RESUMEN
【Objective】 To explore the roles of 1,4,5-trisphosphate receptor (IP3Rs) and ryanodine receptors (RyRs), the two main Ca2+ release channels in sarcoplasmic reticulum (SR), in the regulation of intracellular Ca2+ oscillations in neonatal rat cardiomyocytes (NRCMs). 【Methods】 We isolated and cultured NRCMs for different days, then loaded them with Ca2+ indicator fura-2 and performed real-time fluorescent imaging. To distinguish the effects of IP3Rs and RyRs, NRCMs were pre-treated with phenylephrine (PE, IP3Rs agonist), caffeine (RyRs agonist), 2-APB (IP3Rs antagonist), and tetracaine (RyRs antagonists), respectively. 【Results】 The cultured monolayer NRCMs showed spontaneous synchronized Ca2+ oscillations. PE activation or 2-APB blockade of IP3Rs increased or reduced the frequency of Ca2+ oscillations in NRCMs, accordingly, with no significant effect on the amplitude of Ca2+ oscillations. Activation of RyRs with caffeine increased the frequency of Ca2+ oscillations, but unsynchronized the intercellular rhythm of calcium release and beating pace, while blocking RyRs with tetracaine completely abolished the Ca2+ oscillations and beats in NRCMs. In addition, the effect of PE stimulation on Ca2+ oscillation frequency gradually decreased along with cultured days. 【Conclusion】 IP3Rs regulate the rhythm of calcium oscillations, whereas RyRs are the main channel for bulky store Ca2+ release.
RESUMEN
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.