Donna Briggs Boedtkjer

Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca2+-activated Cl− currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca2+-activated Cl− current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a “classic” (cGMP-independent) Ca2+-activated Cl− current and to have characteristics distinct from those previously known for Ca2+-activated Cl− currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca2+-activated Cl− current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca2+-activated Cl− current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca2+-activated Cl− current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca2+-activated Cl− current, whereas the magnitude of the classic Ca2+-activated Cl− current was not affected. The majority of previous suggestions that bestrophins are a new Cl− channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca2+-activated Cl− current in smooth muscles in the intact vascular wall.

TMEM16A knockdown abrogates two different Ca2+-activated Cl− currents and contractility of smooth muscle in rat mesenteric small arteries

The presence of Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (SMCs) is well established. Their molecular identity is, however, elusive. Two distinct Ca2+-activated Cl− currents (ICl(Ca)) were previously characterized in SMCs. We have shown that the cGMP-dependent ICl(Ca) depends on bestrophin expression, while the “classical” ICl(Ca) is not. Downregulation of bestrophins did not affect arterial contraction but inhibited the rhythmic contractions, vasomotion. In this study, we have used in vivo siRNA transfection of rat mesenteric small arteries to investigate the role of a putative CaCC, TMEM16A. Isometric force, [Ca2+]i, and SMC membrane potential were measured in isolated arterial segments. ICl(Ca) and GTPγS-induced nonselective cation current were measured in isolated SMCs. Downregulation of TMEM16A resulted in inhibition of both the cGMP-dependent ICl(Ca) and the “classical” ICl(Ca) in SMCs. TMEM16A downregulation also reduced expression of bestrophins. TMEM16A downregulation suppressed vasomotion both in vivo and in vitro. Downregulation of TMEM16A reduced agonist (noradrenaline and vasopressin) and K+-induced contractions. In accordance with the depolarizing role of CaCCs, TMEM16A downregulation suppressed agonist-induced depolarization and elevation in [Ca2+]i. Surprisingly, K+-induced depolarization was unchanged but Ca2+ entry was reduced. We suggested that this is due to reduced expression of the L-type Ca2+ channels, as observed at the mRNA level. Thus, the importance of TMEM16A for contraction is, at least in part, independent from membrane potential. This study demonstrates the significance of TMEM16A for two SMCs ICl(Ca) and vascular function and suggests an interaction between TMEM16A and L-type Ca2+ channels.

Interaction Between Na+/K+-Pump and Na+/Ca2+-Exchanger Modulates Intercellular Communication

Ouabain, a specific inhibitor of the Na+/K+-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na+/K+-pump and the Na+/Ca2+-exchanger leading to an increase in the intracellular calcium concentration ([Ca2+]i) in discrete areas near the plasma membrane. [Ca2+]i in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na+/K+-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca2+]i near the membrane, or by ATP depletion. Reduction of Na+/K+-pump activity by removal of extracellular potassium ([K+]o) also uncoupled cells, but only after inhibition of KATP channels. Inhibition of the Na+/Ca2+-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na+ and clamping of [Ca2+]i at low concentrations prevented the uncoupling. The experiments suggest that the Na+/K+-pump may affect gap junction conductivity via localized changes in [Ca2+]i through modulation of Na+/Ca2+-exchanger activity.

Bestrophin is important for the rhythmic but not the tonic contraction in rat mesenteric small arteries

AIMS We have previously characterized a cGMP-dependent Ca(2+)-activated Cl(-) current in vascular smooth muscle cells (SMCs) and have shown its dependence on bestrophin-3 expression. We hypothesize that this current is important for synchronization of SMCs in the vascular wall. In the present study, we aimed to test this hypothesis by transfecting rat mesenteric small arteries in vivo with siRNA specifically targeting bestrophin-3. METHODS AND RESULTS The arteries were tested 3 days after transfection in vitro for isometric force development and for intracellular Ca(2+) in SMCs. Bestrophin-3 expression was significantly reduced compared with arteries transfected with mutated siRNA. mRNA levels for bestrophin-1 and -2 were also significantly reduced by bestrophin-3 down-regulation. This is suggested to be secondary to specific bestrophin-3 down-regulation since siRNAs targeting different exons of the bestrophin-3 gene had identical effects on bestrophin-1 and -2 expression. The transfection affected neither the maximal contractile response nor the sensitivity to norepinephrine and arginine-vasopressin. The amplitude of agonist-induced vasomotion was significantly reduced in arteries down-regulated for bestrophins compared with controls, and asynchronous Ca(2+) waves appeared in the SMCs. The average frequency of vasomotion was not different. 8Br-cGMP restored vasomotion in arteries where the endothelium was removed, but oscillation amplitude was still significantly less in bestrophin-down-regulated arteries. Thus, vasomotion properties were consistent with those previously characterized for rat mesenteric small arteries. Data from our mathematical model are consistent with the experimental results. CONCLUSION This study demonstrates the importance of bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.

New selective inhibitors of calcium-activated chloride channels – T16Ainh-A01, CaCCinh-A01 and MONNA – what do they inhibit?

T16Ainh‐A01, CaCCinh‐A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium‐activated chloride channel (CaCC). The aim of this study was to examine the chloride‐specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride.

Human thoracic duct in vitro: diameter-tension properties, spontaneous and evoked contractile activity

The current study characterizes the mechanical properties of the human thoracic duct and demonstrates a role for adrenoceptors, thromboxane, and endothelin receptors in human lymph vessel function. With ethical permission and informed consent, portions of the thoracic duct (2-5 cm) were resected and retrieved at T(7)-T(9) during esophageal and cardia cancer surgery. Ring segments (2 mm long) were mounted in a myograph for isometric tension (N/m) measurement. The diameter-tension relationship was established using ducts from 10 individuals. Peak active tension of 6.24 +/- 0.75 N/m was observed with a corresponding passive tension of 3.11 +/- 0.67 N/m and average internal diameter of 2.21 mm. The equivalent active and passive transmural pressures by LaPlaces law were 47.3 +/- 4.7 and 20.6 +/- 3.2 mmHg, respectively. Subsequently, pharmacology was performed on rings from 15 ducts that were normalized by stretching them until an equivalent pressure of 21 mmHg was calculable from the wall tension. At low concentrations, norepinephrine, endothelin-1, and the thromboxane-A(2) analog U-46619 evoked phasic contractions (analogous to lymphatic pumping), whereas at higher contractions they induced tonic activity (maximum tension values of 4.46 +/- 0.63, 5.90 +/- 1.4, and 6.78 +/- 1.4 N/m, respectively). Spontaneous activity was observed in 44% of ducts while 51% of all the segments produced phasic contractions after agonist application. Acetylcholine and bradykinin relaxed norepinephrine preconstrictions by approximately 20% and approximately 40%, respectively. These results demonstrate that the human thoracic duct can develop wall tensions that permit contractility to be maintained across a wide range of transmural pressures and that isolated ducts contract in response to important vasoactive agents.

Association between endothelial dysfunction and depression-like symptoms in chronic mild stress model of depression.

Objective Cardiovascular diseases have high comorbidity with major depression. Endothelial dysfunction may explain the adverse cardiovascular outcome in depression; therefore, we analyzed it in vitro. In the chronic mild stress model, some rats develop depression-like symptoms (including “anhedonia”), whereas others are stress resilient. Methods After 8 weeks of chronic mild stress, anhedonic rats reduced their sucrose intake by 55% (7%), whereas resilient rats did not. Acetylcholine-induced endothelium-dependent relaxation of norepinephrine-preconstricted mesenteric arteries was analyzed in nonstressed, anhedonic, and resilient rat groups. Results Small resistance arteries from anhedonic rats were less sensitive to acetylcholine than those of the nonstressed and resilient groups (p = .029). Pathways of endothelium-dependent relaxation were altered in arteries from anhedonic rats. Nitric oxide (NO)–dependent relaxation and endothelial NO synthase expression were increased in arteries from anhedonic rats (0.235 [0.039] arbitrary units and 155.7% [8.15%]) compared with the nonstressed (0.135 [0.012] arbitrary units and 100.0% [8.08%]) and resilient (0.152 [0.018] arbitrary units and 108.1% [11.65%]) groups (p < .001 and p = .002, respectively). Inhibition of cyclooxygenase (COX) activity revealed increased COX-2–dependent relaxation in the anhedonic group. In contrast, endothelial NO synthase– and COX-independent relaxation to acetylcholine (endothelium-dependent hyperpolarization–like response) was reduced in anhedonic rats (p < .001). This was associated with decreased transcription of intermediate-conductance Ca2+-activated K+ channels. Conclusions Our findings demonstrate that depression-like symptoms are associated with reduced endothelium-dependent relaxation due to suppressed endothelium-dependent hyperpolarization–like relaxation despite up-regulation of the NO and COX-2–dependent pathways in rat mesenteric arteries. These changes could affect peripheral resistance and organ perfusion in major depression.

Chronic mild stress-induced depression-like symptoms in rats and abnormalities in catecholamine uptake in small arteries.

Objective Major depression and cardiovascular diseases have a strong comorbidity; however, the reason for this is unknown. In the chronic mild stress (CMS) model of depression, only a fraction of rats develop a major feature of depression—anhedonia-like behavior, whereas other rats are stress resilient. Previous studies suggested that CMS rats also have increased total peripheral vascular resistance. Methods On the basis of CMS-induced changes of sucrose intake, a reliable measure for anhedonia, rats were divided into “resilient” and “anhedonic” groups. An interaction between hedonic status and vascular function was studied after 4 and 8 weeks of CMS exposure in vitro in wire myograph on saphenous arteries and mesenteric small arteries (MSAs) from these rats. Results When comparing the different experimental rat groups, arterial sensitivities to noradrenaline (NA) were similar under control conditions, but in the presence of the neuronal reuptake inhibitor cocaine, arteries from anhedonic rats were more sensitive to NA. No change in perivascular innervation was found, but elevated expression of neuronal NA transporter was detected. Inhibition of extraneuronal uptake with corticosterone (1 &mgr;M) suggests that this transport is diminished in MSAs after CMS. The corticosterone-sensitive transporter organic cation cotransporter 2 was shown to be reduced in MSAs after CMS. No CMS-induced changes in the corticosterone-sensitive transport were found in saphenous arteries. Conclusions Our results indicate that CMS-induced depression-like symptoms in rats are associated with changes in catecholamine uptake pathways in the vascular wall, which potentially modulates the effect of sympathetic innervation of resistance arteries. Abbreviations ANOVA = analysis of variance; CMS = chronic mild stress; GABA = &ggr;-aminobutyric acid; MSAs = mesenteric small arteries; NA = noradrenaline; NET = neuronal noradrenaline transporter; OCT = organic cation cotransporter; SAs = saphenous arteries; TPR = total peripheral vascular resistance

The human thoracic duct is functionally innervated by adrenergic nerves

Lymphatic vessels from animals have been shown to be innervated. While morphological studies have confirmed human lymphatic vessels are innervated, functional studies supporting this are lacking. The present study demonstrates a functional innervation of the human thoracic duct (TD) that is predominantly adrenergic. TDs harvested from 51 patients undergoing esophageal and cardia cancer surgery were either fixed for structural investigations or maintained in vitro for the functional assessment of innervation by isometric force measurements and electrical field stimulation (EFS). Electron microscopy and immunohistochemistry suggested scarce diffuse distribution of nerves in the entire vessel wall, but nerve-mediated contractions could be induced with EFS and were sensitive to the muscarinic receptor blocker atropine and the α-adrenoceptor blocker phentolamine. The combination of phentolamine and atropine resulted in a near-complete abolishment of EFS-induced contractions. The presence of sympathetic nerves was further confirmed by contractions induced by the sympathomimetic and catecholamine-releasing agent tyramine. Reactivity to the neurotransmitters norepinephrine, substance P, neuropeptide Y, acetylcholine, and methacholine was demonstrated by exogenous application to human TD ring segments. Norepinephrine provided the most consistent responses, whereas responses to the other agonists varied. We conclude that the human TD is functionally innervated with both cholinergic and adrenergic components, with the latter of the two dominating.

Identification of Interstitial Cajal-Like Cells in the Human Thoracic Duct

Interstitial Cajal-like cells (ICLCs) are speculated to be pacemakers in smooth muscle tissues. While the human thoracic duct (TD) is spontaneously active, the origin of this activity is unknown. We hypothesized that ICLCs could be present in the TD and using histological techniques, immunohistochemistry and immunofluorescence we have investigated the presence of ICLCs, protein markers for ICLCs and the cellular morphology of the human TD. Transmission electron microscopy was employed to investigate ultrastructure. Methylene blue staining, calcium-dependent fluorophores and confocal microscopy were used to identify ICLCs in live tissue. Methylene blue stained cells with morphology suggestive of ICLCs in the TD. Immunoreactivity localized the ICLC protein markers c-kit, CD34 and vimentin to many cells and processes associated with smooth muscle cells (SMCs): coexpression of c-kit with vimentin or CD34 was observed in some cells. Electron microscopy analysis confirmed ICLCs as a major cell type of the human TD. Lymphatic ICLCs possess caveolae, dense bands, a patchy basal lamina, intermediate filaments and specific junctions to SMCs. ICLCs were ultrastructurally differentiable from other interstitial cells observed: fibroblasts, mast cells, macrophages and pericytes. Lymphatic ICLCs were localized to the subendothelial region of the wall as well as in intimate association with smooth muscle bundles throughout the media. ICLCs were morphologically distinct with multiple processes and also spindle shapes. Confocal imaging with calcium-dependent fluorophores corroborated cell morphology and localization observed in fixed tissues. Lymphatic ICLCs thus constitute a significant cell type of the human TD and physically interact with lymphatic SMCs.