Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon)

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 Ca(2+) in pressurized RA-IMCAs and, as previously shown in human RA-IMCAs, hyperpolarized porcine arteries. Immunolabelling showed small- and intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)) present in the endothelium of both species, and concentration-dependent vasodilation to bradykinin followed activation of these K(Ca) 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 (K(ATP)) and inwardly rectifying (K(IR)) 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

Research of accessibility to inclusive medicine through the prism of marketing analysis

Представлено частину результатів дослідження, проведеного авторами з метою виявлення потреб осіб з обмеженими можливостями стосовно медичних послуг. Аналіз наукових праць виявив брак досліджень щодо сучасного стану доступності медичних послуг для осіб з особливими потребами на рівні регіону. Мета статті – виявлення рівня доступності медичних послуг для осіб з особливими потребами на рівні регіону та вироблення рекомендацій за результатами дослідження. Використано методи: аналіз вторинної інформації; опитування за допомогою анкети; статистичний метод; метод компаративного аналізу; синтез; узагальнення; спостереження (аналіз інформації, розміщеної на веб-сайтах та офіційних сторінках у соціальних мережах досліджуваних суб’єктів); групування. Аналіз отриманої в ході спостереження та опитування інформації виявив, що найвищий рівень доступності будівель для осіб з особливими потребами в досліджених ОТГ притаманний медичним установам (0,9), найнижчий рівень доступності – освітнім установам ОТГ (0,5). У ході спостереження виявлено, що більшість амбулаторій досліджених територіальних громад обладнані доступними туалетами (здебільшого, міських тергромад), пандуси і сходи відповідають вимогам, наявні інформаційні позначки. Разом з тим невирішеними є питання забезпечення паркомісцями, достатнього простору у коридорах для безперешкодного переміщення маломобільних осіб, достатньої ширини пройому дверей до кабінетів спеціалістів, наявності кнопки виклику й табличок шрифтом Брайля, звукового сповіщення для осіб з проблемами слуху та зору, справності ліфтів у більш ніж одноповерхових медичних закладах. Половина опитаних осіб з особливими потребами, що проживають у досліджуваних ОТГ, вважає, що доступність якісної медичної допомоги для осіб з інвалідністю є рівною в їхній громаді та надається своєчасно. Отже ще є потенціал для покращення і медичних послуг, і їхньої доступності. Для поліпшення доступності послуг для осіб з обмеженими можливостями респонденти вказали на важливість наявності доступного транспорту у територіальній громаді та розширення асортименту медичних послуг для людей з обмеженими можливостями. Подальші дослідження будуть присвячені аналізу доступності освітніх закладів територіальних громад для осіб з особливими потребами.Part of the results of the research conducted by the authors with the aim of identifying the needs of persons with disabilities in relation to medical services are presented. The analysis of scientific works revealed a lack of research on the current state of accessibility of medical services for persons with special needs at the regional level. The purpose of the article was to identify the level of availability of medical services for persons with special needs at the regional level and to develop the recommendations based on the results of the study. The following methods were used: analysis of secondary information; survey using a questionnaire; statistical method; method of comparative analysis; synthesis; generalization; observation (analysis of information posted on the websites and official pages in social networks of the subjects under study); grouping. The analysis of the information obtained during the observation and survey revealed that the highest level of accessibility of buildings for persons with special needs in the studied UTC is characteristic of medical institutions (0.9), the lowest level of accessibility - educational institutions of UTC (0.5). In the course of the observation, it was found that the majority of dispensaries in the investigated territorial communities are equipped with accessible toilets (mostly, urban territorial communities), the ramps and stairs meet the requirements, and there are informational signs. At the same time, the issues of providing parking spaces, sufficient space in the corridors for the unhindered movement of people with limited mobility, sufficient width of the doorways to specialist offices, the presence of a call button and signs in Braille, sound notification for people with hearing and vision problems, the serviceability of elevators in more than single-story medical facilities. Half of the interviewed persons with special needs living in the studied UTC believe that the availability of quality medical care for persons with disabilities is equal in their community and is provided in a timely manner, so there is still potential for improving both medical services and their accessibility. In order to improve the accessibility of services for people with disabilities, the respondents indicated the importance of having accessible transport in the community and expanding the range of medical services for people with disabilities. Further research will be devoted to the analysis of the accessibility of educational institutions of territorial communities for persons with special needs

Human coronary microvascular contractile dysfunction associates with viable synthetic smooth muscle cells

Aims Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study. Methods and results Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial (RA) appendage and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor (<10%) MT yet retained cell viability and an ability to raise cytoplasmic Ca2+ in response to vasoconstrictor agents. Comparing across human heart chambers and species, we found that based on patient medical history and six tests, the strongest predictor of poor MT in IMCAs was increased expression of the synthetic marker caldesmon relative to the contractile marker SM-myosin heavy chain. In addition, high resolution imaging revealed a distinct layer of longitudinally aligned SMCs between ECs and radial SMCs, and we show poor MT was associated with disruptions in these cellular alignments. Conclusion These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction

How calcium signals in myocytes and pericytes are integrated across in situ microvascular networks and control microvascular tone

The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca(2+) signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca(2+) signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca(2+) signalling and contractility of myocytes and pericytes. Ca(2+) signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca(2+) signals arise from Ca(2+) release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca(2+) release and not via ryanodine receptors or Ca(2+) entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca(2+) signals between arteriolar and venular networks dependent on gap junctions and Ca(2+) entry via L-type Ca(2+) channels. Endothelial Ca(2+) signalling inhibits intracellular Ca(2+) oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca(2+) signals via EDHF. Increases of Ca(2+) in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes

Molecular expression and calcium signalling roles of native TRP channels in vascular cells

In the vasculature, multiple members of the TRP-superfamily of non-selective cation channels (NSCCs) are expressed. These channels mediate diverse non-voltage-gated Ca2+-entry pathways and functions, which involve both vascular myocytes and communicating endothelial cells. Here, we provide an overview of recent progress in this area of research and discuss several specific examples of the important roles of vascular TRP channels in calcium signalling and electrophysiological responses. We especially focus on the recently discovered signal transduction mechanisms involving formation of specific complexes between TRP proteins and other better studied proteins that regulate cell calcium homeostasis, such as voltage-gated calcium channels and ryanodine receptors. Finally, we provide an overview of the progress in our understanding of TRPM8, which is known as the principal neuronal cold receptor, expression, localisation and function in the vasculature. We conclude that this channel is likely involved in complex thermal behaviour of blood vessels, better understanding of which is relevant to hypothermic and cardiovascular surgery conditions, therefore further research in this area is needed

Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFβig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFβig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFβig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFβig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling

High spatial and temporal resolution Ca2+ imaging of myocardial strips from human, pig and rat

Ca2+ handling within cardiac myocytes underpins coordinated contractile function within the beating heart. This protocol enables high spatial and temporal Ca2+ imaging of ex vivo multicellular myocardial strips. The endocardial surface is retained, and strips of 150–300-µm thickness are dissected, loaded with Ca2+ indicators and mounted within 1.5 h. A list of the equipment and reagents used and the key methodological aspects allowing the use of this technique on strips from any chamber of the mammalian heart are described. We have successfully used this protocol on human, pig and rat biopsy samples. On use of this protocol with intact endocardial endothelium, we demonstrated that the myocytes develop asynchronous spontaneous Ca2+ events, which can be ablated by electrically evoked Ca2+ transients, and subsequently redevelop spontaneously after cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, in both healthy and diseased tissue

Chemerin-conditioned medium increases cell adhesion, proliferation and migration.

<p><i>A</i>. Increased adhesion of MSCs (1x10⁵) after treatment with chemerin (Ch) for 30 min. <i>B</i>. Conditioned medium (CM) from MSCs treated with chemerin increased adhesion of normal gastric myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. <i>C</i>. CM from MSCs treated with chemerin stimulated migration of myofibroblasts in Boyden chambers and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. <i>D</i>. CM from MSCs treated with chemerin stimulated proliferation of myofibroblasts and addition of the ChemR23 antagonist CCX832 only slightly reduced the response. Means ± SE, n = 3; horizontal arrows, p<0.05.</p