The intermittent rise in intracellular Ca2+ concentration ([Ca2+]i oscillation) has been observed in many types of isolated cells, yet it has not been demonstrated whether it plays an essential role during nerve stimulation in situ. We used confocal microscopy to study Ca2+ transients in individual smooth muscle cells in situ within the wall of small arteries stimulated with perivascular sympathetic nerves or noradrenaline. We show here that the sympathetic adrenergic regulation of arterial smooth muscle cells involves the oscillation of [Ca2+]i that propagates within the cell in the form of a wave. Ca2+ release from intracellular stores plays a key role in the oscillation because it is blocked after the store depletion by ryanodine treatment. Ca2+ influx through the plasma membrane sustains the oscillation by replenishing the Ca2+ stores. These results demonstrate the involvement of [Ca2+]i oscillations in the neural regulation of effector cells within the integrated system.

While subtypes 1 and 2 of the ryanodine receptor (RyR) function as intracellular Ca2+ release channels, little is known about the function of the third subtype (RyR-3), first identified in brain. Myocytes from mice homozygous for a targeted mutation in the RyR-1 gene (dyspedic mice) can now be used for a study on the function of RyR-3, which is predominantly expressed in these cells according to our reverse transcriptionpolymerase chain reaction analysis. We here demonstrate in these myocytes caffeine-, ryanodine-and adenine nucleotide-sensitive Ca>+-induced Ca2> release with -10 times lower sensitivity to Ca2+ than that of RyR-1. Although RyR-3 does not mediate excitationcontraction coupling of the skeletal muscle type, we propose that RyR-3 may induce intracellular Ca2+ release in response to a Ca2+ rise with a high threshold.

Many important cell functions are controlled by Ca 2+ release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP 3 R), which requires both IP 3 and Ca 2+ for its activity. Due to the Ca 2+ requirement, the IP 3 R and the cytoplasmic Ca 2+ concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP 3 -induced Ca 2+ release and to play an important role in the generation of spatiotemporal patterns of Ca 2+ signals such as Ca 2+ waves and oscillations. Here we show that glutamate 2100 of rat type 1 IP 3 R (IP 3 R1) is a key residue for the Ca 2+ requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca 2+ sensitivity without other effects on the properties of the IP 3 R1. Agonist-induced Ca 2+ responses are greatly diminished in cells expressing the E2100D mutant IP 3 R1, particularly the rate of rise of initial Ca 2+ spike is markedly reduced and the subsequent Ca 2+ oscillations are abolished. These results demonstrate that the Ca 2+ sensitivity of the IP 3 R is functionally indispensable for the determination of Ca 2+ signaling patterns.

Mobilization of intracellular Ca 2 þ stores regulates a multitude of cellular functions, but the role of intracellular Ca 2 þ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca 2 þ release from intracellular stores of central neurons, and thereby promote prolonged Ca 2 þ signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca 2 þ release. NO-induced Ca 2 þ release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.