Toshiko Yamazawa

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5‐trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio‐temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R‐1, ‐2 and ‐3) are expressed in a tissue‐ and development‐specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+‐signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+‐signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R‐2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B‐cell receptors. IP3R‐1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R‐3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3‐mediated Ca2+ signalling.

Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ.

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.

Ca2+‐sensor region of IP3 receptor controls intracellular Ca2+ signaling

Many important cell functions are controlled by Ca2+ release from intracellular stores via the inositol 1,4,5‐trisphosphate receptor (IP3R), which requires both IP3 and Ca2+ for its activity. Due to the Ca2+ requirement, the IP3R and the cytoplasmic Ca2+ concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP3−induced Ca2+ release and to play an important role in the generation of spatiotemporal patterns of Ca2+ signals such as Ca2+ waves and oscillations. Here we show that glutamate 2100 of rat type 1 IP3R (IP3R1) is a key residue for the Ca2+ requirement. Substitution of this residue by aspartate (E2100D) results in a 10‐fold decrease in the Ca2+ sensitivity without other effects on the properties of the IP3R1. Agonist‐induced Ca2+ responses are greatly diminished in cells expressing the E2100D mutant IP3R1, particularly the rate of rise of initial Ca2+ spike is markedly reduced and the subsequent Ca2+ oscillations are abolished. These results demonstrate that the Ca2+ sensitivity of the IP3R is functionally indispensable for the determination of Ca2+ signaling patterns.

Kinetic diversity in the fusion of exocytotic vesicles

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense‐core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+‐dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.

A Region of the Ryanodine Receptor Critical for Excitation-Contraction Coupling in Skeletal Muscle

Ca2+ release mediated by the ryanodine receptor (RyR) regulates many important cell functions including excitation-contraction (E-C) coupling in skeletal muscle, by which membrane depolarization controls the opening of RyR via the dihydropyridine receptor. Among the three RyR subtypes, RyR-1 mediates skeletal muscle E-C coupling, whereas RyR-2 and RyR-3 cannot substitute for RyR-1. We carried out expression experiments using cultured mutant skeletal myocytes not having intrinsic intracellular Ca2+ release channels to study the structure-function relationship of amino acid residues 1303–1406 in RyR-1 (D2 region). In this region the amino acid sequences are highly divergent between RyR-1 and RyR-2, and the corresponding sequence is lacking in RyR-3. Expression of RyR-1 but not of RyR-2 rescued E-C coupling in the mutant cells. Deletion of either the entire D2 region or its N-terminal half from RyR-1 preserved the function of RyR-1 as a Ca2+ release channel but resulted in the loss of E-C coupling. Substitution of the D2 region for the corresponding sequence of RyR-2 had no effect on the function of RyR-1. These results indicate that the presence of the D2 region is critical for E-C coupling in skeletal muscle, although the D2 region alone cannot determine the functional difference between RyR-1 and RyR-2.

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5‐trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist‐stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine‐sensitive Ca(2+)‐induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)‐dependent feedback control of InsP3‐induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all‐or‐none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum.

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca2+ indicator, fluo‐3. We observed rhythmic [Ca2+]i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca2+ transients, we found brief Ca2+ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca2+]i transients in both cell types was partially inhibited by nicardipine, an L‐type Ca2+ channel antagonist, suggesting that the rhythmic [Ca2+]i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca2+ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca2+]i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.

Nitric oxide‐induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca2+ stores regulates a multitude of cellular functions, but the role of intracellular Ca2+ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca2+ release from intracellular stores of central neurons, and thereby promote prolonged Ca2+ signalling in the brain. Reversible S‐nitrosylation of type 1 RyR (RyR1) triggers this Ca2+ release. NO‐induced Ca2+ 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.

Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling.

In excitable cells membrane depolarization is translated into intracellular Ca2+ signals. The ryanodine receptor (RyR) amplifies the Ca2+ signal by releasing Ca2+ from the intracellular Ca2+ store upon receipt of a message from the dihydropyridine receptor (DHPR) on the plasma membrane in striated muscle. There are two distinct mechanisms for the amplification of Ca2+ signalling. In cardiac cells depolarization‐dependent Ca2+ influx through DHPR triggers Ca2+‐induced Ca2+ release via RyR, while in skeletal muscle cells a voltage‐induced change in DHPR is thought to be mechanically transmitted, without a requirement for Ca2+ influx, to RyR to cause it to open. In expression experiments using mutant skeletal myocytes lacking an intrinsic subtype of RyR (RyR‐1), we demonstrate that RyR‐1, but not the cardiac subtype (RyR‐2), is capable of supporting skeletal muscle‐type coupling. Furthermore, when RyR‐2 was expressed in skeletal myocytes, we observed depolarization‐independent spontaneous Ca2+ waves and oscillations, which suggests that RyR‐2 is prone to regenerative Ca2+ release responses. These results demonstrate functional diversity among RyR subtypes and indicate that the subtype of RyR is the key to Ca2+ signal amplification.

Presence of functionally different compartments of the Ca2+ store in single intestinal smooth muscle cells

Studies in smooth muscle bundles have shown the presence of functionally different compartments of Ca2+ store, one (Sα) sensitive to both caffeine and inositol 1,4,5‐trisphosphate (IP3), and the other (Sβ) sensitive only to IP3, Ca2+ release in isolated single smooth muscle cells from guinea pig taenia caeci was studied to see if both compartments exist within a cell. Responses to caffeine and carbachol were consistently observed but were abolished after treatment with ryanodine, while intracellular application of IP3 induced Ca2+ release after the treatment, albeit smaller in size than control. Thus Sα and Sβ coexist in a single smooth muscle cell and agonist‐induced Ca2+ release requires whole store to be loaded with Ca2+.