Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP3R) serve to discharge Ca2+ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP3R isoforms and protect cells from apoptosis, reducing ER Ca2+ release. However, it has not been elucidated which IP3R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP3R I, strongly suppresses IP3-mediated Ca2+ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca2+ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca2+ release. Moreover, regulating Ca2+ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP3R III, leading to diminished Ca2+ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt

Basal calcium entry in vascular smooth muscle

Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring Ca uptake into cultured rat aortic smooth muscle cells. Wash solution (0°C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular Ca retention more effectively than EGTA (0.2 mM). Basal Ca entry was 1.45×10 Ca ·cell ·min . This translated to ∼250 μmol ·min given cell volumes of 4-15 pl as determined by 3-D image reconstruction. Gadolinium (100 μM) blocked 80% of the leak and exhibited a biphasic concentration-response relation (IC s=1 μM and 2 mM). Organic ion channel blockers also inhibited ∼80% of the leak; 45% by nifedipine (10 μM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H- imidazole) (50 μM) and 23% was exclusively sensitive to 2-aminoethoxy- diphenylborate (2-APB, 75 μM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca entry is mainly due to a basal open probability of excitable Ca -channels. © 2004 Elsevier B.V. All rights reserved. 45 2+ 45 2+ 2+ 9 2+ -1 -1 -1 -1 2+ 2+ 5

Erratum: Endothelin-1-Mediated Wave-Like [Ca²⁺]_i Oscillations in Intact Rabbit Inferior Vena Cava

Endothelin-1 (ET1) is an endogenous vasoconstrictor released by the vascular system to regulate the contractility of vascular smooth muscle cells (VSMC). It is implicated in the pathogenesis of hypertension and diabetic vasculopathy. In rabbit inferior vena cava (IVC), 10 n<i>M</i> ET1 induces tonic contraction mainly via type A endothelin receptor activation. Using confocal imaging of Fluo-3 loaded in thein situ VSMC within the intact IVC, we found that ET1 elicited [Ca²⁺]_i oscillations with an average frequency of 0.31 ± 0.01 Hz. These [Ca²⁺]_i oscillations occurred as repetitive Ca²⁺ waves traveling along the longitudinal axis of the cells with an average velocity of 29 ± 3 µm/s. The Ca²⁺ waves were not synchronized between neighboring VSMC nor were they propagated between them. Nifedipine (10 µ<i>M</i>) inhibited the tonic contraction by 27.0 ± 5.0% while SKF96365 (50 µ<i>M</i>) abolished the remaining contraction. In a parallel Ca²⁺ study, nifedipine reduced the frequency of the oscillations to 0.22 ± 0.01 Hz while SKF96365 abolished the remaining [Ca²⁺]_i oscillations. Subsequent application of 25 m<i>M</i> caffeine elicited no further Ca²⁺ signal. Thus, we conclude that ET1 stimulates tonic contraction in the rabbit IVC by inducing [Ca²⁺]_i oscillations and that stimulated Ca²⁺ entry through both the L-type voltage-gated Ca²⁺ channels and a nifedipine-resistant and SKF96365-sensitive pathway is crucial for the maintenance of [Ca²⁺]_i oscillations and tonic contraction