Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in controlling blood pressure; however, there is little information on cellular mechanisms underlying Ang II-evoked vasoconstrictor responses. The aim of the present study is to investigate the effect of Ang II on cation conductances in freshly dispersed rabbit mesenteric artery myocytes at the single-channel level using patch-clamp techniques. In cell-attached patches, bath application of low concentrations of Ang II (1 nM) activated cation channel currents (I cat1 ) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited I cat1 but evoked another cation channel (I cat2 ) with a conductance of approximately 2 pS. Ang II-evoked I cat1 and I cat2 were inhibited by the AT 1 receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced I cat1 which was subsequently inhibited to reveal I cat2 . The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 µM) activated I cat1 and I cat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 µM) potentiated Ang II-evoked I cat1 and inhibited I cat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 µM) reduced Ang II-induced I cat1 but activated I cat2 . Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated I cat1 . These data indicate that PKC inhibits I cat1 but stimulates I cat2 . Agents that deplete intracellular Ca 2+ stores also activated cation channel currents with similar properties to I cat2 . Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited I cat1 and I cat2 , respectively. Also flufenamic acid and zero external Ca 2+ concentration, respectively, potentiated and reduced Ang II-evoked I cat1 . Immunocytochemical studies showed TRPC6 and TRPC1 expression with TRPC6 preferentially distributed in the plasma membrane and TRPC1 expression located throughout the myocyte. These results indicate that Ang II activates two distinct cation conductances in mesenteric artery myocytes by stimulation of AT 1 receptors linked to PLC. I cat1 is activated by DAG via a PKC-independent mechanism whereas I cat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit I cat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced I cat1 and I cat2 , respectively.

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (approximately 100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone.

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca 2+ chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca 2+ ] o the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca 2+ ] o the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

BackgroundBlended learning is a combination of online and face-to-face learning and is increasingly of interest for use in undergraduate medical education. It has been used to teach clinical post-graduate students pharmacology but needs evaluation for its use in teaching pharmacology to undergraduate medical students, which represent a different group of students with different learning needs.MethodsAn existing BSc-level module on neuropharmacology was redesigned using the Blended Learning Design Tool (BLEnDT), a tool which uses learning domains (psychomotor, cognitive and affective) to classify learning outcomes into those taught best by self-directed learning (online) or by collaborative learning (face-to-face). Two online courses were developed, one on Neurotransmitters and the other on Neurodegenerative Conditions. These were supported with face-to-face tutorials. Undergraduate students’ engagement with blended learning was explored by the means of three focus groups, the data from which were analysed thematically.ResultsFive major themes emerged from the data 1) Purpose and Acceptability 2) Structure, Focus and Consolidation 3) Preparation and workload 4) Engagement with e-learning component 5) Future Medical Education.ConclusionBlended learning was acceptable and of interest to undergraduate students learning this subject. They expressed a desire for more blended learning in their courses, but only if it was highly structured, of high quality and supported by tutorials. Students identified that the ‘blend’ was beneficial rather than purely online learning.