with inhibitors (10Panx1, A438079, or 10Panx1 + A438079, P < 0.05). The immunoreactive signals of tight junction protein zonula occludens-1 (ZO-1) were abundant in all control tissues but were significantly disrupted and lost in the cytokines-only group (P < 0.01). The diminished ZO-1 immunoreactivity induced by cytokines was prevented in the presence of 10Panx1 (P = 0.04). Likewise, 10Panx1 significantly attenuated the cytokines-evoked increase in paracellular permeability of Caco-2 cells. Although the inhibition of P2X7R activity by A438079 diminished cytokines-induced crypt damage, its effect on the maintenance of ZO-1 immunoreactivity and Caco-2 epithelial cell integrity was less evident. Conclusion: The blockade of Panx1 and P2X7R reduced the inflammatory cytokines-induced crypt damage, loss of tight junctions and increase in cell permeability. Thus, Panx1 and P2X7R may have roles in causing mucosal damage, a common clinical feature of inflammatory bowel disease.

Abdominal aortic aneurysm and omega-3 polyunsaturated fatty acids: Mechanisms, animal models, and potential treatment.

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with macrophage accumulation in the adventitia, oxidative stress, medial elastin degradation and aortic dilation. Progression of AAA is linked to increased risk of rupture, which carries a high mortality rate. Drug therapies trialled to date lack efficacy and although aneurysm repair is available for patients with large aneurysm, peri-surgical morbidity and mortality have been widely reported. Recent studies using rodent models of AAA suggest that long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) and their metabolites can moderate inflammation and oxidative stress perpetuated by infiltrating macrophages and intervene in the destruction of medial elastin. This review examines evidence from these animal studies and related reports of inhibition of inflammation and arrest of aneurysm development following prophylactic supplementation with LC n-3 PUFAs. The efficacy of LC n-3 PUFAs for management of existing aneurysm is unclear and further investigations involving human clinical trials are warranted.

Transient receptor potential canonical type 3 channels: Interactions, role and relevance - A vascular focus.

Transient receptor potential canonical type 3 channels (TRPC3) are expressed in neural, cardiac, respiratory and vascular tissues, with both similarities and differences between human and animal models for the same cell types. In common with all members of the six subfamilies of TRP channels, TRPC3 are non-voltage gated, non-selective cation channels that are mainly permeated by Ca2+, and have distinct molecular, biophysical, anatomical and functional properties. TRP channels are present in excitable and non-excitable cells where they sense and respond to a wide variety of physical and chemical stimuli. TRPC3 are expressed in the endothelium and/or smooth muscle of specific intact arteries, such as mesenteric, cerebral and myometrial, where they are critical for the control of vascular tone, and show altered activity in development and disease. In artery endothelium, TRPC3 contributes to endothelium-derived hyperpolarization and nitric oxide-mediated vasodilation. In artery smooth muscle, TRPC3 contributes to constrictor mechanisms. In both endothelium and smooth muscle, TRPC3 contributes to function via caveolae-caveolin dependent and independent mechanisms. In different cell types and states, like other TRP channels, TRPC3 can form complexes with other TRP proteins and associated channels and accessory proteins, including those associated with endo(sarco)plasmic reticulum (ER/SR), thereby facilitating Ca2+ channel activation and/or refilling ER/SR Ca2+ stores. The diversity of TRPC3 interactions with other vascular signaling components is a potential target for artery specific control mechanisms. This brief perspective highlights recent advances in understanding the functional diversity of TRPC3, with an emphasis on vascular health and disease.

Vascular structural and functional changes: their association with causality in hypertension: models, remodeling and relevance.

Essential hypertension is a complex multifactorial disease process that involves the interaction of multiple genes at various loci throughout the genome, and the influence of environmental factors such as diet and lifestyle, to ultimately determine long-term arterial pressure. These factors converge with physiological signaling pathways to regulate the set-point of long-term blood pressure. In hypertension, structural changes in arteries occur and show differences within and between vascular beds, between species, models and sexes. Such changes can also reflect the development of hypertension, and the levels of circulating humoral and vasoactive compounds. The role of perivascular adipose tissue in the modulation of vascular structure under various disease states such as hypertension, obesity and metabolic syndrome is an emerging area of research, and is likely to contribute to the heterogeneity described in this review. Diversity in structure and related function is the norm, with morphological changes being causative in some beds and states, and in others, a consequence of hypertension. Specific animal models of hypertension have advantages and limitations, each with factors influencing the relevance of the model to the human hypertensive state/s. However, understanding the fundamental properties of artery function and how these relate to signalling mechanisms in real (intact) tissues is key for translating isolated cell and model data to have an impact and relevance in human disease etiology. Indeed, the ultimate aim of developing new treatments to correct vascular dysfunction requires understanding and recognition of the limitations of the methodologies used.

Excitability and Synaptic Transmission in the Enteric Nervous System: Does Diet Play a Role?

Changes in diet are a challenge to the gastrointestinal tract which needs to alter its processing mechanisms to continue to process nutrients and maintain health. In particular, the enteric nervous system (ENS) needs to adapt its motor and secretory programs to deal with changes in nutrient type and load in order to optimise nutrient absorption.The nerve circuits in the gut are complex, and the numbers and types of neurons make recordings of specific cell types difficult, time-consuming, and prone to sampling errors. Nonetheless, traditional research methods like intracellular electrophysiological approaches have provided the basis for our understanding of the ENS circuitry. In particular, animal models of intestinal inflammation have shown us that we can document changes to neuronal excitability and synaptic transmission.Recent studies examining diet-induced changes to ENS programming have opted to use fast imaging techniques to reveal changes in neuron function. Advances in imaging techniques using voltage- or calcium-sensitive dyes to record neuronal activity promise to overcome many limitations inherent to electrophysiological approaches. Imaging techniques allow access to a wide range of ENS phenotypes and to the changes they undergo during dietary challenges. These sorts of studies have shown that dietary variation or obesity can change how the ENS processes information-in effect reprogramming the ENS. In this review, the data gathered from intracellular recordings will be compared with measurements made using imaging techniques in an effort to determine if the lessons learnt from inflammatory changes are relevant to the understanding of diet-induced reprogramming.

TRPV3 expression and vasodilator function in isolated uterine radial arteries from non-pregnant and pregnant rats.

This study investigated the expression and function of transient receptor potential vanilloid type-3 ion channels (TRPV3) in uterine radial arteries isolated from non-pregnant and twenty-day pregnant rats. Immunohistochemistry (IHC) suggested TRPV3 is primarily localized to the smooth muscle in arteries from both non-pregnant and pregnant rats. IHC using C' targeted antibody, and qPCR of TRPV3 mRNA, suggested pregnancy increased arterial TRPV3 expression. The TRPV3 activator carvacrol caused endothelium-independent dilation of phenylephrine-constricted radial arteries, with no difference between vessels from non-pregnant and pregnant animals. Carvacrol-induced dilation was reduced by the TRPV3-blockers isopentenyl pyrophosphate and ruthenium red, but not by the TRPA1 or TRPV4 inhibitors HC-030031 or HC-067047, respectively. In radial arteries from non-pregnant rats only, inhibition of NOS and sGC, or PKG, enhanced carvacrol-mediated vasodilation. Carvacrol-induced dilation of arteries from both non-pregnant and pregnant rats was prevented by the IKCa blocker TRAM-34. TRPV3 caused an endothelium-independent, IKCa-mediated dilation of the uterine radial artery. NO-PKG-mediated modulation of TRPV3 activity is lost in pregnancy, but this did not alter the response to carvacrol.

Endothelium-Derived Hyperpolarization and Coronary Vasodilation: Diverse and Integrated Roles of Epoxyeicosatrienoic Acids, Hydrogen Peroxide, and Gap Junctions.

Myocardial perfusion and coronary vascular resistance are regulated by signaling metabolites released from the local myocardium that act either directly on the VSMC or indirectly via stimulation of the endothelium. A prominent mechanism of vasodilation is EDH of the arteriolar smooth muscle, with EETs and H(2)O(2) playing important roles in EDH in the coronary microcirculation. In some cases, EETs and H(2)O(2) are released as transferable hyperpolarizing factors (EDHFs) that act directly on the VSMCs. By contrast, EETs and H(2)O(2) can also promote endothelial KCa activity secondary to the amplification of extracellular Ca(2+) influx and Ca(2+) mobilization from intracellular stores, respectively. The resulting endothelial hyperpolarization may subsequently conduct to the media via myoendothelial gap junctions or potentially lead to the release of a chemically distinct factor(s). Furthermore, in human isolated coronary arterioles dilator signaling involving EETs and H(2)O(2) may be integrated, being either complimentary or inhibitory depending on the stimulus. With an emphasis on the human coronary microcirculation, this review addresses the diverse and integrated mechanisms by which EETs and H(2)O(2) regulate vessel tone and also examines the hypothesis that myoendothelial microdomain signaling facilitates EDH activity in the human heart.

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback.

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca(2+) to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.