Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe.

The cytosolic free Ca(2+) transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca(2+)] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca(2+) dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca(2+)] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca(2+)] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca(2+)] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca(2+), both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca(2+) reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1-5, and 0.3 s(-1) (at 26 °C), reflecting activity of the SR Ca(2+) pump and interplay of luminal and cytosolic Ca(2+) buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

A potential role for the vanilloid receptor TRPV1 in the therapeutic effect of curcumin in dinitrobenzene sulphonic acid-induced colitis in mice.

A protective role of the transient potential vanilloid receptor 1 (TRPV1) in intestinal inflammation induced by dinitrobenzene sulphonic acid (DNBS) has been recently demonstrated. Curcumin, the major active component of turmeric, is also able to prevent and ameliorate the severity of the damage in DNBS-induced colitis. We evaluated the possibility that curcumin (45 mg kg(-1) day p.o. for 2 days before and 5 days after the induction of colitis) was able to reduce DNBS-induced colitis in mice, by acting as a TRPV1 agonist. Macroscopic damage score, histological damage score and colonic myeloperoxidase (MPO) activity were significantly lower (by 71%, 65% and 73%, respectively; P < 0.01), in animals treated with curcumin compared with untreated animals. Capsazepine (30 mg kg(-1), i.p.), a TRPV1 receptor antagonist, completely abolished the protective effects of curcumin. To extend these data in vitro, Xenopus oocytes expressing rat TRPV1 were examined. Capsaicin-evoked currents (3.3 micromol L(-1)) disappeared subsequent either to removal of the agonist or subsequent to the addition of capsazepine. However, curcumin (30 micromol L(-1)) was ineffective both as regard direct modification of cell membrane currents and as regard interference with capsaicin-mediated effects. As sensitization of the TRPV1 receptor by mediators of inflammation in damaged tissues has been shown previously, our results suggest that in inflamed, but not in normal tissue, curcumin can interact with the TRPV1 receptor to mediate its protective action in DNBS-induced colitis.

Stimulation of Ca²+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.