Acetylcholine rescues two-cell block through activation of IP3 receptors and Ca2+/calmodulin-dependent kinase II in an ICR mouse strain

Acetylcholine (ACh) causes early activation events in mouse oocytes, but little is known about its precise role in the early embryonic development of mice. We aimed to determine whether and how ACh is capable of rescuing two-cell block in an in vitro culture system. ACh evoked different transient Ca²⁺ patterns showing a higher Ca²⁺ peak in the two-cell stage embryos (two-cells) than observed in mature oocytes. In early two-cells subjected to an in vitro two-cell block, xestospongin C (Xes-C), an IP3 receptor antagonist, significantly decreased the level of the ACh-induced Ca²⁺ increase. The reduction in the ACh-induced Ca²⁺ increase by Xes-C in late two-cells was lower than that in early two-cells. Furthermore, KN62 and KN93, both CaMKII inhibitors, were found to reduce the magnitude of the ACh-induced Ca²⁺ increase in early two-cells. The addition of ACh to the culture medium showed an ability to rescue in vitro two-cell block. However, the addition of ACh together with both Xes-C and CaMKII inhibitors or with either inhibitor separately had no effect on the rescue of two-cell block. Long-term exposure of late two-cells to ACh decreased morula and early blastocyst development and ACh had a differential effect on early and late two-cells. These results indicate that ACh likely rescues the in vitro two-cell block through activation of IP3R- and/or CaMKII-dependent signal transduction pathways.     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

Sustained elevation of calcium induces Ca(2+)/calmodulin-dependent protein kinase II clusters in hippocampal neurons

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75-175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076-3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl-D-aspartate (250 microM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca(2+). Indeed, intracellular Ca(2+) monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca(2+). Moreover, CaMKII does not cluster when the rise in intracellular Ca(2+) is prevented by the omission of extracellular Ca(2+) during CCCP treatment, confirming that clustering is Ca(2+)-dependent. A lag period of 1-2 min between the onset of high intracellular Ca(2+) levels and the formation of CaMKII clusters suggests that a sustained increase in Ca(2+) level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected.These results indicate that pathological conditions that promote sustained episodes of Ca(2+) overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.     

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release

Aim: To investigate the importance and contribution of calmodulin‐dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca²⁺‐release in response to different work intensities in single, intact muscle fibres. Methods: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3‐I or the pharmacological inhibitor KN‐93. The effect on tetanic force production and [Ca²⁺]_i was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. Results: Using a standard protocol to induce fatigue (50× 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 ± 3 contractions in control to 33 ± 3 with AC3‐I. KN‐93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 ± 3. Tetanic [Ca²⁺]_i was 100 ± 11%, 97 ± 11% and 67 ± 11% at the end of stimulation in control, AC3‐I and KN‐93 respectively. A similar inhibition was obtained using a high intensity protocol (20× 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25× 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca²⁺]_i or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca²⁺ release. Conclusion: Our results show that CaMKII is crucial for maintaining proper SR Ca²⁺ release and that this is regulated in a work intensity manner.     

Distinct roles of CaM and Ca(2+)/CaM -dependent protein kinase II in Ca(2+) -dependent facilitation and inactivation of cardiac L-type Ca(2+) channels

L-type Ca(2+) channels have two opposing forms of autoregulatory feedback, Ca(2+) -dependent facilitation (CDF) and Ca(2+) -dependent inactivation (CDI), in response to increases in intracellular Ca(2+) concentration. Calmodulin (CaM) has been reported to mediate the two feedbacks. Although both the direct binding of CaM and the phosphorylation mediated by Ca(2+)/CaM -dependent protein kinase II (CaMKII) have been suggested as underlying mechanisms, the detailed features remain to be clarified. In this study, we investigated the effects of CaM and CaMKII inhibitors on CDF and CDI with patch clamp cell-attached recordings in guinea-pig ventricular myocytes. We confirmed that a high-K(+) and high-Ca(2)(+) could induce an increase of the intracellular Ca(2+) concentration and subsequent CDF and CDI. We then found that CDF and CDI were both depressed and were finally abolished by treatment with a CaM inhibitor chlorpromazine (1-100 microM) in a concentration-dependent manner. Another CaM antagonist calmidazolium (1 microM) showed a similar effect. In contrast, CaMKII inhibitors, KN-62 (0.1-3 microM) and autocamtide 2 -related inhibitory peptide (1 microM), delayed the development of CDF and CDI significantly, but they did not depress either CDF or CDI. These results imply that CaM is necessary and possibly sufficient for the two mechanisms. We propose a hypothesis that CaM is a key molecule to bifurcate the Ca(2+) signal to CDF and CDI and that CaMKII plays a modulatory role in them both.     

Amplification of odor-induced Ca(2+) transients by store-operated Ca(2+) release and its role in olfactory signal transduction

A critical role of Ca(2+) in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca(2+) stores in this process remains unclear. Using confocal Ca(2+) imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca(2+) that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca(2+) stores are spatially restricted; emptying produces compartmentalized Ca(2+) release and capacitative-like Ca(2+) entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca(2+) mobilization. This odor-induced Ca(2+) release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca(2+) transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca(2+) transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca(2+) wave from the cilia to the soma. We show that this amplification process depends on Ca(2+)-induced Ca(2+) release. The results indicate that stimulation of ORNs with odorants can produce Ca(2+) mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca(2+) mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.     

Increased expression of Ca2+/calmodulin-dependent protein kinase II alpha during chronic morphine exposure

The chronic administration of morphine and related opioid drugs results in tolerance and dependence which limits the clinical utility of these agents. Neuronal plasticity is probably responsible in large part for tolerance and dependence. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in the neuroplastic events underlying memory formation and other phenomena. However, the role of this kinase in morphine tolerance remains unclear. To clarify this issue we explored mRNA and protein expression of CaMKIIalpha in spinal cord tissue from control and morphine treated mice using real-time polymerase chain reaction, Western blot analysis and confocal microscopy. Our chronic exposure paradigm involved the subcutaneous implantation of morphine pellets for 6 days prior to tissue analysis. The results indicate that the levels of CaMKIIalpha mRNA and protein were robustly increased in spinal cord tissue from morphine-treated mice. Confocal microscopy demonstrated that the increase in CaMKIIalpha expression was primarily localized to superficial laminae of the dorsal horn. In addition, the abundance of phosphorylated CaMKIIalpha was increased in spinal cord tissue from morphine-treated mice.We conclude that enhanced CaMKIIalpha expression and activity in spinal cord tissue may contribute to the development of morphine tolerance in mice. The involvement of this enzyme in opioid tolerance suggests other parallels may exist between the neuroplastic events related to memory formation and those related to opioid tolerance or pain.     

Differential expression of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse hippocampus induced by various nociceptive stimuli

In the present study, we characterized differential expressions of phosphorylated Ca(2+)/calmodulin-dependent protein kinase IIalpha (pCaMKIIalpha) and phosphorylated extracellular signal-regulated protein (pERK) in the mouse hippocampus induced by various nociceptive stimuli. In an immunoblot study, s.c. injection of formalin and intrathecal (i.t.) injections of glutamate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1 beta) significantly increased pCaMKIIalpha expression in the hippocampus, but i.p. injections of acetic acid did not. pERK1/2 expression was also increased by i.t. injection of glutamate, TNF-alpha, and IL-1beta but not by s.c. injections of formalin or i.p. injections of acetic acid. In an immunohistochemical study, we found that increased pCaMKIIalpha and pERK expressions were mainly located at CA3 or the dentate gyrus of the hippocampus. In a behavioral study, we assessed the effects of PD98059 (a MEK 1/2 inhibitor) and KN-93 (a CaMKII inhibitor) following i.c.v. administration on the nociceptive behaviors induced by i.t. injections of glutamate, pro-inflammatory cytokines (TNF-alpha or IL-1beta), and i.p. injections of acetic acid. PD98059 as well as KN-93 significantly attenuated the nociceptive behavior induced by glutamate, pro-inflammatory cytokines, and acetic acid. Our results suggest that (1) pERKalpha and pCaMK-II located in the hippocampus are important regulators during the nociceptive processes induced by s.c. formalin, i.t. glutamate, i.t. pro-inflammatory cytokines, and i.p. acetic acid injection, respectively, and (2) the alteration of pERK and pCaMKIIalpha in nociceptive processing induced by formalin, glutamate, pro-inflammatory cytokines and acetic acid was modulated in a different manner.     

Immunohistochemical localization of Ca(2+)/calmodulin-dependent protein kinase kinase beta in the rat central nervous system

We examined regional and intracellular distribution of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaM-KK beta), which activated Ca(2+)/calmodulin-dependent protein kinase I and IV (CaM-K I and IV) immunohistochemically in the central nervous system of the rat by light and electron microscopy. Although most neurons in the brain and spinal cord exhibited the immunoreactivity, no labeled neurons were observed in the globus pallidus or entopeduncular nucleus, and only a small number of neurons showed weak immunoreactivity in the substantia nigra pars reticulata. In general, the immunoreactivity was observed both in the cytoplasm and cellular nucleus, although the immunoreactivity was not found in the cellular nucleus in some large neurons such as in the mesencephalic trigeminal nucleus, lateral vestibular nucleus or gigant cellular reticular formation. As to motoneurons of the cranial nerve nuclei and the anterior horn of the spinal cord, they revealed the immunoreactivity both in the cytoplasm and nucleus. The reaction product appeared as fine granules in the cytoplasm and nucleus under light microscopy. Electron microscopic observations confirmed that the reaction product was localized mainly on the Golgi apparatus or on the nuclear chromatin. Immunolabeling for antibody against CaM-KK beta was discussed with the distribution of CaM-K I, IV and another CaM-KK, CaM-KK alpha, in the central nervous system.     

Bcl10 is phosphorylated on Ser138 by Ca2+/calmodulin-dependent protein kinase II

Ordered assembly of scaffold proteins Carma1-Bcl10-Malt1 determines NF-kappaB activation following T cell receptor (TCR) engagement. Carma1-Bcl10 interaction and the signaling pathway are controlled by Carma1 phosphorylation, which are induced by PKCtheta and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition to Carma1 phosphorylation, previous studies have demonstrated that Bcl10 is phosphorylated in the C-terminal Ser/Thr rich region following TCR engagement. However the kinases that phosphorylate Bcl10 are incompletely understood. Here we show that CaMKII phosphorylates Bcl10 on Ser138. Furthermore, a CaMKII inhibitor, KN93, and CaMKII siRNA substantially reduce Bcl10 phosphorylation induced by phorbol myristate acetate/ionomycin. S138A mutation prolongs Bcl10-induced NF-kappaB activation, suggesting that Bcl10 phosphorylation is involved in attenuation of NF-kappaB activation. These findings suggest that CaMKII modulates NF-kappaB activation via phosphorylating Bcl10 as well as Carma1.     

长期甲状腺素刺激对大鼠心肌Ca /钙调蛋白依赖性蛋白激酶Ⅱ的影响

目的: 通过观察长期甲状腺素刺激对心肌Ca²⁺/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKII)的影响,探讨CaMKII是否参与甲亢性心脏病的发生发展。方法: 将20只SD大鼠随机分为甲状腺素刺激组和对照组,每组各10只。以0.2 mg·kg^-1·d^-1的剂量腹腔注射甲状腺素或等体积生理盐水3个月(每次给药前称体重),造模后3个月处死大鼠。以心重(HW)、心重与体重之比(HW/BW)、左心室重与体重之比(LVW/BW)及心肌细胞直径大小反映心肌肥厚;以心肌血管周围胶原面积(PVCA)/血管腔面积(VA)的比值反映心肌纤维化。用实时定量RT-PCR和Western blotting分别从mRNA水平和蛋白表达水平反映CaMKII的变化。结果: 造模3个月后与对照组相比,甲状腺素刺激组的HW/BW、LVW/BW、心肌细胞直径大小和心肌纤维化程度(PVCA/VA)均明显高于对照组,分别是对照组的1.87、1.84、2.15和1.94倍,差异均显著(P<0.05,P<0.01);甲状腺素刺激组心肌细胞中CaMKII mRNA表达水平、蛋白含量与对照组相比均降低,分别是对照组的40%和79%,但CaMKII的活性较对照组增加1.58倍,其差异均显著(P<0.05)。结论: 长期甲状腺素刺激诱导大鼠心肌肥厚模型中,甲状腺素降低心肌CaMKII的表达,但心肌CaMKII的活性增加,CaMKII可能参与甲状腺素诱导的甲亢性心脏病的发生发展。     

Heparin inhibits phosphorylation and autonomous activity of Ca(2+)/calmodulin-dependent protein kinase II in vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca(2+) regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the delta-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca(2+)/calmodulin-independent CaM kinase II activity. This effect was less evident in heparin-resistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Finally, the effects of pharmacological inhibitors of phosphatases like okadaic acid, calyculin, and tautomycin suggest that heparin inhibits CaM kinase II phosphorylation by activating protein phosphatases 1 and 2A. These findings support the hypothesis that alterations in calcium-mediated mitogenic signaling pathways may be involved in the antiproliferative mechanism of action of heparin.     

Expression of Ca2+/calmodulin-dependent protein kinase (CaMK) Ibeta2 in developing rat CNS

We observed the onset time and distribution pattern of beta2 isoform of Ca2+/calmodulin-dependent protein kinase I (CaMKIbeta2) in the CNS of the rat during the embryonic period until birth using an immunohistochemical method. The expression of CaMKIbeta2 started at embryological day 10 when the three primary brain vesicles and neural tube are generated from the neural plate. During the embryonic period, highly immunoreactive products were ubiquitously detected in neurons in the CNS, although neurons in the caudate-putamen and globus pallidus were faintly immunostained or immunonegative. High expression of CaMKIbeta2 persisted in the olfactory bulb, lymbic system, neocortex, septal nuclei, amygdala complex, some hypothalamic nuclei, pontine nuclei, Purkinje cells and granule cells in the cerebellar cortex through the developing period. At the subcellular level, CaMKIbeta2 was strongly expressed in nuclei of neurons but faintly in their cytoplasm, suggesting that this protein has an important role in the nuclear signaling pathway.This study demonstrates that expression of CaMKIbeta2 begins at the earliest developmental stage of the rat CNS and persists through the developing period.     

Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating

The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.     

Ca(2+) entry through L-type Ca(2+) channels helps terminate epileptiform activity by activation of a Ca(2+) dependent afterhyperpolarisation in hippocampal CA3

In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation.We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and -insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network excitability during epileptiform discharges.