Co-ordination of Ca(2+) signalling in mammalian cells by the new Ca(2+)-releasing messenger NAADP

Ca(2+) signalling is one of the most important means in mammalian cells of relaying the action of hormones and neurotransmitters. The great diversity of agonist-induced Ca(2+) signatures, visualized by optical imaging techniques, can be explained by the production of intracellular messengers triggering Ca(2+) release from internal stores and/or by different coupling of Ca(2+) release to Ca(2+) entry. Several messengers, such as inositol trisphosphate and cyclic ADP-ribose, have been identified to date. More recent studies have reported the important role of a newly discovered Ca(2+) releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). These studies have shown important interactions of these messengers in the generation of specific Ca(2+) signals. NAADP acts at a very low concentration and seems to have a key role in sensitising cyclic ADP-ribose and inositol trisphosphate receptors. These points will be discussed in the present review.     

Effects of age and spatial learning on adenylyl cyclase mRNA expression in the mouse hippocampus

Adenylyl cyclase (AC) subtypes have been implicated in memory processes and synaptic plasticity. In the present study, the effects of aging and learning on Ca2+/calmodulin-stimulable AC1, Ca2+-insensitive AC2 and Ca2+/calcineurin-inhibited AC9 mRNA level were compared in the dorsal hippocampus of young-adult and aged C57BL/6 mice using in situ hybridization. Both AC1 and AC9 mRNA expression were downregulated in aged hippocampus, whereas AC2 mRNA remained unchanged, suggesting differential sensitivities to the aging process. We next examined AC mRNA expression in the hippocampus after spatial learning in the Morris water maze. Acquisition of the spatial task was associated with an increase of AC1 and AC9 mRNA levels in both young-adult and aged groups, suggesting that Ca2+-sensitive ACs are oppositely regulated by aging and learning. However, aged-trained mice had reduced AC1 and AC9, but greater AC2, mRNA levels relative to young-trained mice and age-related learning impairments were correlated with reduced AC1 expression in area CA1. We suggest that reduced levels of hippocampal AC1 mRNA may greatly contribute to age-related defects in spatial memory.     

Specific involvement of Ca(2+)-calmodulin kinase II in cholinergic modulation of neuronal responsiveness.

1. Muscarinic agonists when applied in the hippocampus at low concentrations suppress intrinsic controls on neuronal excitability through the block of Ca(2+)-activated K conductance(s), gK (Ca), underlying the adaptation of firing and slow afterhyperpolarization (sAHP) in CA1 and CA3 neurons. Carbachol, for example, is effective at 0.1-0.3 microM suggesting activation of a relatively high-affinity receptor. 2. We have examined the mechanism of this action by using a new, highly specific, peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as other kinase inhibitors and show that the muscarinic block of gK (Ca) relies on CaMKII activation in both CA1 and CA3 neurons. Thus phosphorylation of these channels or of an intermediary protein causes the channels to remain closed in the presence of Ca2+ and depolarization. 3. The very similar electrophysiological effects of serotonergic and glutamatergic agonists are mediated either through other kinases or by entirely different processes. 4. Block of intrinsic phosphatase activity by okadaic acid also reduced adaptation and sAHP, and muscarinic agonists had no further effect on these quantities. 5. The removal of presynaptic cholinergic inputs to the hippocampus in animals has a deleterious effect on the performance of tasks requiring spatial memory and is also implicated as a cause of cognitive disorders in humans. By increasing Ca2+ accumulation during electrical activity and promoting CaMKII activity, muscarinic input provides parallel reinforcing pathways for the induction of long-term potentiation, an important cellular memory mechanism. This suggests a possible link between behavioral and cellular approaches to the analysis of learning and memory.     

Ca(2+)-dependent regulation of synaptic vesicle endocytosis

Action potentials, when arriving at presynaptic terminals, elicit Ca(2+) influx through voltage-gated Ca(2+) channels. Intracellular [Ca(2+)] elevation around the channels subsequently triggers synaptic vesicle exocytosis and also induces various protein reactions that regulate vesicle endocytosis and recycling to provide for long-term sustainability of synaptic transmission. Recent studies using membrane capacitance measurements, as well as high-resolution optical imaging, have revealed that the dominant type of synaptic vesicle endocytosis at central nervous system synapses is mediated by clathrin and dynamin. Furthermore, Ca(2+)-dependent mechanisms regulating endocytosis may operate in different ways depending on the distance from Ca(2+) channels: (1) intracellular Ca(2+) in the immediate vicinity of a Ca(2+) channel plays an essential role in triggering endocytosis, and (2) intracellular Ca(2+) traveling far from the channels has a modulatory effect on endocytosis at the periactive zone. Here, I integrate the latest progress in this field to propose a compartmental model for regulation of vesicle endocytosis at synapses and discuss the possible roles of presynaptic Ca(2+)-binding proteins including calmodulin, calcineurin and synaptotagmin.     

Organization and Ca2+ regulation of adenylyl cyclases in cAMP microdomains

The adenylyl cyclases are variously regulated by G protein subunits, a number of serine/threonine and tyrosine protein kinases, and Ca(2+). In some physiological situations, this regulation can be readily incorporated into a hormonal cascade, controlling processes such as cardiac contractility or neurotransmitter release. However, the significance of some modes of regulation is obscure and is likely only to be apparent in explicit cellular contexts (or stages of the cell cycle). The regulation of many of the ACs by the ubiquitous second messenger Ca(2+) provides an overarching mechanism for integrating the activities of these two major signaling systems. Elaborate devices have been evolved to ensure that this interaction occurs, to guarantee the fidelity of the interaction, and to insulate the microenvironment in which it occurs. Subcellular targeting, as well as a variety of scaffolding devices, is used to promote interaction of the ACs with specific signaling proteins and regulatory factors to generate privileged domains for cAMP signaling. A direct consequence of this organization is that cAMP will exhibit distinct kinetics in discrete cellular domains. A variety of means are now available to study cAMP in these domains and to dissect their components in real time in live cells. These topics are explored within the present review.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

Possible role for Ca(2+) calmodulin-dependent protein kinase II as an effector of the fertilization Ca(2+) signal in mouse oocyte activation

The present study shows that Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) is physiologically activated in fertilized mouse oocytes and is involved in the Ca(2+) response pathways that link the fertilization Ca(2+) signal to meiosis resumption and cortical granule (CG) exocytosis. After 10 min of insemination, CaM kinase II activity increased transiently, then peaked at 1 h and remained elevated 30 min later when most of the oocytes had completed the emission of the second polar body. In contrast, in ethanol-activated oocytes the early transient activation of CaM kinase II in response to a monotonic Ca(2+) rise was not followed by any subsequent increase. Inhibition of CaM kinase II by 20 micromol/l myristoylated-AIP (autocamtide-2-related inhibitory peptide) negatively affected MPF (maturing promoting factor) inactivation, cell cycle resumption and CG exocytosis in both fertilized and ethanol-activated oocytes. These results indicate that the activation of CaM kinase II in mouse oocytes is differently modulated by a monotonic or repetitive Ca(2+) rise and that it is essential for triggering regular oocyte activation.     

二氢麦角碱升高血管性痴呆小鼠海马cAMP和腺苷环化酶

目的观测血管性痴呆小鼠海马组织环磷酸腺苷(cAMP)和腺苷环化酶(AC)水平及二氢麦角碱对其的影响,探讨血管性痴呆发病的分子生物学机制。方法通过双侧颈总动脉线结、连续3次缺血-再灌注,制作血管性痴呆动物模型,并设立假手术组、二氢麦角碱组;术后29 d、30 d分别测试学习和记忆成绩;应用放射免疫法检测小鼠海马组织cAMP水平,应用原位杂交技术检测AC水平。结果与假手术组比较,模型组学习和记忆成绩均降低(P<0.05),且海马组织cAMP水平也降低(P<0.05),海马CA1区AC mRNA阳性神经元比例明显降低(P<0.05);而与模型组比较,二氢麦角碱组学习和记忆成绩均改善(P<0.05),且海马组织cAMP水平也升高(P<0.05),海马CA1区AC mRNA阳性神经元比例明显增加(P<0.05)。结论海马组织cAMP和AC水平降低可能参与了血管性痴呆的分子生物学发病机制;二氢麦角碱可以升高其cAMP和AC水平而改善临床症状。     

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.     

Acceleration of Ca(2+) repletion in the junctional sarcoplasmic reticulum and alternation of the Ca(2+)-induced Ca(2+)-release mechanism in hypertensive rat (SHR) cardiac muscle

We estimated the time taken for a repletion of the junctional sarcoplasmic reticulum (JSR) Ca(2+) stores from a family of mechanical restitution curves after twitches of various magnitudes in the cardiac muscle of hypertensive rats (SHR), using a method described previously (Tameyasu et al. Jpn J Physiol. 2004;54:209-19), to evaluate abnormality in Ca(2+) handling by cardiac JSR in hypertension. We found no differences in contractility or in the time course of mechanical restitution between SHR and the controls (WKY) at 3 weeks of age. In comparison to WKY, 7- and 20-week-old SHR showed a greater rested state contraction (RST) and similar or smaller rapid cooling contracture, suggesting that their JSR contains a similar amount of Ca(2+) at saturation, but releases more Ca(2+) upon stimulation. The adult SHR and WKY showed similar mechanical restitution time courses, but the adults had longer pretwitch latencies. The function G(t) representing the time course of JSR Ca(2+) store repletion in adult SHR exceeded the WKY value at t < or = 0.5 s, but the function H(t) representing JSR [Ca(2+)] change corresponding to the mechanical restitution after RST was smaller in the adult SHR at t < or = 0.5 s, resulting in smaller H(t)/G(t) in adult SHR at t < or = 0.5 s. Deviations of G(t), H(t), and H(t)/G(t) from WKY were greater at 20 weeks than at 7. The results suggest an acceleration of JSR Ca(2+) store repletion and an alternation of the Ca(2+)-induced release of Ca(2+ )from the JSR in young adult SHR.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Ca(2+) entry caused by depolarization in acetylcholine-stimulated antral mucous cells of guinea pig: G protein regulation of Ca(2+) permeable channels

The effects of depolarizing conditions resulting from increasing extracellular K(+) concentration or nystatin treatment on intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in guinea pig antral mucous cells following acetylcholine (ACh) stimulation. ACh stimulation evoked a biphasic increase in [Ca(2+)](i), that is, an initial transient increase followed by a plateau. Depolarizing conditions reduced the [Ca(2+)](i) in the plateau phase during ACh stimulation. However, pertussis toxin (PTX, a G protein inhibitor) treatment caused [Ca(2+)](i) in the ACh-evoked plateau phase to increase under depolarizing conditions, while it had no effect on [Ca(2+)](i) under hyperpolarized conditions. Based on these observations, Ca(2+) permeable channels are regulated by a G protein which is activated by depolarized conditions and inhibited by hyperpolarized conditions and PTX; activation of the G protein (depolarization) causes Ca(2+) permeable channels to inhibit, and in turn, inhibition of the G protein (hyperpolarization) causes them to activate.     

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.     

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.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Age-related decline in hypocretin (orexin) receptor 2 messenger RNA levels in the mouse brain

The hypocretin (Hcrt; also known as orexin) system has been implicated in arousal state regulation and energy metabolism. We hypothesize that age-related sleep problems can result from dysfunction of this system and thus measured messenger RNA (mRNA) levels of preprohcrt in the hypothalamus, and hcrt receptor 1 (hcrtr1) and hcrt receptor 2 (hcrtr2) in eight brain regions of 3, 12, 18 and 24 months old C57BL/6 mice. Expression of preprohcrt and the colocalized prodynorphin did not change with age. Whereas an age-related change in hcrtr1 mRNA expression was observed only in the hippocampus, hcrtr2 mRNA levels declined in the hippocampus, thalamus, pons, and medulla; these reductions ranged from 33 to 44%. Declining trends (P < 0.1) in hcrtr2 mRNA levels were also observed in the cortex, basal forebrain and hypothalamus. These results are consistent with the hypothesis that an age-related deterioration occurs in the Hcrt system that may contribute to age-related sleep disorders.     

Overexpression of type-1 adenylyl cyclase in mouse forebrain enhances recognition memory and LTP

Cyclic AMP is a positive regulator of synaptic plasticity and is required for several forms of hippocampus-dependent memory including recognition memory. The type I adenylyl cyclase, Adcy1 (also known as AC1), is crucial in memory formation because it couples Ca(2+) to cyclic AMP increases in the hippocampus. Because Adcy1 is neurospecific, it is a potential pharmacological target for increasing cAMP specifically in the brain and for improving memory. We have generated transgenic mice that overexpress Adcy1 in the forebrain using the Camk2a (also known as alpha-CaMKII) promoter. These mice showed elevated long-term potentiation (LTP), increased memory for object recognition and slower rates of extinction for contextual memory. The increase in recognition memory and lower rates of contextual memory extinction may be due to enhanced extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling, which is elevated in mice that overexpress Adcy1.     

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.