Thirty three rabbits from five litters that were weaned at the age of 5 weekswere observed. The animals were kept in pens that were enriched with anelevation made of bricks. In total, 150 h of observations made at feedingtime  were analysed. Anumber of affiliative, exploratory, comfort, eating, resting and locomotorbehaviours were observed. Agonistic behaviour was not observed. Rabbitsshowed companion and location preferences: 56 % of animals had apreferred companion, and 84 % preferred a particular place in the pen.Significant effects of group size and time of day on the frequency of someforms of behaviour were found, e.g. rabbits performed comfort behaviours moreoften in the morning. Sex did not influence the rabbits' behaviour.Correlations were also found between different forms of behaviour,e.g. animals that performed more exploratory behaviours also showed morelocomotor behaviours and affiliative interactions. Wild rabbits live ingroups comprised of one to three males and one to five females. Previousstudies have compared wild and domestic rabbits, and have shown thatdomestic rabbits in semi-natural conditions exhibit territoriality, dominance and socialbehaviours that are similar to wild rabbits , e.g.they communicate by marking an area with buccal glands as wild rabbits do. Dueto territorial behaviour, rabbits also often exhibit aggression towards one anotherafter puberty. Castration decreases interpersonal aggression in rabbitgroups .Rabbits can be kept singly, in pairs or in groups. A rabbit kept alone isunable to interact with other individuals, and the risk of stereotypy isincreased ; however, the positive result of thiskind of husbandry is that there is no aggression, and rabbits are notexposed to physical injury, e.g. bites. Keeping the animals in groups isimportant for adolescent rabbits. The risk ofaggression is very low (before puberty), and rabbits can play together anddisplay the entire spectrum of social behaviour. Studies show that at a density of morethan 16 rabbits/mWeaning is a stressful situation for all domestic animals. Weaned rabbitsshould be kept in litter groups or in mixed groups of animals of the same age . In intensive breeding, rabbits are weaned at the age of 21 days. Rabbits should be weaned at 4–6 weeks of age. It isideal to transfer the mother to another cage and leave the kits in theoriginal cage (where they lived until weaning), although this is rarely applied under breeding conditions.Kits can be kept together until the age of 3 months, or until they reach puberty.Social stimulation is very important in rabbits owing to their social nature.Studies in semi-natural systems indicate the occurrence of ahierarchy between rabbits and behaviours such as allogrooming, lying andeating in the company of another individual .Enriching the social life of rabbits by placing at least two individualstogether introduces more benefits than the use of objects to diversify theenvironment. This also prevents the occurrence of stereotypy as it introducesmore unforeseen situations due to the presence of another individual . The role of environmental enrichment in captive rabbits'lives is enormous. Studies have shown that rabbits kept in cages withchewing sticks, shelters where they can hide and/or elevations show lessstereotypy, more social behaviour and less aggression toward other animalsin the group .Previous studies have also reported that the housing system ,the group and cage size, and the density  influence the behaviour of growing rabbits.The aim of this study was to characterize behaviours expressed by youngrabbits after weaning and to determine the occurrence of company and/orlocationpreferences. Moreover, the potential influence of factors such as sex, bodyweight, group size, and time of day/week of observation on the frequency ofobserved behaviours was investigated.22.1The observations took place in July/August 2013 at the “ZielonaChata” agro-tourism farm in Cichowa, Wielkopolska Province, Poland.A total of 33 rabbits  from five litters were studied. There werenine animals in two litters and four, five and six animals in other three respective litters.During observations two individuals died. One of the animals(from a litter consisting of six siblings) died early in the experiment and was not included infurther analysis. In spite of the death of rabbit no. 8 from the fifth group a daybefore the final weighting, the observations of its behaviour were includedin this paper. Thus, data from 32 individuals were analysed. The litterswere from five females that were crossed with two males (Table 1). Each litter was placedin a purpose-built enclosure. Each pen was the same size – 0.85 m (length) 2.2Observations began the day after weaning to get the rabbits acquaintedwith the new environment. Observations were carried out over 2 weeks at the end ofJuly and beginning of August 2013. Each group was observed from 07:30 to 10:00 am andfrom 18:00 to 20:30 pm, 6 days a week. In total, 150 h of observations were madeby one observer (A.C.). The continuous recording method and the sampling of alloccurrences of behaviours method were used . Observations ofgroups 3 and 4 were carried out directly on site, while the rest of the observations  were noteddown from video recordings. Both the behaviour and the position of the rabbitswere recorded. An ethogram was developed based on previous ethograms used . The pens were split into four virtual squares in order to calculatelocationpreference . In square number 2 there was a bowl of food, the elevationwas located in square number 3 , and abowl of water was in square number 4. Every 30 min the position of each individual was noted.The observation stand was near squares number 3 and 4.2.3The data were recorded and analysed using SPSS 20.0 (for Windows), and each category ofbehaviour was analysed using the Pearson correlation coefficient and a univariateANOVA (sex and litter size). A paired Location preference was analysed using a Chi square test.3The classification and descriptive information regarding the observed forms of behaviourare shown in Table 2. Vocalization, agonistic behaviours, chin marking,thumping and sexual behaviour were not observed. Fights while eating anddrinking or attempts to defend food bowls were not observed.Rabbits did not chase other animals away,and they were not aggressive toward one another . Defecation was not reported due to the difficulty involved with observing thisbehaviour. The most frequent affiliative behaviour involving another animalwas allogrooming, and the least frequent affiliative behaviour was nose touching .The most frequent comfort behaviour was grooming . The most frequentbehaviour on the elevation was sitting. All forms of behaviour observed onthe elevation were also recorded in other parts of the pen .3.1Some rabbits in each group showed a companion preference . These individualspreferred the company of one particular rabbit (only two rabbits had twopreferred siblings). Twelve out of twenty-one preferences were reciprocal.No specific traits that could have affected the companion preference werefound. Some rabbits avoided the company of particular siblings . Each of these animals avoidedonly one sibling. Four of eleven avoidance relationships were reciprocal.3.2The majority of rabbits exhibited a location preference. Table 3presents the number of animals that showed a location preference in detail.A preference for virtual square number 2 was seen in every group, specifically ingroups number 1 and 2. Rabbits in these groups notably rested in the food bowl,while rabbits in the other groups preferred to relax invirtual square number 1. Rabbits from group 3 gladly used the elevation.3.3Rabbits that displayed more behaviours on the elevation showed more exploratorybehaviours .Litter size did not impact the occurrence of behaviours on the elevation.3.7The analysis did not a show significant impact of the observation week on the frequency ofany form of behaviour  could suggest that being on the elevationprevented group leisure  or that individualspreferred resting together somewhere other than on the elevation (e.g. near the food bowl).https://www.pzhk.pl/wp-content/uploads/Rozporzadzenie_Dz.U.2010.116.778.pdf,last access: 20 April 2014); however, according to body weight guidelines atotal of 22 kg in each pen would have been suitable. Therefore, owing to theyoung age and low body weight of the animals used, the pen was an appropriatesize and there was no overcrowding. Morisse and Maurice (1997) showed thatrabbits kept at a density that is too high exhibit less social and locomotorybehaviours and more comfort and exploratory behaviours. At a density of over20 rabbits/mAnimal density is more important in rabbit husbandry than the use of theappropriate bedding . In this study there weresignificantly less social, exploratory, ingestion, comfort, locomotory andresting behaviours in groups of nine individuals . This may have been caused by an animal density that was too high. Inthe experimental pen designed for this study . Furthermore, rabbits in bigger groups consume less food.When the number of rabbits exceeds seven, feed conversion efficiency is reduced. Studiesconfirm that it is important to select a sufficiently large groupand to keep rabbits at the correct density, as specified by the standards, so that rabbitbreeding is as effective as possible without harm to the animals. Breeding rabbits inlarge groups has more disadvantages  than it has advantages . The best conditions for rabbits are groups of 4–5 individuals anda density of 15–17 rabbits/mIngestion and rest behaviours are often performed in the company ofanother individual. This displays the social bond between rabbits. In this study, animalswere close to each other while lying down, sitting, drowsing, drinkingand eating. The fact that these behaviours are performed together has also beenevidenced by previous studies such as Held et al. (1995) and Zucca et al. (2012).Locomotory and exploratory behaviours are more often performed alone. Jordanet al. (2010) showed that rabbits prefer to eat and drink in thecompany of other individuals. Under these conditions, they ingest smaller amounts more ofteninstead of long periods of grazing. During this research, rabbits also stayedtogether on the elevation; however, this was correlated with the rabbits' weights.According to our results, rabbits showed a companion preference for anindividual with whom they performed the above-mentioned activities. Rabbitsweaned at a young age (21 days) tend to stay in groups. Matics et al. (2004)observed (in his free choice system) that rabbits are morelikely to choose the company of other individuals than to be alone afterweaning: rabbits chose small cages, where the density reached 50–70 rabbits/mNo agonistic behaviours were observed in this study. Vervaecke et al. (2010)showed that rabbits form a linear hierarchy at the age of 10 weeks. The fact that observations endedat the age of 7 weeks explains the lack of agonistic behaviours, which made it impossible toestablish a hierarchy in the groups of rabbits studied. Rabbits should be slaughtered before reaching 80 days due to increased aggression . Aggression is also correlated with the number of individuals kept: themore rabbits, the higher the probability of aggressive behaviour . The results of a study by Trocino and Xiccato (2006) showed thatthe occurrence of aggressive acts increases with the age and the size of thegroup.The correlations between exploratory behaviours, affiliative interactions andlocomotory behaviours were highly significant in this research.More active individuals were more likely to exhibit social behaviours, which isin agreement with the above-mentioned results published by Trocino andXiccato (2006): rabbits with the ability to exhibit locomotor behaviours (in a suitablecage or enclosure) displayed more inter-individual contact behaviours. It wasobserved that comfort behaviour was correlated with resting in thecompany of another individual. During this research, it was possible toobserve the frequent occurrence of both behaviours alternately.Jordan et al. (2010) found that grooming is the most common comfort behaviour. Thehigh incidence of comfort behaviours may also be related to the bedding used.Clean fur is important for rabbits, so they groom it often.Morisse et al. (1999) showed that the dirtier the deep beddingbecomes, the more often rabbits exhibit grooming.The impact of sex on the occurrence of behaviours was not observed due to theyoung age of the rabbits. It is said that behavioural differences occur afterpuberty .The rabbits studied most often stayed in the second virtual square of the pen.These results probably arise from the fact that rabbits were eatingduring the observation periods (time of feeding),and that the bowls were placed in virtual square number 2.However, the location preference observed may also be relatedto the fact that rabbits chose resting places near the eating site. This fact also explains the preference for the firstvirtual square shown by some individuals. The preference for thefirst and second squares could also be explained by the tendency of rabbitsto select places near walls to rest. Rabbits are less likely to rest incentral areas. Buijs et al. (2011b) reported a high occurrence of rabbits resting nearthe walls, and proposed that rabbits do not fear attack from predatorsin such places. The rabbits in this study also often rested beside the elevation. The introduction of the brickconstruction (the elevation) meant that rabbits were often observed next to it and not justnear the walls. In all groups, animals defecation was mainly observed in thecorner of virtual square 3, next to the elevation.The selection of a specific place for excretions is also observed in the wild. Wildrabbits leave their droppings outside the burrow, near its entrance, oftennear a molehill if there is one close to the burrow .Rabbits are considered to be nocturnal or crepuscular animals. Studies ontheir daily activity show that resting behaviours most frequently occur duringthe day and that all activities  occur at night. The observed rabbits mostly showed eatingbehaviour in the evening. This may be related to observation time,as the evening observation period was closer to sunset than the morning observation period was tosunrise. Domestic rabbits eat in the evening and at night, as do wild rabbits.Feeding in the evening reduces the occurrence of stereotypy in rabbits .Buijs et al. (2011a) observed that rabbits consumed most of their food during the period around sunset.This study has brought us to several conclusions. Time of day and littersize influenced some behaviours. Rabbits displayed more ingestion behaviours inthe evening and more comfort and resting behaviours  in the morning.The highest number of behaviours was observedin groups of four or five individuals; in bigger groups  the frequency ofbehaviours decreased. There was no significant effect of sexon the frequency of the behaviours observed. Weaned rabbits did not showagonistic behaviour due to their young age (before puberty). Rabbits  exhibited companion and location preferences. The factor/s influencingthe choice of companion was not determined. Different forms of behaviourwere related, e.g. the correlations between exploratory and affiliative behaviourswith other individual and locomotor behaviours were highly significant.Further studies such as this one could provide more important information on behaviour in weaned rabbits.


The calcium sensing receptor (CaSR) was first identified in parathyroid glands, and its primary role in controlling systemic calcium homeostasis by the regulation of parathyroid hormone (PTH) secretion has been extensively described in literature. Additionally, the receptor has also been investigated in cells and tissues not directly involved in calcium homeostasis, e.g., the nervous system (NS), where it plays crucial roles in early neural development for the differentiation of neurons and glial cells, as well as in the adult nervous system for synaptic transmission and plasticity. Advances in the knowledge of the CaSR’s function in such physiological processes have encouraged researchers to further broaden the receptor’s investigation in the neuro-pathological conditions of the NS. Interestingly, pre-clinical data suggest that receptor inhibition by calcilytics might be effective in counteracting the pathomechanism underlying Alzheimer’s disease and ischemia, while a CaSR positive modulation with calcimimetics has been proposed as a potential approach for treating neuroblastoma. Importantly, such promising findings led to the repurposing of CaSR modulators as novel pharmacological alternatives for these disorders. Therefore, the aim of this review article is to critically appraise evidence which, so far, has been yielded from the investigation of the role of the CaSR in physiology of the nervous system and to focus on the most recent emerging concepts which have reported the receptor as a therapeutic target for neurodegeneration and neuroblastic tumors. The Cagonists . The VFTdulators ,8. The fdulators ,11.2+, including the polyvalent cations Mg2+, Al3+, Sr2+, Mn2+, Gd3+ and some antibiotics such as neomycin, gentamycin and tobramycin [2+ [2+ metabolism [3) and diacyl glycerol (DAG) [3 promotes intracellular Ca2+ mobilizations, while DAG activates protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade [i/o pathway, which inhibits cyclic adenosine monophosphate (cAMP) production [12/13 which, in turn, stimulates the rho kinase signaling [In line with other GPCRs, the CaSR mediates a biased-signaling that is the capacity of the receptor to respond to a number of agonists that activate distinct intracellular signaling cascades in a tissue-dependent manner ,13,14. Ibramycin . Moreovebramycin . In contycin [2+ . Synthettabolism . Such cool (DAG) . As a co cascade . Moreoveoduction , and theignaling . In addiignaling  and reguignaling .2+ and in maintaining Ca2+ homeostasis by regulating the secretion of PTH [2+ increases, the activated CaSR inhibits PTH secretion. Conversely, in the case of hypocalcemia, the receptor is inactive, and the consequent release of parathyroid hormone restores Ca2+ concentration within the physiological range by promoting Ca2+ resorption from kidney and bone and Ca2+ absorption in the intestine [2+ mobilization, has been identified in parathyroid cells [Being mainly expressed in the chief cells of the parathyroid glands, the CaSR exerts its primary role in sensing changes in extracellular Can of PTH ,25. Indentestine . To negantestine ,28. Moreid cells , and thiid cells . Along wid cells , whereasid cells . Moreoveid cells  and keraid cells . Additioid cells , while iid cells . Such evid cells . Afterwaid cells ,38,39, t2+ signals are crucial regulators of the specification of neural versus epidermal ectoderm. Studies in Xenopus laevis embryos have demonstrated that spontaneous elevations of intracellular Ca2+ are restricted to the dorsal ectoderm cells, which originate the neural progenitors and never occur in ventral ectoderm cells, which give rise to the epidermis [Calcium signaling is extremely important for the development of the nervous system. In vertebrates, Capidermis ,41. In apidermis .2+ relevance for the NS, an involvement of the CaSR in the development of the nervous system appeared to be likely, and several in vitro and in vivo approaches have been used to test this hypothesis. At this point, an important premise regarding CaSR in vivo models is necessary. The murine knock-out (KO) model of the CaSR (CaSR-/-), which is a model of human neonatal severe hyperparathyroidism [Consistent with the Cayroidism , displayHowever, such lethal effects can be normalized by the constitutive genetic ablation of PTH. Indeed, mice with the CaR-/-/PTH-/- genotype survive to adulthood with no obvious difference in size or appearance relative to controls ,45 allow2+ or with the calcimimetic NPS R-467 significantly increased axonal growth [2+ on neurite outgrowth. A similar effect of the CaSR was also observed in dendritic extensions, as hippocampal neurons from DNCaSR mouse at post-natal day four (P4) developed significantly shorter dendrites than cells from wild-type animals [2+. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 efficiently blocked the Ca2+ mediated neurite growth, demonstrating that the CaSR mediated axonal growth depends on the ERK1/2 activated pathway [The role of the CaSR in regulating the axonal and dendritic growth of neurons in the peripheral and central nervous system has been extensively studied. In sympathetic neurons isolated from the mice superior cervical ganglion (SCG), CaSR mRNA reaches a peak of expression at embryonic day 18 (E18), which coincides with the time when the axons grow to innervate their targets . By usinl growth . On the  animals . More re animals . In part pathway . 2+, while such effect was attenuated when cells were transfected with DNCaSR [2+ and spermine led to the secretion of the monocyte chemoattractant protein-1 (MCP-1), which is known to support migration of rat neural stem cells [2+ [Similar to what has been mentioned for osteoblasts and immune cells, in vitro and in vivo reports have suggested the involvement of the CaSR in neuronal migration and chemotaxis. Two gonadotropin-releasing hormone (GnRH) neuronal cell lines, GN11 and GT1-7, showed an increased chemotaxis when stimulated with high extracellular Cah DNCaSR . Moreoveem cells . Conversells [2+ . Additioells [2+ .Another example of CaSR involvement in neuronal chemotaxis and migration was recently reported in the developing cerebellum . CerebelAlong with the described function in the neurons, several studies report the CaSR being involved in the maturation of oligodendrocytes. A CaSR genetic mutation, causing neonatal severe hyperparathyroidism (NSHTP), was associated with a paucity of deep white matter and a delayed myelination in a six-month-old infant . Though 2+ and spermine promoted the proliferation and maturation of oligodendrocytes progenitor cells (OPCs), while such processes were significantly blunted by the expression of a dominant negative CaSR [2+ or neomycin increased the open state probability (Po) of an outward K+ channel, an effect likely mediated by the CaSR [2+ mobilization and inositol phosphate accumulation were observed in GalC-positive oligodendrocytes isolated from 20 days postnatal rat brain upon stimulation with high Ca2+ and with the positive allosteric modulator NPS R-568, demonstrating a CaSR-mediated activation of the phospholipase C pathway, similar to that observed in other cell types [Consistent with this idea, evidence in CaSR-null mice showed that these animals had reduced levels of MBP in the cerebellum during cerebellar development, compared to the wild-type animals . In vitrive CaSR ,55. Inte2+, Gd3+, and neomycin was found to increase cell proliferation, as assessed by H3-thymidine-incorporation, and to activate nonselective cation channels (NCCs) [2+ or by NPS R-467 was responsible to mediate the opening of an outward K+ channel [Whether the CaSR has a comparable function to that described in the proliferation and development for cells of astrocytic lineage is still unresolved. Few studies, aimed to address this issue, have been carried out in glial-derived tumors cell lines. In U373 astrocytoma, the positive stimulation of the CaSR with increasing Cas (NCCs) . Further channel . However2+ homeostasis is a crucial process for any cell, especially for such excitable cells as the neurons. The most important processes occurring in the nervous system, such as neurotransmission, synaptic plasticity, and excitability, depend on highly sophisticated machinery that finely control the intra- and extra-cellular Ca2+ level [The maintenance and regulation of Ca2+ level . A role 2+ level . Moreove2+ level . A follo2+ level . However2+ level .2+, neomycin, or spermine significantly increased the opening state probability of nonselective cation channels in WT neurons but not in cells isolated from CaR-/- mice [o of NCCs resulted to be enhanced by high Ca2+ stimulation in HEK-293 cells stably transfected with the CaSR but not in non-transfected cells, confirming a role of the receptor in the regulation of NCCs [2+-activated K+ channels (CAKCs), as stimulation with high extracellular Ca2 and neomycin increased the Po of these channels in wild-type neurons but not in CaSR-deficient neurons. Authors hypothesized that the receptor’s mediated-increase of intracellular Ca2+ levels activated these K+ channels, leading to membrane hyperpolarization [2 and transduce them into changes in neuronal excitability. More recent reports have introduced new insights in this field [2+ at the synaptic cleft, as it occurs during synaptic activity, activated nonselective cation channels, while increases in an extracellular Ca2+ stimulated CaSR and reduced the channel currents [2+ activated an NALCN which mediated a depolarizing current and increased neuronal excitability. The activation of an NALCN occurred through a cascade which involved a Ca2+-sensing GPCR, presumably the CaSR, and two intracellular proteins, UNC-79 and UNC-80 [2+ in nerve terminals and by measuring miniature EPSCs (mEPSCs) in cultured mouse neocortical neurons, it was observed that the stimulation of the CaSR with calindol and cinacalcet  increased spontaneous vesicle fusion and mEPSC frequency, with a consequent glutamate release. As a confirmation, the frequency of spontaneous synaptic transmission and glutamate secretion were decreased in neurons isolated from CaSR-/- mice [Several in vitro and in vivo studies have attempted to investigate the function of the CaSR in neuronal calcium signaling and synaptic transmission. In this regard, few works indicated a role of the receptor in regulation of NCCs. The patch-clamp of rat hippocampal neurons demonstrated that the activation of the CaSR with high extracellular Ca-/- mice ,64. Simi of NCCs . At the rization . These fis field ,68,69. Icurrents ,68. In acurrents . Authorsd UNC-80 ,71. Altod UNC-80 . Indeed,-/- mice . Such co-/- mice . To sum 2+ ions; these include several metabotropic glutamate receptors (mGluRs) and γ-amino isobutyric acid B receptors (GABABRs) [2+ at physiological concentrations and that a single amino acid residue is responsible to determine the sensitivity to extracellular Ca2+ [2+ acts allosterically to potentiate GABA responses at the GABAB receptors in membranes prepared from Chinese hamster ovary (CHO) cells stably expressing the GABABR1/R2 heterodimer as well as in membranes from rat brain cortex [In addition to the CaSR, other GPCRs have been shown to sense and to be activated by extracellular CaGABABRs) ,75. Indelar Ca2+ . Moreoven cortex . Such abn cortex . Similarn cortex . Interesn cortex . By high2+ homeostasis is extremely important for the nervous system, thus its dysfunction can seriously compromise the condition of NS cells. Importantly, disturbances in Ca2+ regulation and signaling can mediate different processes from brain ageing to pathological conditions according to the level of extent [2+ equilibrium, which are mainly represented by increased intracellular Ca2+ transients as well as reduced Ca2+ buffering capacity, especially in mitochondrial compartments. Indeed, mitochondria contribute to this condition, with a decreased ability to sequester excess Ca2+ from cytosol and the augmented production of reactive oxygen species (ROS) in aged brains, as reviewed in the recent work of Muller and colleagues [2+ dysregulation is critically involved in the development of pathological conditions and neurodegenerative disorders. Remarkably, emerging evidences have reported the CaSR as having an impact in neurodegeneration and neuroblastic tumors and the receptor’s allosteric modulators as playing novel potential therapeutics. Such concepts will be widely described in the next sections.As above mentioned, the maintenance of Caf extent . The phylleagues . Togethe2+, which promotes excitotoxicity and neuronal cell death [Ischemia is the interruption of blood flow to cells and tissues, causing decreased oxygen and nutrient consumption and the improper removal of ‘metabolic waste’ at the same time . A consell death . Interestingly, it has been reported that CaSR expression is up-regulated in rat brains after transient focal cerebral ischemia . In this3). Accordingly, apoptosis rate and the expression of apoptosis-related proteins all significantly increased upon H/R, and as expected, GdCl3 further augmented such increase. Conversely, an antagonist of the CaSR, NPS 2390, significantly reversed the H/R-mediated effects. Moreover, the phosphorylation of ERK1/2 was upregulated upon hypoxia/reoxygenation treatment, but it was significantly attenuated by NPS 2390, suggesting that the CaSR is involved in the induction of hippocampus apoptosis during H/R through the phosphorylation of ERK1/2 [2+ mobilization, consequent to the CaSR-mediated PLC activation, was stimulated upon hypoxic stimuli in hippocampal neurons, and the selective blocker of the CaSRCalhex 231inhibited such an increase. In addition, in hippocampal neurons and tissue, hypoxia or GdCl3 increased the expression of beta-secretase 1 (BACE1), an enzyme responsible for amyloid precursor protein (APP) cleavage and for the production of the two main amyloid species, Aβ40 and Aβ42. Interestingly, Calhex 231 or Xesto C (a selective inhibitor of IP3 receptor) partly prevented hypoxia-induced BACE1 overexpression. Because these findings supported a link between CaSR activation and amyloid synthesis (a topic that will be discussed in detail in the next section), authors further analyzed the effect of hypoxia on this process. An evaluation of the Aβ40 and Aβ42 contents of rat hippocampal lysates showed that hypoxia or GdCl3 raised the content of both amyloid species, and that Calhex 231 or Xesto C partly prevented such increases [A similar role for the CaSR has been proposed in exacerbating pathological hypoxic processes. Primary cultures isolated from a rat hippocampus and subjected to hypoxia/reoxygenation (H/R) were assessed for cell viability, apoptosis rate, and the expression level of cell death-related proteins such as caspase-3, Bax, and cytochrome C . This ref ERK1/2 . In linef ERK1/2 . Indeed,f ERK1/2 . Similarf ERK1/2 . Moreovencreases . To summAlzheimer’s disease (AD) is the most common form of dementia, and it accounts for nearly 50 million people worldwide, as recently estimated by the World Health Organization. The biochemical hallmarks of AD are represented by extracellular accumulations of the amyloid peptide Aβ42, known as amyloid plaques  and neur2+, an effect likely mediated by the CaSR, as it was not observed in neurons from mice with constitutive CaSR deletion [Interestingly, several evidences have supported the CaSR being implicated in the AD pathomechanisms. Early evidence of such involvement showed that Aβ peptides (Aβs) stimulated NCCs in a CaSR-dependent fashion. Indeed, in the cultured hippocampal neurons of wild-type mice, Aβs activated NCCs probably via elevation in cytosolic Cadeletion . Afterwadeletion . In the last few years, a function of the CaSR in modifying amyloid production and secretion has been supported . As mentInterestingly, a role of the CaSR in the mechanism underlying phosphorylation of the TAU protein was also proposed. Human astrocytes exposed to fAβ25−35 exhibited an increased activity of glycogen synthase kinase 3 (GSK)-3β responsible for mediating TAU phosphorylation. As a consequence, exogenous fibrillary Aβ significantly increased the production of p-TAU oligomers, which were then released within exosomes. Remarkably, the inhibition of the CaSR with NPS 2143 efficiently abolished the amyloid-mediated effects on GSK-3β and TAU , thus giA further contribution to the field was given by a study aimed at investigating the association of CaSR variations in Alzheimer’s disease susceptibility . By usin2+ dysfunction has been recognized to play a crucial role in AD. Indeed, the activation of the amyloidogenic pathway produces a remodeling of the neuronal Ca2+ signaling, which results in the upregulation of AD and disrupts Ca2+ dependent signaling mechanisms, which are responsible for memory formation and learning. On one side, this is due to an increase Ca2+ entry through the plasma membrane ion-channel receptors, such as NMDA receptors, activated by extracellular accumulated toxic amyloid species. On the other hand, Ca2+ dysfunction is caused by an augmented Ca2+ release from internal stores [2+ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs) [2+ signaling system concurs to disrupt the synaptic mechanisms responsible for learning and memory, thus causing neuronal dysfunction and neurodegeneration through the intrinsic activation of calcium-dependent apoptosis [Taken together, these studies suggest that interest surrounding the potential involvement of the CaSR in AD has emerged especially during the last decade. To date, studies have reported that the receptor’s modulation affects amyloid and TAU secretion  , furtherpoptosis ,108. TheNeuroblastomas are heterogenic tumors of the peripheral nervous system (PNS), originating by the impaired development of neural crest cells. Neuroblastic tumors  account for 7–10% of all tumors in children, and 95% of all neuroblastomas occur in individuals under five years of age . PrimaryMYCN)-amplification and undifferentiated histology [MYCN amplification is the best characterized genetic alteration in human neuroblastomas [MYCN-amplified cell lines [2+ [As previously described in this review, evidence reported the expression of the CaSR in malignancies of the central nervous system (CNS) ,58. In tistology . It is ilastomas , and it ll lines . Furtherines [2+ , which iines [2+ . Altogetines [2+ . In thisines [2+ . TherefoEvidence accumulated since the first report of CaSR expression in the brain suggests that the receptor is involved in several critical processes underlying the physiological development of neuronal and glial cells, as well as in the proper maintenance and function of the adult nervous system. Furthermore, a critical role of the receptor has been highlighted in neuro-pathological conditions, such as ischemia, Alzheimer’s disease, and in neuroblastoma, and preliminary in vitro and in vivo data suggest that receptor’s modulation might provide novel therapeutic approaches for such disorders. Nevertheless, our understanding of the precise role and signaling of the receptor in the nervous system is largely unknown. Similarly, the exact cellular mechanisms mediated by CaSR modulators that resulted in mitigation of such NS-affecting conditions, need still to be better elucidated. One of the main problems comes from the limits of in vivo models. As mentioned before, the constitutive CaSR KO murine model  is charaAn alternative tool for studying the receptor’s relevance for the nervous system might be represented by induced pluripotent stem cell (iPSC) technology . This cu


It belongs to the chemical class of the sesquiterpenes and has been the subject of a number of studies. Here, we evaluated the effects of this compound in airway smooth muscle. The biological activities of trans-caryophyllene were examined in isolated bath organs to investigate the effect in basal tonus. Electromechanical and pharmacomechanical couplings were evaluated through the responses to K+ depolarization and exposure to acetylcholine (ACh), respectively. Isolated cells of rat tracheal smooth muscle were used to investigate trans-caryophyllene effects on voltage-dependent Ca2+ channels by using the whole-cell voltage-clamp configuration of the patch-clamp technique. trans-Caryophyllene showed more efficiency in the blockade of electromechanical excitation-contraction coupling while it has only minor inhibitory effect on pharmacomechanical coupling. Epithelium removal does not modify tracheal smooth muscle response elicited by trans-caryophyllene in the pharmacomechanical coupling. Under Ca2+-free conditions, pre-exposure to trans-caryophyllene did not reduce the contraction induced by ACh in isolated rat tracheal smooth muscle, regardless of the presence of intact epithelium. In the whole-cell configuration, trans-caryophyllene (3 mM), inhibited the inward Ba2+ current (IBa) to approximately 50% of control levels. Altogether, our results demonstrate that trans-caryophyllene has anti-spasmodic activity on rat tracheal smooth muscle which could be explained, at least in part, by the voltage-dependent Ca2+ channels blockade. It is the major chemical constituent (20.6%) of the essential oil of Pterodon polygalaeflorus (EOPp). EOPp blocks the electromechanical excitation-contraction coupling without affecting the pharmacomechanical coupling  were investigated. In segments where epithelial layer was present trans-caryophyllene diminished the ACh-induced contraction (p < 0.05). When contractions with (n = 8) or without (n = 8) intact epithelium were compared between themselves and with their respective controls no statistically significant difference  was observed -induced contraction] could block Ca2+ influx by inhibiting L-type Ca2+ channels in single rat tracheal smooth muscle cells. To provide direct evidence we performed whole-cell patch-clamp in myocytes to test the effects of trans-caryophyllene on L-type Ca2+ channels.Having established that the trans-caryophyllene (gray trace). ICa,L  was reduced by approximately 50% of maximal current. To verify the time-course for inhibition provoked by trans-caryophyllene, ICa,L amplitudes were plotted as function of time . of time B. These 2+  Those dependent on changes in membrane potential  and (2) those independent of the changes in membrane potential  . Throughstituent .trans-Caryophyllene decreased the basal tone in rat trachealis muscle only when epithelial layer was preserved. This observation could be explained by a direct inhibitory effect of trans-caryophyllene on the COX or by causing an imbalance between relaxant and constrictor prostanoids synthesized by epithelial COX. The absence of trans-caryophyllene-induced relaxant activity on preparations without intact epithelium is consistent with these assumptions. Further studies conducted with selective antagonists are needed to clarify this point.It is generally known that prostanoids synthesized by epithelial cyclo-oxygenases (COX) play a major role in the control of airway contractility since COX inhibitors diminish the airway basal tone ,15,16,17+ concentrations induces contraction of airway smooth muscle that is easily relaxed by Ca2+ channel antagonists . The equivalent Tween 80 concentration had a very small effect on pre-contracted tracheal strips .Acetylcholine (ACh) was used at 10 and 60 μM to elicit contractions in 2.0 and in 0.0 mM calcium concentration, respectively. High extracellular Ktrans-caryophyllene, ethylene glycol-bis(aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), tetraethylammonium chloride (TEA), adenosine 5'-triphosphate disodium (Na2-ATP), papain, bovine serum albumin (BSA), dithiothreithol (DTT), collagenase type II and hyaluronidase] were purchased from Sigma Chemical Company  and from Reagen . All drugs and salts were of analytical grade.Drugs [ACh, The animals were sacrificed by cervical dislocation. To study the tracheal muscle relaxation tracheal ring strips were then mounted in a 5 mL tissue bath containing modified Tyrode’s solution maintained at 37 °C. Under baseline tension of 1.0–1.15 gF the isolated tissues were allowed to equilibrate for 1–2 h before the addition of any substance. Experiments were performed on isolated tracheal smooth muscle preparations with or without intact epithelium. Isometric contractions were recorded through a force displacement transducer  coupled to an integrated organ bath system attached to a computer .trans-caryophyllene on tracheal basal tone, isolated tracheal rings with or without intact epithelium were exposed to increasing concentrations of trans-caryophyllene (0.0005–50 mM). The tracheal epithelium was mechanically removed by rubbing with a cotton bud and it was confirmed by measurement of contraction’s magnitude promoted by KCl 60 mM. Contractions above 1300 gF were considered without functional epithelium.In order to assess the effect of the trans-caryophyllene (0.005–50 mM) on the contractile responses to ACh (10 μM) and (0.3–50 mM) to K+ (80 mM) were studied in rat tracheal smooth muscle maintained in Ca2+-containing medium.In this series of experiments, effects of non-cumulative increasing concentrations of 2+ availability from extracellular Ca2+ entry was indirectly evaluated by recording the contraction induced by ACh in Ca2+-free medium (containing 0.2 mM EGTA). After the equilibration period, ACh at 60 μM  was added to the bath and produced a contraction that was established as control response. After washing preparation again with calcium free solution, trans-caryophyllene (10 mM) was added to the preparations. Afterwards, in the presence of trans-caryophyllene the preparation was challenged with ACh (60 μM). As there is a possibility of time-dependent decrease in the ACh elicited smooth muscle contraction we compared trans-caryophyllene effects with the response to a second application of ACh. In all experimental series external controls  were done.Ca2+ physiological salt solution (PSS) in mM: 137 NaCl, 5.6 KCl, 0.44 NaH2PO4, 0.42 Na2HPO4, 4.17 NaHCO3, 3.55 MgCl2, 0.05 CaCl2, 10 HEPES and 5 glucose. The tracheal muscle was dissected away from the cartilage and minced with scalpel blades. Tracheal smooth muscle strips were divided and transferred to 3 mL of dissociation solution containing normal PSS (in mM): 137 NaCl, 5.6 KCl, 0.44 NaH2PO4, 0.42 Na2HPO4, 4.17 NaHCO3, 1 MgCl2, 2.6 CaCl2, 10 Hepes and 5 glucose, papain (0.9 mg/mL), BSA (1 mg/mL), and DTT (0.9 mg/mL). The strips were placed in a 37 °C water bath during 60 min and minced every seven-minute interval. After, tracheal smooth muscle strips were transferred to 3 mL of low-Ca2+ PSS supplemented with collagenase , BSA (1 mg/mL) and hyaluronidase (0.9 mg/mL) for further digestion. The strips were placed in a 37 °C water bath during approximately 30 min and minced every seven-minute interval. All enzymes were obtained from Sigma Chemical Co. The solution was centrifuged at 1,500 revolutions/min for 1 min three times and washed twice with low-Ca2+ PSS. Afterwards, cells were resuspended in normal PSS and triturated with a pipette to release individual smooth muscle cells from the tissue. The solution containing the dissociated cells was poured onto glass coverslips, and the cells were allowed to adhere for 40–60 min at room temperature.A 15-cm segment of trachea was immediately removed and placed at room temperature in a low-Ca2, 0.5 MgCl2, 10 HEPES and 5 glucose (pH 7.4 corrected with NaOH). Currents were compared in the presence and absence of trans-caryophyllene (3 mM). Pipettes were pulled from glass capillaries  with a micropipette puller . They had resistances of 2–4 MΩ (average 3.4 ± 0.3 MΩ) when filled with pipette solution . An Ag–AgCl wire was used as reference electrode.The standard bath solution employed in the whole-cell voltage-clamp experiments contained the following composition (in mM): 130 NaCl, 20 BaCl2+ channel currents. Capacitive currents were compensated electronically and a P/4 protocol was used for linear leak and residual capacitance subtraction. Ca2+ currents were low-pass filtered at 3 kHz and sampled at 10 kHz. Patch clamp experiments were performed on 35-mm Petri dishes using an inverted microscope . The bath was continuously perfused at 1–2 mL/min throughout the experiment. Solutions were gravity fed to the input ports of a solenoid valve mounted close to the bath, which was used to choose between one of two solutions. An EPC-9 patch clamp amplifier  and pulse software were used to record whole-cell Ca2+ channels were evaluated using Ba2+ (IBa) as the charge carrier. The currents were generated by 100 ms steps to 10 mV from a holding potential of −80 mV. To determine current–voltage relationships, cells were held at holding potential of −80 mV and subjected to step depolarizations of 100 ms from −70 to +60 mV in 10 mV increments every 10 s. Data were collected after the whole-cell configuration was obtained and current amplitude had stabilized, usually approximately 3 min after rupture of the cell membrane. Only cells with an input resistance >1 GΩ with no substantial rundown were analyzed. Currents through high-voltage-activated (HVA) Cat test, one-way and two-way analysis of variance (ANOVA). p < 0.05 was considered to be significant.All values were presented as mean ± S.E.M., and statistical comparisons were made using the paired Student’s trans-caryophyllene has anti-spasmodic activity on trachealis muscle. We also demonstrated by means of direct electrophysiological measurements (using the patch-clamp method) that trans-caryophyllene-induced blockade of Ca2+ influx through voltage-dependent Ca2+ channels. The provided information that will certainly enrich the pharmacological profile of trans-caryophyllene, a substance that thus appears as a candidate worthy of more investigations envisaging its pharmacotherapeutic use.In conclusion, we have clearly demonstrated that


C) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aβ oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.The cellular prion protein (PrP Nowadays, neurodegenerative disorders affecting elderly people is one major health problem worldwide, which is aggravated by lack of early diagnostic tools and pharmaceutical approaches. To name a few, they include amyotrophic lateral sclerosis and most common diseases such as Alzheimer’s (AD) and Parkinson′s. Despite its much less occurrence (1–2 cases per million), prion disorders have become renown after appearance of ′mad cow disease′ . RemarkaSc) of the constitutively expressed, α helix-rich prion protein (PrPC). Mostly present in the pre- and post-synaptic neuronal membranes , or simultaneously stimulated by glutamate addition, a picture identical to SOCE-activated neurons was recorded at sub-PM Ca2+ ′hot spots′ and in the cytosol [C augmented Ca2+ permeation (by a mean of 80%) via iGluRs compared to PrP-expressing counterparts. Similar effects were observed in cortical neurons [C regulates iGluRs, while the ways by which PrPC depresses NMDA-sensitive receptors are known  by PrPC [When iGluR-channels of CGN were individually activated by specific agonists [ cytosol . Indeed, neurons . As to h subunit ; modulat subunit , we foun by PrPC . In fact by PrPC , the res by PrPC , which d by PrPC .2+ uptake, we observed that only exposure of CGN to glutamate replicated exactly the effect of PrPC presence/absence on iGluRs, whereby a higher Ca2+ amount entered mitochondria of PrP-KO neurons [2+ entered mitochondria of PrP-expressing neurons. Another intriguing observation, collected by electron microscopy analysis of the two CGN populations, was that, on average, mitochondria of PrP-KO CGN were around 40% more detached from the PM (hence from iGluR-mediated Ca2+ influx) relative to PrP-expressing neurons. Thus, to understand which Ca2+ pool, among those exploited by mitochondria [2+ uptake from the mode of iGluR stimulation, we focused on inositol triphosphate (IP3)- and ryanodine-sensitive channels that discharge Ca2+ from the ER. These channels deliver Ca2+ directly into mitochondria by establishing contacts with the mitochondrial membranes [2+ release by activating IP3-sensitive channels [2+-induced Ca2+-release (CICR) [2+ accumulation observed in the two untreated-CGN types. These results led us to conclude that the much higher Ca2+ quantity flowing into glutamate-stimulated PrP-KO CGN compared to PrP-expressing counterparts potently activates CICR, and the subsequent mitochondrial Ca2+ uptake, in spite of the larger mitochondria-PM distance. Conversely, the much smaller Ca2+ amounts entering via NMDA- or AMPA-stimulated iGluRs [2+ by mitochondria of PrP-expressing neurons, which are positioned much closer to the PM iGluRs.Intriguingly, however, when testing mitochondrial Ca neurons . Conversembranes . After echannels , we conce (CICR) ,64, whicd iGluRs  could haC in controlling major post-synaptic Ca2+ entry routes, which PrPC accomplishes by modulating kinase activities . Furthermore, our data emphasize the capacity of PM-bound PrPC to finely tune Ca2+ transients in the cytosol and, most importantly, in the mitochondrial matrix by regulating CICR as in the case of CGN. In this way, location of PrPC to post-synaptic density [2+ overload in the various neuronal compartments and the detrimental consequences therein, e.g., glutamate excitotoxic insults threatening neuronal integrity. Concurrently, PrPC is likely to play a role in finely regulating key functions attributed to SOCE, which include neuronal excitability and gene expression [2+-dependent transcriptional factors as demonstrated for c-fos and nuclear factor of activated T cells [C primarily controls post-synaptic Ca2+ metabolism and, consequently, Ca2+-linked synaptic plasticity. To accomplish the works described above, our experimental approach implied the use of a PrP-KO model to enlighten, by comparison, PrPC physiology, and to mimic (to a certain extent) the consequences of compromised PrPC functions. Yet, it is well known that the chronic ablation of PrPC does not result in evident neuronal phenotypes, which would be expected from a compromised pre-/post-synaptic cross-talk consequent to the reduced glutamate exocytosis. This contradiction could, however, be only apparent, because the higher activity of iGluRs (and SOCE) in these cells likely compensates for the deficient glutamate release, thereby ensuring that an adequate Ca2+ signal arrives to post-synaptic PrP-KO neurons.Taken together, these observations highlight the central role played by PrP density ,65 stronpression ,67, such T cells . From al2+ influx, we tested whether 1 h-treatment with soluble oligomers of Aβ1-42 peptides  had any neurons , and lik neurons , for CGN1-42 influence on the resting cytosolic Ca2+ concentration  of Aβ-tn of Fyn . Alternauggested ,73. What by BTP2   iGluR sub-types fall under the same kind of control. Likewise, PrPC limits the activity of SOCE.In particular, we have discussed the novel finding that PrPd memory ,101, whoC spans the entire synaptic junction. Although employing different means , the action of PrPC at the two synaptic sides is fully coherent under the notion of PrPC neuroprotective attributes. On one hand, the protein guarantees that the refined molecular machinery deputed to glutamate secretion (and possibly other neurotransmitters) operates optimally. On the other hand, PrPC also acts as a safeguard against excessive neuronal Ca2+ rise at the post-synaptic level .These results clearly imply that the operational framework of PrPC physiologic functions are acutely challenged, the favorable control of the protein over the above pre-/post-synaptic events gets irremediably broken provoking synapse deterioration and loss, which are well established markers of neurodegenerative processes [2+  are als


The [Ca2+]i increase triggers a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming. However, the molecular mechanism of [Ca2+]i modulation controlled by the attractants is not well defined. Here, we examined receptive mechanisms for the sperm attractant, SAAF, in the ascidian, Ciona intestinalis, and identified a plasma membrane Ca2+-ATPase (PMCA) as a SAAF-binding protein. PMCA is localized in sperm flagella membranes and seems to interact with SAAF through basic amino acids located in the second and third extracellular loops. ATPase activity of PMCA was enhanced by SAAF, and PMCA inhibitors, 5(6)-Carboxyeosin diacetate and Caloxin 2A1, inhibited chemotactic behavior of the sperm. Furthermore, Caloxin 2A1 seemed to inhibit efflux of [Ca2+]i in the sperm, and SAAF seemed to competitively reduce the effect of Caloxin 2A1. On the other hand, chemotactic behavior of the sperm was disordered not only at low-Ca2+, but also at high-Ca2+ conditions. Thus, PMCA is a potent candidate for the SAAF receptor, and direct control of Ca2+ efflux via PMCA is a fundamental mechanism to mediate chemotactic behavior in the ascidian spermatozoa.When a spermatozoon shows chemotactic behavior, transient [Ca Even in spermatozoa, intracellular Ca2+ mediates critical events in the fertilization process, namely, activation of sperm motility2, sperm chemotaxis5, capacitation and hyperactivation of motility7 and the acrosome reaction9. Especially, Ca2+ regulates asymmetric property of flagellar beating10, and the concentration of intracellular Ca2+ is precisely controlled for regulating sperm direction.Ca2+ concentration ([Ca2+]i) is regulated by many Ca2+ transport systems in the plasma membrane such as the Ca2+ channels contributing to [Ca2+]i increase, and, Ca2+ exchangers/pumps leading to the [Ca2+]i decrease12. Among them the plasma membrane Ca2+-ATPase (PMCA), which belongs to the P-type ATPase family, seems to act as a fine tuner of Ca2+ efflux13 and maintains Ca2+ homeostasis in different kinds of mammalian cells14. PMCA also plays a role in modulating the signal transduction pathways. Activity of PMCA seems to be regulated by proteins from the membrane-associated guanylate kinase (MAGUK) family, such as Ca2+/calmodulin-activated serine/threonine kinase (CASK)16. Furthermore, PMCA4 deficiency is known to cause reduced sperm motility in mice17.The intracellular Ca2+]i18. In the sperm of the ascidian, Ciona intestinalis, the sperm-activating and attracting factor (SAAF), which is a sulfate-conjugated hydroxysteroid21, mediates both activation of sperm motility and chemotaxis22. The phenomena requires extracellular Ca2+ 24. To activate the sperm and make it motile, SAAF activates the voltage-dependent Ca2+ channel-driven Ca2+ influx and the calmodulin/calmodulin kinase II pathway25. With respect to chemotaxis, the asymmetric waveform of the sperm flagellum, which is important for chemotactic response of the sperm, is mediated by the Ca2+ channels23. Moreover, we have revealed that transient [Ca2+]i increases in the spermatozoon (Ca2+ bursts) and is induced in a SAAF gradient, triggering a series of stereotypic responses of flagellar waveforms that comprise turning and straight-swimming24.The chemotactic behavior of a sperm towards an egg plays an important role in ensuring fertilization. It is a widespread phenomenon that occurs in most life forms from lower plants to mammals. This behavior is also mediated by i modulation by attractants still remain unknown. In the case of the sea urchin and starfish, cGMP produced by the receptor guanylyl cyclase opens the K+-selective cGMP-gated channel, resulting in hyperpolarization29. This seems to activate the Na+/H+ exchanger and leads to alkalization, resulting in Ca2+ increase via the alkalization-gated channel CatSper30. The involvement of cGMP in regulating sperm function is not known in species other than the echinoderm. In the mammalian sperm, CatSper seems to act as a chemoattractant receptor32. In the ascidian, cGMP does not seem to be involved in chemotactic behavior3, and the SAAF receptor may not be the guanylyl cyclase. Thus, identification of the SAAF receptor is required to understand Ca2+ signaling and the molecular mechanisms of ascidian sperm chemotaxis. Furthermore, in the case of any other species, Ca2+ influx and [Ca2+]i increases in the sperm cell are focused in chemotactic behavior, and Ca2+ efflux and [Ca2+]i decreases are scarcely examined; despite the need for prompt [Ca2+]i decrease, it has been observed only in sperm activation of the sea urchin33.In contrast to the knowledge on sperm attractants and the participation of CaC. intestinalis. We show here that the PMCA located on the sperm tail binds to the SAAF and mediates chemotactic behavior in the ascidian. Furthermore, modification of the extracellular Ca2+ concentration ([Ca2+]ex) disrupted the Ca2+ bursts and the sperm chemotactic behavior. Thus, PMCA is a potential candidate for the SAAF receptor on the sperm. Regulation of the flagellar waveform in chemotactic behavior requires precise control of the Ca2+ efflux.In this study, we attempted to identify the SAAF receptor on the sperm of the ascidian, C. intestinalis, we attempted to discover SAAF-binding proteins. The SAAF-binding proteins were purified from the sperm membrane fraction by the pull-down assay using a resin conjugated with bio-SAAF, which is the biotinylated derivative of the C(4)-hydroxy group 34. The most abundant SAAF-binding protein, the 370-kDa protein   in C. intestinalis (KH.C8.156), which seems to diverge from the common ancestral gene of Atp2b1-4  was expressed ubiquitously, and the other one (Atp2b-var.b) was highly expressed in the testis and weakly expressed in the stomach, intestine, and heart  method. PMCA interacts with SAAF and the dissociation constant (KD) of the interaction between SAAF and Atp2b-var.b was calculated as 240 ± 50 nM 35. Using this program, the membrane-spanning regions and the orientation of the protein were predicted -Carboxyeosin diacetate (CEDA), an inhibitor of PMCA via ATPase activity, most of the sperm lost their chemotactic behavior toward the capillary tip  was increased by Caloxin 2A1, 70%  and 50%  of the sperm still showed the same [Ca2+]i baseline as the control (<1.5) , changes in the [Ca2+]i could not be observed. Interestingly, the chemotactic behavior also decreased when the concentration of [Ca2+]ex was higher than 10 mM , but the timing of the response was disrupted , the Ca2+ bursts occur at the Distal Phase24  with many splice variants in mammalian and probably in most vertebrate species  and mediates activity of CatSper by depletion of 2-arachidonoylglycerol46. Thus, the Ca2+ channel CatSper seems to be linked with the receptor of the sperm attractants, and the [Ca2+]i increase which is induced by the attractants controls chemotactic behavior of the sperm in these species. On the other hand, the present study shows that PMCA acts as the attractant receptor, and the Ca2+ efflux induced by the attractant, mediates sperm chemotaxis in the ascidian. These models are contradictory, but our ascidian model fits our previous results. Addition of SAAF does not induce Ca2+ increase, and Ciona sperm seems to sense decreases in SAAF concentration, thus resulting in Ca2+ bursts47.The receptor of sperm attractants has been described in echinoderm species. Specifically, the receptor for sperm attractants in the sea urchin is a transmembrane-type guanylyl cyclase2+ bursts and chemotactic responses were observed only in the presence of 10 mM Ca2+. At low [Ca2+]ex, no [Ca2+]i change was observed and the chemotactic response was reduced. At high [Ca2+]ex, the [Ca2+]i increases were potentiated and the chemotactic response was disrupted 51, BAPTA (160 to 700 nM)52, fluo-4 (345 nM)53, etc. We previously estimated that the SAAF gradient around the capillary containing 1 µM SAAF ranges from 10 to 200 nM24. That is, the affinity between SAAF and PMCA is suitable for sensing the concentration of the sperm attractant SAAF. Probably, echinoderms and ascidians are using a completely different basic molecular design for sperm chemotaxis even though the regulation of flagellar beating by [Ca2+]i is the same. These different results may provide new insights into the specificity and diversity of fertilization mechanisms.To examine the molecular mechanisms of sperm attraction, evaluating the binding affinity between the sperm attractant and its receptor is important. In the sea urchin, the binding affinity of the sperm attractant and its receptor seems to be very high. The K2+ efflux on the plasma membrane, exists on the ascidian sperm flagella and contributes towards the control of transient [Ca2+]i in chemotactic behavior. Inhibition of NCX decreased the alteration of swimming-path curvature in the chemotactic behavior and diminished turning and straight-swimming11. Thus, NCX and PMCA seem to cooperate to maintain [Ca2+]i in the ascidian sperm. On the other hand, the capacitative Ca2+ entry, which is mediated by the store-operated Ca2+ channel, seems to mediate flagellar movements to establish the chemotactic behavior of the ascidian sperm23. The capacitative Ca2+ entry is induced by depletion of internal Ca2+ stores and lasts for a few minutes. Here, we propose a new working hypothesis showing the molecular mechanism of sperm chemotaxis in the ascidian C. intestinalis . When SAAF concentration decreases, SAAF may detach from PMCA and become inactivated, resulting in [Ca2+]i bursts by the capacitative Ca2+ entry (descending phase). Intracellular Ca2+ is excreted by NCX and PMCA. Finally [Ca2+]i reaches low levels (refractory phase). It is still unknown whether Orai1 and Stim1, which compose the store-operated Ca2+ channels, are involved in the sperm chemotaxis system. On the other hand, CatSper, which is the Ca2+ channel having an important role in mammalian sperm, also exists in the ascidian54. Function of CatSper in the ascidian sperm is still unknown. CatSper might act as the Ca2+ channel in the hypothesis instead of the store-operated Ca2+ channel. Furthermore, difference of amino-acid sequences between the two splice variants of PMCA was only found in the C-terminus region  was obtained from the National BioResource Project for Ciona (http://marinebio.nbrp.jp/). Sperm of the ascidian was obtained as described previously22. Artificial seawater (ASW) contained 462 mM NaCl, 9 mM KCl, 10 mM CaCl2, 48 mM MgCl2, and 10 mM HEPES-NaOH (pH 8.2). High and low Ca2+-ASW was prepared as the ASW but with 0.01, 0.1, 1, 50 or 100 mM CaCl2 and 5 mM EGTA. EGTA, Fluo-4 AM, Cremophor EL and 5(6)-Carboxyeosin diacetate succinimidyl ester (CEDA) were purchased from Thermo Fisher Scientific ; Caloxin 2A1 was purchased from Peptide Institute Inc. ; Fluo-8H was purchased from Nacalai Tesque, Inc. . SAAF and its derivatives were synthesized as described previously55.The ascidian Bio-SAAF was immobilized on streptavidin-coated beads by using the affinity of biotin for streptavidin . Binding capacity of the beads was 30 nmol of biotin/mL. In general, 150 nmol of bio-SAAF was added to 10 mL of 50% slurry of streptavidin-coated beads. The beads were previously obtained by washing with 10 mL of PBS three times and incubating for 30 min at room temperature on a rotary mixer at low speed. The unbound material was carefully removed, and the beads were washed with 10 mL of PBS three times and immediately used or stored at 4 °C. For simplicity, the SAAF bound to the beads is referred to as the SAAF-resin and the beads bound with biotin as the control resin.https://www.ncbi.nlm.nih.gov/protein/). Sequence alignments and phylogenetic analysis for the collected sequence data were carried out by GENETYX  with ClustalW . Accession numbers of the used sequences: Human ATP2B1 isoform 1a [Homo sapiens], NP_001001323.1; Human ATP2B2 isoform 1 [Homo sapiens], NP_001001331.1; Human ATP2B3 isoform 3b [Homo sapiens], NP_001001344.1; Human ATP2B4 isoform 4a [Homo sapiens], NP_001001396.1; Mouse Atp2b1 [Mus musculus], NP_080758.1; Mouse Atp2b1 isoform 1 [Mus musculus], NP_001031761.1; Mouse Atp2b1 isoform 2 [Mus musculus], NP_001297466.1; Mouse Atp2b1 isoform a [Mus musculus], NP_001161421.1; Rat Atp2B3 [Rattus norvegicus], NP_579822.1; Chicken ATP2B1 [Gallus gallus], NP_001161474.1; Crow Atp2B1 isoform X1 [Corvus cornix cornix], XP_019135366.1; Lizard predicted Atp2b4 isoform X1 [Anolis carolinensis], XP_008107875.1; Bearded dragon Atp2b1 isoform X2 [Pogona vitticeps], XP_020633124.1; Xenopus predicted ATP2B1 isoform X1 [Xenopus tropicalis], XP_017947410.1; Xenopus Atp2b3 [Xenopus laevis], NP_001087020.1; Goby Atp2b1 [Boleophthalmus pectinirostris], XP_020796624.1; Medaka fish Atp2b1 isoform X1 [Oryzias latipes], XP_020569770.1; Zebrafish Atp2b2 [Danio rerio], NP_001116710.1; Coelacanth predicted Atp2b3 isoform X1 [Latimeria chalumnae], XP_006002568.1; Shark Atp2b1 isoform X1 [Rhincodon typus], XP_020391438.1; Amphioxus predicted PMCA2-like isoform X1 [Branchiostoma belcheri], XP_019624340.1; Sea Urchin sperm PMCA [Strongylocentrotus purpuratus], NP_001028822.1; Drosophila PMCA isoform Q [Drosophila melanogaster], NP_001188516.1.Protein sequences of the PMCA family were obtained from the NCBI database  using N2 cavitation . Cell lysates were centrifuged at 13,200 g for 10 min at 4 °C to obtain the post-nuclear supernatant. The post-nuclear supernatant was centrifuged at 543,000 g for 1 hr at 4 °C to obtain the membrane fractions (pellet). The membrane fractions were resuspended in PBS containing 1% NP-40, sonicated, and then used for SAAF pull-down assay, molecular interaction assays, and for ATPase activity assays.Sperm or ® Ruby (Thermo Fisher Scientific) according to the manufacturers’ instructions. Protein bands were excised and subjected to for MALDI-TOF/MS as described previously56. Briefly, gels were dehydrated and then re-hydrated in 20 ng/μL trypsin or lysyl endopeptidase in 100 mM NH4HCO3 for 12 h at 37 °C. Peptides were subjected to MALDI-TOF/MS and analyzed by Autoflex II system . Proteins were identified by peptide mass fingerprinting (PMF) using MASCOT search with the lab-made protein databases of C. intestinalis56.The SAAF-resin was mixed with the membrane fractions at 4 °C for 1 h with rotation, washed with three bed volumes of PBS, and eluted with the elution buffer . The membrane fractions (input), the washes, and the elutes were collected in NuPAGE LDS sample buffer and resolved on NuPAGE SDS–PAGE gel . The resulting gel was stained with SYPROAtp2b were amplified from RNA, which was isolated from the C. intestinalis testis by reverse transcription-polymerase chain reaction (RT-PCR) using the following primers: forward, 5′-TAGCGTTACTGCTGCT-3′, and reverse, 5′-TGCCAGCCATGATGTTTTCAGAG-3′. The PCR products were subcloned into the pCR®8 TOPO® TA vector (Thermo Fisher Scientific). The construct was controlled by sequencing. Site-directed mutagenesis was carried out to obtain the mutant clone in the appropriate vector. Atp2b-var.b was used as the target, and experiments were performed according to the manufacturers’ standard protocol . The following primers were used: mutation1 (W395A_R396A_K409A) 5′-GGT TTT ACA gcc gcg AGC GAA TGC ACT TCA GTT TAC ATC C-3′ (forward) and 5′-AT AAT GAA GAA cgc GAC AAA ATA TTG GAT GTA AAC TGA AG-3′ (reverse), mutation2 (R869A_K874A_W880A) 5′-C GCC TGC ATC CTC gcg GAC AGT CCC CTG gcc GCA GTC CAA-3′ (forward) and 5′-C CAT GAT AAG ATT CAC cgc TAA CAT TTG GAC TGC cgc CAG-3′ (reverse), mutation3 (R951A_H956A_H962A) 5′-GAC ATC GAC AGC GGA gcc GGG CTC AGT CTT gcc TCA GCT C-3′ (forward), and 5′-AA GAC GAT GGT GAA cgc CTG GGT GGG AGC TGA ggc AAG AC-3′ (reverse). The nucleotides that determine the mutation are indicated in lowercase letters. Baculoviruses expressing wild-type and mutant Atp2b-var.b were generated using the BaculoDirect Baculovirus Expression System (Thermo Fisher Scientific) according to the manufacturers’ instructions.The cDNAs of 18 primer and random hexamer primer. The 50-µl reaction mixture contained 32 µl nuclease-free water, 1.0 µl cDNA, 1.5 µl (10 µmol/l) each primer, 5 µl (2 mM) dNTPs, 3 µl (25 mM) MgSO4, 5 µl (10×) KOD plus ver.2 buffer and 1 µl KOD plus . The primers were used for detecting the 20th and 22nd exons: 5′-CCC ATC TTT TGC ACA ATC CT-3′ (forward) and 5′-TTC TTC TTC CTC GTC GTC GT-3′ (reverse), for detecting the 20th and 21st exons: 5′-GGC ACG CGT TCT ATC AAC TT-3′ (forward) and 5′-TTT ACT GCA GCA GCA GAT GG-3′ (reverse) were used. For the household gene, glyceraldehyde-3-phosphate dehydrogenase (Gapdh): 5′-ACC CAG AAG ACA GTG GAT GG -3′ (forward) and 5′- CAG GAC ACC AGC TTC ACA AA -3′ (reverse). The amplification cycle started with denaturation at 94 °C for 2 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, and 68 °C for 1 min. PCR products were analyzed on 2% (wt/vol) agarose gels stained with 0.5 µg/ml ethidium bromide and were visualized under UV light. Images of the gels were captured by using a gel imaging device . Acquired images were processed by Adobe Photoshop and Adobe Illustrator .Total RNA was extracted from each tissue using RNeasy Mini Kit  and the concentration of RNA was determined by absorbance at 260 nm in relation to absorbance at 280 nm. One microgram of total RNA was reverse transcribed to cDNA by Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics) using anchored-oligo(dT)Sf9 cells, these solubilized samples were treated with Benzonase Nuclease . Proteins were separated by an SDS-PAGE (Laemmli) or by NuPAGE SDS-PAGE Gel System using 3–8% Tris-Acetate Gels  and transferred to PVDF membranes. The anti-pan PMCA antibody [5F10], , horse radish peroxidase (HRP)-conjugated anti-mouse IgG , and ECL Prime  were used to detect the PMCA. To detect C. intestinalis Atp2b-var.a or Atp2b-var.b, polyclonal antibodies specific to each variant were developed using rabbits . The epitope sequences for Atp2b-var.a and Atp2b-var.b were ASEENSTENNQTQNSVA and ARHHSSHHAHLEPV, respectively. For detecting the anti-Atp2b-var.a and Atp2b-var.b antibodies, HRP-conjugated anti-rabbit IgG , and ECL Prime (GE) were used. Luminescence was detected using an X-ray film  and images were acquired using a scanner GT-X770   or the NuPAGE LDS sample buffer . In the case of spermatozoa or an) Fig.  or imagean) Fig.  and 3. A57. Acquired data was processed by Adobe Photoshop and Adobe Illustrator (Adobe Systems).For immunostaining, the sperm suspension was put onto a coverslip and fixed with 4% paraformaldehyde for 10 min, after which it was washed twice with ASW for 5 min. The fixed sperm was permeabilized with 0.1% NP-40 for 15 min, blocked with 1% BSA for 1 h and incubated with 5 µg/mL 5F10 or x1/500 anti-Atp2b-var.b antibody with 1% BSA in PBS for 45 min. The sperm was washed 3 times with PB and incubated with x1/2000 Alexa 488-conjugated anti-mouse IgG(H + L) (ThermoThermo Fisher Scientific) or x1/2000 DyLight550-conjugated anti-rabbit IgG (Abcam) for 30 min, respectively. After washing two times with PBS, the sperm was observed using a fluorescent microscope . Images were recorded on a PC connected to a digital CCD camera  using an imaging application (TI workbench)Sf9 membranes (5 mg/mL of proteins) suspended in PBS (pH 7.4) containing 0.1% of NP-40. The second channel of the same sensor chip was coated with Sf9 membranes (5 mg/mL of proteins), as a reference. The sensor chip was washed three times with PBS, placed into the chamber, perfused with PBS until the frequency was stabilized. Then, the sensor chip was perfused with SAAF (125 µM) dissolved in PBS, and the change in frequency was recorded. In order to examine SAAF and mutant PMCA interactions, the first channel of a sensor chip was coated with mutant PMCA-expressing Sf9 membranes. The second channel on the same sensor chip was coated with PMCA-expressing Sf9 membranes, as a reference. After washing, perfusion, and blocking the sensor chip, it was perfused with SAAF, and the change in frequency was recorded. All experiments were performed at 20 °C, with a flow rate of 50 μL/min. To evaluate the binding affinity, changes in the frequency of cumulative perfusion were analyzed. Affinities were evaluated by the Michaelis-Menten equation, and fitted curves were obtained by calculating the average of three experimental values. The dissociation constant (KD) was calculated with NAPiCOS Analysis software (Nihon Dempa Kogyo).Interaction between SAAF and PMCA was investigated by a highly sensitive 30-MHz quartz crystal microbalance . The first channel of a sensor chip was coated with PMCA-expressing Ciona sperm and Sf9 membranes was determined by measuring the inorganic phosphate produced using a coupled enzyme assay kit  according to the manufacturers’ instructions. In brief, membranes were incubated for 5 min at 25 °C in a reaction mixture containing 50 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 0.2 mM 2-Amino-6-mercapto-7-methylpurine riboside (MESG) as a substrate, 1 U/mL purine nucleoside phosphorylase (PNP) as a converting enzyme, and 100 µM CaCl2. The enzymatic reaction was initiated by addition of 1 mM ATP. ATPase activity was calculated based on the difference in ATP hydrolysis between samples incubated in the presence and absence of 10 µM SAAF or bio-SAAF. Enzymatic conversion of MESG by PNP was measured for 5 min at room temperature at a wavelength of 360 nm in a recording spectrophotometer .Enzyme activity of PMCA from 24. Briefly, semen was diluted 104 to 105 times in the medium for experiment . Theophylline  was added to the suspension in a final concentration of 1 mM for the induction of motility activation which is mediated by an increase in intracellular cAMP3, except in a series of Caloxin 2A1 experiments. Series of Caloxin 2A1 experiments including control were performed with the sperm suspension not containing theophylline, since the Caloxin 2A1-treated theophylline-activated sperm completely adhered to the glass slide and there was no free-swimming sperm. The activated-sperm suspension was placed in the observation chamber, and sperm movement around the micropipette tip containing SAAF was recorded. The position of the sperm head was analyzed with Bohboh software 58. The parameters of chemotactic activity  were calculated as described previously19.Sperm chemotaxis was examined as described previously2+ imaging, Fluo-8H AM , and captured on a PC connected to a digital CCD camera  at 32.5 frames/sec using Aquacosmos (Hamamatsu Photonics), or a digital CCD camera  at 50 frames/sec using an imaging application (TI workbench57), as described previously59. For fluorescence illumination, a stroboscopic lighting system with a power LED was used as described24. Fluorescent signal intensity and sperm flagellar bending were also analyzed using the Bohboh software58.For Ca AM Fig.  or Fluo-t-test; P < 0.05 was considered significant.All experiments were repeated at least three times with different specimens. Data is expressed as the mean ± SD. Statistical significance against control Figs  and 7, oSupplemental Figures and Table


The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca The heart is a highly specialized machine that requires fine regulation: intracellular changes and cell-to-cell communication disturbances could destroy this balance. Depending on the type of cardiac arrhythmia, a series of events may occur: intracellular dysfunction‒tissue remodeling‒impaired cell‒cell connection–electrical dysfunction. Loss of cardiac tissue integrity could be both a cause and an effect of arrhythmic phenotype. A typical example is arrhythmogenic cardiomyopathy (ACM), a cardiac disorder in which mutations in proteins of the desmosome may be present, causing cell-to-cell discontinuity, arrhythmic events, and myocardial fibro-adipose substitution ,2,3.2+) signals drive excitation‒contraction (EC) coupling and are, therefore, necessary for the heart to effectively pump blood into the pulmonary artery and aorta. It is, therefore, not surprising that any defect in the Ca2+ cycling machinery or in the complex network of Ca2+-related proteins that maintain cardiac Ca2+ homeostasis under tight control may severely compromise cardiac function. Alterations or mutations in the Ca2+ handling machinery have been associated with a number of severe cardiac diseases, including cardiac hypertrophy, dilated cardiomyopathy, and heart failure i) induced by membrane depolarization according to a process known as EC coupling i, which activates a cluster (7–20) of type 2 Ryanodine receptors (RYR2) in a process known as Ca2+-induced Ca2+ release (CICR) and gives rise to significant Ca2+ release events from the junctional SR . The temporal summation of these local events by the propagating action potential results in a regenerative Ca2+ transient that is detected by troponin C to initiate the sliding of the thick (myosin) and thin (actin) filaments, cell shortening, and hence pressure development within each ventricle and ejection of blood into the circulation during cardiac systole [2+]i drops and mechanical force relaxes quickly to pre-systolic levels during cardiac diastole, which is essential to enable cardiac chambers to refill with blood. The increase in [Ca2+]i is indeed short-lived as cytosolic Ca2+ is extruded across the plasma membrane by the Na+/Ca2+ exchanger (NCX1) and the plasmalemmal Ca2+-ATPase (PMCA) and sequestered back into the SR by the sarco-endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) i and DADs in Timothy syndrome i transient amplitudes, lower SR Ca2+ stores, and elevated diastolic [Ca2+]i, consistent with the presence of a CPVT-related SR Ca2+ leak . It. ItJUP greported . Conversse model .PKP2, encoding for plakophilin-2, is the most frequently mutated gene in ACM patients [patients . It has patients , idiopatpatients . PKP2 ispatients . The maipatients . Differepatients .Pkp2 homozygous knockout (Pkp2-cKO) mouse model correlated the lack of PKP2 with transcriptional alteration of Ca2+ homeostasis [2+ concentration were downregulated in Pkp2-cKO hearts. The low level of transcripts relevant to Ca2+ cycling, i.e., Ryr2, Ank2, Cacna1c, and Trdn, accompanied a decreased expression of corresponding proteins and the impairment of the EC mechanism [Ank2) and triadin (encoded by Trdn), which contribute to maintaining the structural integrity of dyadic junctions [Pkp2-cKO-derived cardiomyocytes showed decreased ICa,L density and a slower rate of current inactivation, which is consistent with the reduced expression of Cacna1c [2+ current was decreased, the total Ca2+ charge  was unaltered as compared to wild-type cardiomyocytes because of the slower inactivation rate [2+ leak due to the downregulation of both Ryr2 and Casq2 [2+ content was remarkably increased in Pkp2-cKO cardiomyocytes, which exhibited an increase in the amplitude and frequency of spontaneous Ca2+ release events  and were therefore more prone to release SR Ca2+ during the EC coupling [Pkp2-cKO cardiomyocytes were paced at increasing rates, they displayed both early and delayed after-transients, which were sufficient to generate ventricular arrhythmogenic events during β-adrenergic stimulation with isoproterenol [2+ handling machinery in RV-derived PKP2-cKO (Pkp2-cKO-RV) cardiomyocytes isolated 14 days after tamoxifen injection [2+ release and RyR2-mediated Ca2+ sparks due to the remarkable elevation in the SR Ca2+ load that was caused by a Cx43-dependent increase in membrane permeability [2+ influx [2+ store in a SERCA2A-dependent manner. In addition, RyR2’s eagerness to release Ca2+ may be boosted by the observed phosphorylation in Thr2809, an amino acid residue near the consensus sequence for CaMKII and PKA [2+ handling machinery precedes overt ultrastructural alterations and manifestations of ACM.Data from a cardiomyocyte-specific tamoxifen-activated eostasis . This inechanism . Accordiunctions ,100, sigunctions . In addi Cacna1c . It shouion rate . Notablynd Casq2   and a risk of sudden death. This condition was first described in 1988 and classified as arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2): a clinically different form because of its particular effort-induced ventricular arrhythmias, high penetrance, and 1:1 male: female ratio among the affected subjects [RYR2 were detected in ARVD2 patients [RYR2 mutations observed in ARVD2 patients enhance Ca2+ leakage and increase spontaneous SR Ca2+ release, thereby leading to DADs and ventricular arrhythmia [2+ release events in both paced and non-paced isolated cardiomyocytes [2+-induced molecular rearrangement of RYR2, thereby increasing the fractional Ca2+ release [2+ overload and stimulate apoptosis [The  beating ,110,111.subjects . Four mipatients  resultinpatients . The mutpatients . A functpatients . It is, rhythmia . In agremyocytes . Notably release . Besidespoptosis , which hpoptosis .RYR2, mapping the locus to chromosome 1q42‒q43 [RYR2 domains [2+ channel have been associated so far with CPVT or sudden death [Six further rare missense variants have been identified in 1q42‒q43 ,51 in fo domains . Several domains ,121,122.en death .RYR2 gene, causing non-conservative amino acid exchanges, i.e., G1885E and G1886S, have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) [RYR2 mutations have been detected, are responsible for creating a putative protein kinase C phosphorylation site known to be involved in cardiac contractility and propensity to heart failure [RYR2 activity revealed that the combination of these polymorphisms in a single individual led to a leaky SR channel under diastolic conditions, thereby triggering arrhythmia and sudden cardiac death.Two common single nucleotide polymorphisms in exon 37 of the human y (ARVC) . These p failure . Genetic2+ cycling and an effector of β-adrenergic stimulation. PLN is sensitive to PKA-dependent phosphorylation and PLN expression levels and its phosphorylation state is a key to fine-tuning SERCA activity. The de-phosphorylated PLN interacts with SERCA, resulting in a low Ca2+ affinity state that limits Ca2+ sequestration into SR lumen; when it is phosphorylated, the inhibitory activity on SERCA is abolished and the affinity of SERCA to Ca2+ is restored, so that a larger amount of Ca2+ is pumped back into the SR [2+ content and the major determinant of cardiac contractility and relaxation, depending on its phosphorylation levels: increased phosphorylation improves contractility; Vice versa, the de-phosphorylation state promotes heart failure [PLN is one of the main regulators of Cao the SR .Therefor failure .PLN gene were firstly associated with dilated cardiomyopathy (DCM) [PLNArg14Del mutation. This is a deletion of Arg-14 in the coding region and has been identified in a family in which heterozygous carriers exhibited inherited DCM and died by middle age. Notably, a mouse model with cardiac-specific expression of the heterozygous PLNArg14Del mutant recapitulates the human phenotype, such as depressed cardiac function, fibrosis, and premature death [PLNArg14Del mutation prevents the phosphorylated PLN from relieving SERCA inhibition, thereby causing a reduction in SR Ca2+ concentration [PLNArg14Del mutant in HEK-293 cells, Ca2+ uptake into microsomal vesicles was severely impaired [2+ transport in cardiac homogenates isolated from transgenic mice expressing the PLNArg14Del mutation was impaired as compared to wild-type samples [PLNArg14Del mutation further revealed that plakoglobin is downregulated at cell junctions, mostly in ACM samples [PLN total mRNA, total and phosphorylated protein levels are significantly increased compared to healthy controls and patients with DCM [Mutations in the human hy (DCM)  Patientshy (DCM) , as for re death . At molentration . Accordiimpaired . Likewis samples . Immunoh samples . Moreovewith DCM . This fiwith DCM .PLNArg14Del mutation is likely to result in reduced SR Ca2+ content. In agreement with this hypothesis, a recent investigation revealed that the intracellular Ca2+ dynamics were perturbed in human iPSC-CM deriving from a patient affected by DCM and bearing the PLNArg14Del mutation [2+ pool was significantly reduced, there was an increase in the diastolic Ca2+ levels, beating rate, and frequency of irregular Ca2+ waves [PLNArg14Del mutant was introduced into a PLN null mouse model, it did not associate with SERCA2a, but translocated to the plasma membrane to physically interact with the Na+/K+ ATPase [PLNArg14Del mutation and ACM could be better interpreted by considering another DCM-associated PLN mutation, i.e., R25C [2+ cycling machinery. Indeed, when R25C-PLN was overexpressed in rat adult cardiomyocytes, it suppressed SERCA2a activity, thereby reducing SR Ca2+ content and the amplitude of voltage-dependent Ca2+ transients, with impairment of contractile activity following [2+ levels was found to recruit CaMKII, which in turn phosphorylated RYR2 to induce an increase in SR Ca2+ leakage, spontaneous Ca2+ sparks, and resultant arrhythmias [PLN mutation carriers more often showed T-wave inversion and are associated with additional left ventricle (LV) involvement and low-voltage electrocardiogram. However, as reported for desmosomal mutation carriers, PLN mutation carriers also showed RV disease, fibrofatty replacement, and arrhythmogenic phenotype [The pathological mechanisms of the PLN mutation underlying ACM are still unclear, but, in general, the mutation . Indeed,2+ waves . Interes+ ATPase . As a co+ ATPase . The caue., R25C . This muollowing . The reshythmias . Comparehenotype ,131. In henotype .2+ dysregulation can be considered as a possible causative event based on its involvement in the cells’ adhesive property and its pro-arrhythmic ability. The desmosomes’ adhesive state is Ca2+-dependent and their structural stability consists of a PKC-mediated mechanism. Moreover, defects in the Ca2+ cycling machinery, also identified among the ACM-causative genetic mutations, are responsible for dysregulated Ca2+ homeostasis, leading to cardiac arrhythmia. Of note, a loss of PKP2, most frequently mutated in ACM patients, correlates with an alteration of Ca2+ machinery [2+ handling machinery, not only limited to myocytes, ultimately leading to arrhythmias, but also in the stromal compartment, which is actively involved in the pathological remodeling [ACM is classified as a genetically determined cardiac disease that is clinically characterized by arrhythmic events. Different pathways have been associated with ACM and were mostly correlated with enhanced adipogenesis and electrical dysfunction. Despite the identification of different molecular mechanisms, a full understanding of the ACM pathogenesis is still lacking. Caachinery . These omodeling .2+ homeostasis, such as calcium antagonists and flecainide, are used to treat arrhythmias. However, confirming the involvement of Ca2+ signaling and understanding the molecular underpinnings of Ca2+ signaling in the pathogenesis of ACM could be pivotal to finding targeted therapies (cell-specific and/or molecule-specific) that could be beneficial for the treatment of this life-threatening disease.To date, pharmacological treatments generically targeting Ca