Ca(2+)-induced Ca2+ Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic beta-cells is triggered by the influx of Ca2+ through voltage-dependent Ca2+ channels. The resulting elevation of intracellular calcium ([Ca2+]i) triggers additional Ca2+ release from internal stores. Less well understood are the mechanisms involved in Ca2+ mobilization from internal stores after activation of Ca2+ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic beta-cell line, INS-1 cells. To measure cytosolic and stored Ca2+, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca2+]i was repetitively increased by caffeine stimulation in normal Ca2+ buffer. However, peak [Ca2+]i was only observed after the first caffeine stimulation in Ca2+ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca2+]i were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca2+ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca2+ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca2+ release from internal stores was activated by caffeine, Ca2+, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic beta-cells.

Differential Expression of Ryanodine Receptors in the Developing Rat Cochlea / 听力学及言语疾病杂志

Objective Ryanodine receptor (RyR) is one of the Ca2+ release channels on the intracellular Ca2+ stores. RyR induced-Ca2+ release is activated by the voltage-dependent Ca2+ entry, that is, calcium-induced calcium release (CICR). Intracellular free Ca2+ concentration (ECa2+]i) plays a key role on cochlear function. Our study is to investigate the differential expression of RyR in the developing rat cochlea, and to analyze the relationship between the expression of RyR and auditory functional development. Methods Immature SD rats, which were 1 day (P1), 5 days (P5), 10 days (P10), 14 days (P14), 28 days (P28) after parturition, and adutl rats(5 rats for each age) were included in the study. Frozen cochlea sectioning and immunofluorescence were applied to observe the differential expression of RyR. Results The RyR expression in the Corti's organ increased during the cochlear development. It's not significant that the stain was observed on the hair cells and supporting cells in the Corti's organ of P1 and P5 rats. The appearance of the Corti's organ of P10 rats trended to maturity. In P14 rats the RyR expression on hair cells located in the synaptic area against the afferent or efferent nerve, and the strain on supporting cells was extensive. There was little different between the strain on cochlea of P14 and P28 rats. In postmature rat spiral ganglion neurons (SGN), the RyR expression verged gradually from extensive whole cell soma to the synaptic area near to the plasma membrane. Conclusion The RyR expression peaked the 14th day after parturition, which was close to that in the mature cochlea. The time course of the RyR expression during the cochlear development was coincident with that of the auditory functional development. The RyR expression on both hair cells and SGNs located in the synaptic area near to the plasmolemma, implying that RyR induced-CICR was related to the auditory functions such as neurotransmission. Extensive RyR expression in the soma of SGNs at the early stage possibly involved in apoptosis of SGNs during neuron development.

Characteristics of Ca2+ release mechanisms from an intracellular Ca2+ store in rabbit coronary artery

To elucidate the Ca2+ release mechanisms in the rabbit coronary artery, arterial preparations were permeabilized with beta-escin and changes in tension were measured under varying experimental conditions. Additionally, we investigated properties and distribution of two kinds of Ca2+ release mechanisms, Ca2+-induced Ca2+ release (CICR) and IP3-induced Ca2+ release (IICR). The results obtained were summarized as follows; 1. When a rabbit coronary artery was incubated in a relaxing solution containing 30 microM beta-escin for 40 min. sensitivity to externally added Ca2+ was much higher in beta-escin permeabilized muscle than in intact preparations. The contractile effect of IP3 in beta-escin permeabilized muscle was also demonstrated; 2. Caffeine and IP3 contracted coronary arteries were permeabilized with beta-escin, but the amplitude of contraction was much larger in the presence of caffeine than of IP3. 3. Intracellular heparin completely inhibited the contractions induced by IP3, but not those by caffeine. On the other hand, procaine inhibited the responses to caffeine, but not those to IP3. Ryanodine inhibited both the caffeine- and IP3-induced contractions. 4. The amplitude of contractile responses was much larger to the maximal stimulation of CICR by applying caffeine than to the maximal stimulation of IICR by applying IP3. After the maximal CICR stimulation by caffeine, the activation of IICR by IP3 without the reloading of Ca2+ could no longer evoke contraction. On the other hand, after the maximal IICR activation, the activation of CICR could still evoke contraction although the amplitude of the contraction was smaller when compared with the case without the initial IICR stimulation. 5. Acetylcholine contracted coronary artery smooth muscles were permeabilized with beta-escin. However, in the absence of added guanosine triphosphate (GTP), the responses were very small. Acetylcholine-induced contraction was inhibited by heparin, but not by procaine. From the above results, it may be concluded that there are two kinds of mechanisms of Ca2+ release, CICR and IICR, in the rabbit coronary artery smooth muscle cell. Also, whereas the CICR mechanism distributes on the membrane of the whole smooth muscle Ca2+ store, the IICR mechanism distributes only on a part of it.