I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

TRPV6 Determines the Effect of Vitamin D3 on Prostate Cancer Cell Growth

Despite remarkable advances in the therapy and prevention of prostate cancer it is still the second cause of death from cancer in industrialized countries. Many therapies initially shown to be beneficial for the patients were abandoned due to the high drug resistance and the evolution rate of the tumors. One of the prospective therapeutical agents even used in the first stage clinical trials, 1,25-dihydroxyvitamin D3, was shown to be either unpredictable or inefficient in many cases. We have already shown that TRPV6 calcium channel, which is the direct target of 1,25-dihydroxyvitamin D3 receptor, positively controls prostate cancer proliferation and apoptosis resistance (Lehen'kyi et al., Oncogene, 2007). However, how the known 1,25-dihydroxyvitamin D3 antiproliferative effects may be compatible with the upregulation of pro-oncogenic TRPV6 channel remains a mystery. Here we demonstrate that in low steroid conditions 1,25-dihydroxyvitamin D3 upregulates the expression of TRPV6, enchances the proliferation by increasing the number of cells entering into S-phase. We show that these pro-proliferative effects of 1,25-dihydroxyvitamin D3 are directly mediated via the overexpression of TRPV6 channel which increases calcium uptake into LNCaP cells. The apoptosis resistance of androgen-dependent LNCaP cells conferred by TRPV6 channel is drastically inversed when 1,25-dihydroxyvitamin D3 effects were combined with the successful TRPV6 knockdown. In addition, the use of androgen-deficient DU-145 and androgen-insensitive LNCaP C4-2 cell lines allowed to suggest that the ability of 1,25-dihydroxyvitamin D3 to induce the expression of TRPV6 channel is a crucial determinant of the success or failure of 1,25-dihydroxyvitamin D3-based therapies

Two isoforms of Trpm8 in rat vas deferens

Trpm8 is a nonselective Ca2+-permeable ion channel activated by temperatures below 28 °C and cooling chemical compounds such as menthol and icilin. Trpm8 is expressed in sensory neurons where it functions for temperature detection. However, Trpm8 is also expressed in various internal organs where temperature is stably higher than 36 °C, that is much higher than cool temperature needed for Trpm8 activation. This opens possible makes roles for Trpm8, such as nociception. Trpm8 mRNA is expressed in vas deferens (VD), a smooth muscle organ of male reproductive system. However, no Trpm8-mediated currents were previously registered in the myocytes. VD is located outside of testes, and it consists of smooth muscle tube covered by the epithelium. It actively contracts and transfers sperm from testes to ejaculatory ducts prior to ejaculation. Temperature in VD is stably high, thus, Trpm8 role there could be outside of cold detection. The objective of this study was to analyze Trpm8 mRNA and protein expression in rat VD, as well as splice variant analysis. Trpm8 mRNA expression in VD was confirmed with RT-PCR, and Trpm8 protein was detected by the Western-blot analysis. Additionally, we found that both mRNA and protein of shorter non-classical isoform, as well as canonical isoform of Trpm8 in VD. We isolated smooth muscle cells from VD and performed a multi-cell PCR. This technique makes possible non-myocytic mRNA detection that could be isolated from e.g. sensory neurons termini where Trpm8 is expressed at much higher levels. Interestingly, in the isolated smooth muscle cells, no canonical Trpm8 transcript was found, though the non-classical isoform was present. We propose the shorter isoform could be formed as a result of alternative splicing. This would account for a difference in Trpm8 function in VD, i.e. no Trpm8-mediated currents registered in the myocytes. A shorter isoform could have a truncated N-terminal domain, that is consistent with known human Trpm8 isoforms sM8-6 and sM8-18

Гемодинаміка при ішемії-реперфузії головного мозку щурів при застосуванні деяких інфузійних розчинів

In experiments on rats with modeled apoplectic attack (bilateral 20-minutes ischemia of the internal carotid arteries with further reperfusion), was established that the administration of HAES-LX-5% and Voluven at a dose of 2,5 ml/kg 2 times a day (5,0 mL/kg per day), every day, every 12 hours during the 7 days was accompanied by improved blood circulation in cerebral vessels (p<0,05) compared with animals treated with 0,9% NaCl solution and also to some extent inhibits the development of cerebrocardiac syndrome which manifested in the stabilization and restoration of arterial and central venous pressure.В опытах на крысах с модельным острым нарушением мозгового кровотока (двусторонняя 20-минутная ишемия внутренних сонных артерий с последующей реперфузией) установлено, что введение HAES-LX-5% и волювена в дозе 2,5 мл/кг 2 раза/день (5,0 мл/кг сутки) ежесуточно через каждые 12 ч в течение семи суток сопровождалось улучшением кровообращения по церебральным сосудам (р<0,05) по сравнению с животными, которых лечили 0,9 % раствором NaCl, а также в некоторой степени торможением развития цереброкардиального синдрома, что проявилось в стабилизации и восстановлении артериального и центрального венозного давления.У дослідах на щурах з модельним гострим порушенням мозкового кровотоку (двобічна 20-хвилинна ішемія внутрішніх сонних артерій з подальшою реперфузією) встановлено, що уведення HAESLX-5 % та волювену в дозі 2,5 мл/кг 2 рази/день (5,0 мл/кг добу) щодоби через кожні 12 год протягом семи діб супроводжувалося поліпшенням кровообігу по церебральних судинах (р<0,05) порівняно з тваринами, яких лікували 0,9 % розчином NaCl, а також у деякій мірі гальмуванням розвитку цереброкардіального синдрому, що проявилось у стабілізації і відновленні артеріального і центрального венозного тиску

Особливості морфології головного мозку щурів при гострій церебральній ішемії на тлі уведення волювену

Morphological study of rat brain with acute cerebral ischemia showed that ischemia-reperfusion of the brain provokes the appearance of many specific pathological changes in the wall of a blood vessel microcirculation of the sensomotor cortex. In this case the course therapy of rats with colloidal and isoosmolar solution Voluven did not lead to a stable full restoration of the structure of the cerebral cortex sensomotor areas, but significantly reduced the negative impact of hypoxia within 7 days of observation and improved reparative and compensatory mechanisms of microglia and neurons, mainly due to the stabilization of hemodynamics in the vessels of the microcirculation.Проведенное морфологическое исследование головного мозга крыс с острой церебральной ишемией показало, что ишемия-реперфузия головного мозга провоцирует появление многих специфических патоморфологических изменений в стенке микроциркуляторного русла сенсомоторной коры больших полушарий. При этом курсовая терапия крыс коллоидно-изоосмолярным раствором волювен не приводила к полному стабильному восстановлению структуры коры больших полушарий сенсомоторной зоны, но заметно снизила негативное влияние гипоксии на протяжении семи суток наблюдения и улучшила репаративно-компенсаторные механизмы микроглии и нейронов, преимущественно за счет стабилизации гемодинамики в сосудах микроциркуляторного русла.Проведене морфологічне дослідження головного мозку щурів з гострою церебральною ішемією показало, що ішемія-реперфузія головного мозку провокує появу багатьох специфічних патоморфологічних змін у судинній стінці мікроциркуляторного русла сенсомоторної кори великих півкуль. При цьому курсова терапія щурів колоїдно-ізоосмолярним розчином волювен не призводила до повного стабільного відновлення структури кори великих півкуль сенсомоторної зони, але помітно знизила негативний вплив гіпоксії на протязі семи діб спостереження та покращила репаративно-компенсаторні механізми мікроглії та нейронів,переважно за рахунок стабілізації гемодинаміки в судинах мікроциркуляторного русла

Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death

International audienceThe growing number of studies suggested that inhibition of autophagy enhances the efficacy of Akt kinase inhibitors in cancer therapy. Here, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, myosin light-chain kinase (MLCK) and stromal interaction molecule 1 (STIM1), represents the 'two-in-one' compound that stimulates autophagosome formation (by downregulating Akt/mammalian target of rapamycin (mTOR) pathway) and inhibits their degradation (by acting like a lysosomotropic agent and increasing lysosomal pH). We show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. Further, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. Altogether, our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy