A cross-disciplinary approach to learning medical physiology and behavioral skills involving drama students performing as simulated patients.

The early years of physiology education in medical curricula provide unique challenges. As well as inculcating concepts that are seen as difficult, modern curricula require that students learn in context in case-based learning courses. Additionally, regulating bodies stress that the soft skills of compassion, communication, and empathy are embedded throughout curricula. This has driven work in our organization involving drama and final-year medicine students during which they collaborate in realistic simulations of doctor/patient interactions. We adapted this transdisciplinary approach to second-year physiology tutorials. This emphasized the holistic importance of physiology to patient care, while also embedding "human factors" skills from the very earliest stages of the curriculum. After preparing by attending acting classes based on aspects of Konstantin Stanislavski's "System," the authors supervised tutorials in which drama students participated in a "physiology of hypofertility" session for second-year medical students, playing a 34-year-old woman with premature menopause (or their partner). Opinion (from all students) was evaluated by Likert questionnaires (which included open questions). A focus group of drama students was also interviewed, and the conversation was recorded for thematic analysis. Positive Likert scores were recorded for the authenticity of the tutorials, skills development, fostering empathy, and motivating students to improve. All participants evaluated the tutorial as highly enjoyable. These scores are reflected in positive open commentary on the questionnaires and in the focus group interviews. The results suggest that even basic science tutorials give opportunities for interdisciplinary study and enhancement of behavioral skills while gaining enthusiastic student acceptance.NEW & NOTEWORTHY This work details how physiology tutorials for early years medical students are transformed by taking the clinical case off the two-dimensional page and instead having the case scenario acted by drama students. This adds context and authenticity. The benefits are twofold: emphasizing the importance of physiology to the budding clinician and embedding empathy and compassion from the earliest points in a clinician's career.

Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

Ion channels and myogenic activity in retinal arterioles.

Retinal pressure autoregulation is an important mechanism that protects the retina by stabilizing retinal blood flow during changes in arterial or intraocular pressure. Similar to other vascular beds, retinal pressure autoregulation is thought to be mediated largely through the myogenic response of small arteries and arterioles which constrict when transmural pressure increases or dilate when it decreases. Over recent years, we and others have investigated the signaling pathways underlying the myogenic response in retinal arterioles, with particular emphasis on the involvement of different ion channels expressed in the smooth muscle layer of these vessels. Here, we review and extend previous work on the expression and spatial distribution of the plasma membrane and sarcoplasmic reticulum ion channels present in retinal vascular smooth muscle cells (VSMCs) and discuss their contribution to pressure-induced myogenic tone in retinal arterioles. This includes new data demonstrating that several key players and modulators of the myogenic response show distinctively heterogeneous expression along the length of the retinal arteriolar network, suggesting differences in myogenic signaling between larger and smaller pre-capillary arterioles. Our immunohistochemical investigations have also highlighted the presence of actin-containing microstructures called myobridges that connect the retinal VSMCs to one another. Although further work is still needed, studies to date investigating myogenic mechanisms in the retina have contributed to a better understanding of how blood flow is regulated in this tissue. They also provide a basis to direct future research into retinal diseases where blood flow changes contribute to the pathology.

Molecular mechanisms mediating the beneficial metabolic effects of [Arg4]tigerinin-1R in mice with diet-induced obesity and insulin resistance.

The frog skin host-defense peptide tigerinin-1R stimulates insulin release in vitro and improves glucose tolerance and insulin sensitivity in animal models of type 2 diabetes. This study extends these observations by investigating the molecular mechanisms of action underlying the beneficial metabolic effects of the analogue [Arg4]tigerinin-1R in mice with diet-induced obesity, glucose intolerance and insulin resistance. The study also investigates the electrophysiological effects of the peptide on KATP and L-type Ca2+ channels in BRIN-BD11 clonal ß cells. Non-fasting plasma glucose and glucagon concentrations were significantly (p<0.05) decreased and plasma insulin increased by twice daily treatment with [Arg4]tigerinin-1R (75 nmol/kg body weight) for 28 days. Oral and intraperitoneal glucose tolerance were significantly (p<0.05) improved accompanied by enhanced secretion and action of insulin. The peptide blocked KATP channels and, consistent with this, improved beta cell responses of isolated islets to a range of secretagogues. Peptide administration resulted in up-regulation of key functional genes in islets involved insulin secretion (Abcc8, Kcnj11, Cacna1c and Slc2a2) and in skeletal muscle involved with insulin action (Insr, Irs1, Pdk1, Pik3ca, and Slc2a4). These observations encourage further development of tigerinin-1R analogues for the treatment of patients with type 2 diabetes.

TNF-α-induced p38MAPK activation regulates TRPA1 and TRPV4 activity in odontoblast-like cells.

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Exendin-4 protects against post-myocardial infarction remodelling via specific actions on inflammation and the extracellular matrix.

Glucagon-like peptide-1 (GLP-1) is an insulin-releasing hormone clinically exploited for glycaemic control in diabetes, which also confers acute cardioprotection and benefits in experimental/clinical heart failure. We specifically investigated the role of the GLP-1 mimetic, exendin-4, in post-myocardial infarction (MI) remodelling, which is a key contributor to heart failure. Adult female normoglycaemic mice underwent coronary artery ligation/sham surgery prior to infusion with exendin-4/vehicle for 4 weeks. Metabolic parameters and infarct sizes were comparable between groups. Exendin-4 protected against cardiac dysfunction and chamber dilatation post-MI and improved survival. Furthermore, exendin-4 modestly decreased cardiomyocyte hypertrophy/apoptosis but markedly attenuated interstitial fibrosis and myocardial inflammation post-MI. This was associated with altered extracellular matrix (procollagen IαI/IIIαI, connective tissue growth factor, fibronectin, TGF-ß3) and inflammatory (IL-10, IL-1ß, IL-6) gene expression in exendin-4-treated mice, together with modulation of both Akt/GSK-3ß and Smad2/3 signalling. Exendin-4 also altered macrophage response gene expression in the absence of direct actions on cardiac fibroblast differentiation, suggesting cardioprotective effects occurring secondary to modulation of inflammation. Our findings indicate that exendin-4 protects against post-MI remodelling via preferential actions on inflammation and the extracellular matrix independently of its established actions on glycaemic control, thereby suggesting that selective targeting of GLP-1 signalling may be required to realise its clear therapeutic potential for post-MI heart failure.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in patients with severe asthma.

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated. OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways. METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1. RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current. CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K(+) channel K(ir)2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3'UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of K(ir)2.1 expression, and was used to investigate the targeting of the K(ir)2.1 3'UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and K(ir)2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Pharmacological profiling of store-operated Ca2+ entry in retinal arteriolar smooth muscle.

OBJECTIVE: Pharmacological profiling of SOCE and molecular profiling of ORAI and TRPC expression in arterioles. METHODS: Fura-2-based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression was screened using RT-PCR. RESULTS: The SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2 and 1.4 µm, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21 and 42 nm, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6 µm). 2APB inhibited SOCE (IC(50) = 3.7 µm) but enhanced basal influx (>100 µm). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (≥0.1 µm). The RT-PCR demonstrated transcript expression for ORAI 1, 2, and 3, and TRPC1, 3, 4, and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. CONCLUSIONS: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared with other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded.

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch­dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch­dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle.

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis.

Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis. Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording. Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model. Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Actions of PGLa-AM1 and its [A14K] and [A20K] analogues and their therapeutic potential as anti-diabetic agents.

PGLa-AM1 (GMASKAGSVL10GKVAKVALKA20AL.NH2) was first identified in skin secretions of the frog Xenopus amieti (Pipidae) on the basis of its antimicrobial properties. PGLa-AM1 and its [A14K] and [A20K] analogues produced a concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells without cytotoxicity at concentrations up to 3 µM. In contrast, the [A3K] analogue was cytotoxic at concentrations ≥ 30 nM. The potency and maximum rate of insulin release produced by the [A14K] and [A20K] peptides were significantly greater than produced by PGLa-AM1. [A14K]PGLa-AM1 also stimulated insulin release from mouse islets at concentrations ≥ 1 nM and from the 1.1B4 human-derived pancreatic ß-cell line at concentrations > 30 pM. PGLa-AM1 (1 µM) produced membrane depolarization in BRIN-BD11 cells with a small, but significant (P < 0.05), increase in intracellular Ca2+ concentrations but the peptide had no direct effect on KATP channels. The [A14K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of the peptide. [A14K]PGLa-AM1 (1 µM) protected against cytokine-induced apoptosis (p < 0.001) in BRIN-BD11 cells and augmented (p < 0.001) proliferation of the cells to a similar extent as GLP-1. Intraperitoneal administration of the [A14K] and [A20K] analogues (75 nmol/kg body weight) to both lean mice and high fat-fed mice with insulin resistance improved glucose tolerance with a concomitant increase in insulin secretion. The data provide further support for the assertion that host defense peptides from frogs belonging to the Pipidae family show potential for development into agents for the treatment of patients with Type 2 diabetes.

Esculentin-2CHa(1-30) and its analogues: stability and mechanisms of insulinotropic action.

The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1-30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca2+ and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1-30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg7,d-Lys15,d-Lys23]-esculentin-2CHa(1-30) and Lys15-octanoate-esculentin-2CHa(1-30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca2+ omission inhibited insulin secretion induced by the esculentin-2CHa(1-30) analogues suggesting an action on KATP and Ca2+ channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca2+ Evaluation with fluorescent-labelled esculentin-2CHa(1-30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of KATP channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1-30) may prove useful for glycaemic control in obesity-diabetes.

Diabetes Impairs the Aldehyde Detoxifying Capacity of the Retina.

PURPOSE: We studied whether the accumulation of advanced lipoxidation end-products (ALEs) in the diabetic retina is linked to the impairment of lipid aldehyde detoxification mechanisms. METHODS: Retinas were collected from nondiabetic and diabetic rats and processed for conventional and quantitative RT-PCR (qRT-PCR), Western blotting, immunohistochemistry, and aldehyde dehydrogenase (ALDH) activity assays. The effect of the ALDH1a1 inhibitor, NCT-501, on ALE accumulation and cell viability in cultured Müller glia also was investigated. RESULTS: The rat retina expressed a range of lipid aldehyde detoxifying ALDH and aldo-keto reductase (AKR) genes. In diabetes, mRNA levels were reduced for 5 of 9 transcripts tested. These findings contrasted with those in the lens and cornea where many of these enzymes were upregulated. We have reported previously accumulation of the acrolein (ACR)-derived ALE, FDP-lysine, in retinal Müller glia during diabetes. In the present study, we show that the main ACR-detoxifying ALDH and AKR genes expressed in the retina, namely, ALDH1a1, ALDH2, and AKR1b1, are principally localized to Müller glia. Diabetes-induced FDP-lysine accumulation in Müller glia was associated with a reduction in ALDH1a1 mRNA and protein expression in whole retina and a decrease in ALDH1a1-immunoreactivity specifically within these cells. No such changes were detected for ALDH2 or AKR1b1. Activity of ALDH was suppressed in the diabetic retina and blockade of ALDH1a1 in cultured Müller glia triggered FDP-lysine accumulation and reduced cell viability. CONCLUSIONS: These findings suggest that downregulation of ALDH and AKR enzymes, particularly ALDH1a1, may contribute ALE accumulation in the diabetic retina.

TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles.

PURPOSE: Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina. METHODS: Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs). RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, Δ9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone. CONCLUSIONS: Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.

Biodentine Reduces Tumor Necrosis Factor Alpha-induced TRPA1 Expression in Odontoblastlike Cells.

INTRODUCTION: The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression. METHODS: Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies. RESULTS: Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha-induced TRPA1 responses after Biodentine treatment. CONCLUSIONS: In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses.

A-type potassium current in retinal arteriolar smooth muscle cells.

PURPOSE: By their control of membrane potential and intracellular free Ca(2+) ([Ca(2+)](i)), K(+) currents are pivotal in the regulation of arterial smooth muscle tone. The goal of the present study was to identify and characterize the A-type K(+) current in retinal microvascular smooth muscle (MVSM) and to examine its role in modulating membrane potential and cellular contractility. METHODS: Whole-cell perforated patch-clamp recordings were made from MVSM cells within intact isolated arteriolar segments. Before patch-clamping, retinal arterioles were anchored in the physiological recording bath and perfused with an enzyme cocktail to remove surface basal lamina and to uncouple electrically the endothelial cells from the overlying MVSM cells. RESULTS: K(+) currents were activated by depolarizing steps from -80 to +100 mV in 20-mV increments. A dominant, noninactivating current was elicited by depolarization to potentials positive of -50 mV. Inhibition of this current by 100 nM of the Ca(2+)-activated K(+) channel blocker, Penitrem A, revealed a rapidly inactivating K(+) current that resembled an A-type current. The A-type current was insensitive to tetraethylammonium (TEA) at 1 mM, but was partially suppressed by higher concentrations (10 mM). 4-Aminopyridine (10 mM; 4-AP) completely blocked the A-type current. The 4-AP-sensitive transient current was activated at a potential of -60 mV with peak current densities averaging 29.7 +/- 5.68 pA/pF at +60 mV. The voltage of half-inactivation was -28.3 +/- 1.9 mV, and the time constant for recovery from inactivation at +60 mV was 118.7 +/- 7.9 ms. Under current-clamp conditions 4-AP depolarized the membrane potential by approximately 3 to 4 mV and triggered small contractions and relaxations of individual MVSM cells within the walls of the arterioles. CONCLUSIONS: A-type current is the major voltage-dependent K(+) current in retinal MVSM and appears to play a physiological role in suppressing cell excitability and contractility.