after partial hepatic destruction in experimental or clinical context , the initial mass of the organ is restored through compensatory growth of the remnant liver .
a complex and yet incompletely elucidated network of cellular interactions ( including paracrine , autocrine , endocrine , or nervous pathways ) orchestrates the regulation of regeneration , through both proliferative and hepatoprotective signalling cascades .
a number of agonists constituting this network drive intracellular ca movements , in particular through the formation of inositol 1,4,5 trisphosphate which binds on its receptor in the membrane of the endoplasmic reticulum , and release the ca stored in this organelle .
such agonists include some of the main comitogenic  as noradrenalin , arginine vasopressin ( avp ) , and adenosine triphosphate ( atp ) and mitogenic factors , as epidermal growth factor ( egf ) , hepatocyte growth factor ( hgf ) , and insulin .
the resulting increase in ionized cytosolic calcium concentration generally consists of a regular succession of ca peaks ( oscillations )   that can be transmitted to other cells ( intercellular calcium waves ) which mechanisms and functions are not fully known [ 8 , 9 ] .
the impact of calcium signalling on liver regeneration has , however , only been scarcely studied .
the first calcium oscillations were reported in hepatocytes , and numerous studies , both experimental and theoretical , have been conducted after that to decipher , in the hepatocyte , the machinery by which agonists generated cytosolic calcium signals .
however , the functions of hepatocyte calcium signalling remain far less understood . in hepatocytes , as in most nonexcitable cells , ca oscillations originate from the periodic opening of ca channels located in the er membrane , following activation of the phosphoinositide cascade .
the binding of an agonist to a membrane - bound receptor activates the g-subunit of a g - protein complex coupled to the receptor .
the latter enzyme catalyzes the hydrolysis of the membrane - bound phosphatidyl - inositol bisphosphate ( pip2 ) into diacyl - glycerol and inositol trisphosphate ( insp3 ) .
ca release from the internal stores is ensured by the insp3r , an homotetramer that can bind up to 4 insp3 molecules , forming a ca channel which equilibrium open probability presents a bell - shaped dependence on cytosolic ca .
the decrease of [ ca]i in the cytosol is due to the activity of the ca atpases ( serca pumps ) , which actively transports ca from the cytosol into the er . ca - regulated insp3rs and ca atpases are together sufficient to generate ca oscillations . in most cases , hormone - induced ca oscillations in hepatocytes take the form of repetitive , sharp spikes sometimes preceded by a slower , pacemaker - like elevation in the cytosolic ca concentration .
these periodic increases in the level of free ca in the cytosol from about 0.1  m up to 1  m have been observed in hepatocytes in response to stimulation by a large number of agonists such as noradrenalin , vasopressin , phenylephrin , angiotensin ii , adenosine triphosphate ( atp ) , histamine , and thrombin , the shape of the oscillations being agonist dependent .
the oscillation frequency increases with the agonist concentration , a phenomenon known as  frequency encoding  , and is affected by external [ ca]and thus by the rate of ca entry into the cell through plasma membrane ca channels .
intracellular ca waves do not result from a simple diffusion of ca itself  which is quickly buffered  but from the spreading of insp3which is more soluble in the cytosol  that mobilizes ca from storage compartments throughout the cell . a particular spatial pattern of subcellular insp3r distribution was reported to support the direction of intracellular ca waves , starting from the canalicular region containing the most abundant and affine insp3r isoform ( type ii insp3r ) and spreading toward the other regions of the cytosol , less sensitive to insp3 . as in many other cell types ,
intracellular movements of ca in hepatocytes , induced by hormones and neurotransmitters , may be propagated from cell to cell .
our group demonstrated in multicellular rat hepatocyte systems ( couplets and triplets ) that agonists such as vasopressin or noradrenalin induce tightly coordinated and sequentially ordered intracellular ca increases [ 8 , 1416 ] .
such signals were also observed in the intact perfused liver in which vasopressin elicits waves of [ ca]i increase running along hepatocyte plates across the lobules [ 1719 ] .
we demonstrated that unidirectional ca waves resulted from a gradually decreasing cellular sensitivity to hormonal stimuli from the first to the last responding cell , and that this cell to cell heterogeneity was due to a lobular gradual distribution of hormonal receptors density . moreover , insp3 has been shown to flow through gap junctions and thereby coordinate ca spiking among adjacent hepatocytes .
such a configuration in which the most responsive hepatocytes drive the response of the less sensitive cells is similar to the cell to cell triggering of cardiac pacemaker cells [ 8 , 21 , 22 ] . in general terms , ca oscillations in hepatocytes optimize the effect of hormonal stimulation , thanks to enzymes decoding   their frequency [ 24 , 25 ] , as proposed for glycogen metabolism in hepatocytes . since the early data showing that production of glucose by the liver was at least in part mediated by hormone - induced intracellular ca increases , most recent studies have deciphered the molecular mechanisms linking intracellular ca to glucose metabolism in hepatocytes [ 2830 ] . in particular , the serine - threonine kinase  calcium calmodulin - dependent kinase ii  ( camkii ) , a major mediator of ca signalling in different cell types , has been found to play essential roles in the regulation of glycogenolysis and gluconeogenesis in hepatocytes , not only during physiological fasting , but also in the pathophysiological setting of obesity [ 28 , 30 ] .
it is moreover well established that ca oscillations in hepatocytes coordinate intramitochondrial atp synthesis with cellular energy demand , maintaining cell homeostasis and viability .
it has also been demonstrated that   the temporal pattern of calcium signals was of major impact as to the expression of transcription factors in lymphocytes , but this aspect has never been investigated in hepatocytes .
many events related to bile secretion are also regulated by cytosolic ca , such as vesicular trafficking and canalicular exocytosis of bile acid transporters [ 33 , 34 ] , permeability of tight junctions , or canalicular contraction [ 9 , 36 ] .
intracellular calcium waves , as described above , starting from the canaliculus to the basolateral poles may have physiological impact on secretion , as it has been shown in pancreatic acinar cells , although direct evidence in hepatocytes is lacking . moreover , interhepatocyte calcium waves have been reported to support canalicular peristaltism and thereby to regulate bile flow , in the normal and regenerating rat liver [ 3 , 38 ] .
as emphasized in the following , all these ca - regulated physiological processes may impact the course of liver regeneration .
it is well established that intracellular ca is crucial for tissue homeostasis through regulation of cell cycle and apoptosis . in particular , intracellular calcium has been reported to regulate cell proliferation at multiple steps of the cell cycle , from immediate early genes activation , toward g1-s and g2-m transitions , as well as during mitosis .
also , modifications of intracellular calcium homeostasis during liver regeneration have been reported , concerning ca - binding proteins , membrane ca - atpases , or the insp3 receptor [ 43 , 44 ] .
it has been also suggested that the alteration of the insp3 and camobilisation pathway could alter liver regeneration in the rat .
subsequently , it has been shown in nonhepatocytic cell lines that the spatiotemporal organisation of ca signals was determined for the activation of transcription factors like creb , nf-b , or nf - at , and for immediate early genes like c - fos or c - jun [ 4648 ] .
it is also well established that the activation of the ras pathway is controlled by [ ca]i oscillations .
moreover , intra - nuclear calcium signals , which have been well documented , have a major impact on gene transcription [ 5153 ] and can result either from the diffusion of cytosolic calcium to the nucleus , or from an insp3 -mediated calcium release in the nucleus itself ( see below ) [ 6 , 54 ] . finally , our previous work demonstrated that hepatocyte calcium signalling was deeply remodelled during liver regeneration in the rat , contributing to the regulation of bile flow and cell proliferation [ 3 , 55 ] . in a recent study
, we examined the physiological involvement of cytosolic calcium during liver regeneration in the rat .
we interfered with calcium signalling before ph by expressing parvalbumin ( pv ) in the liver , a calcium - binding protein expressed in muscle cells and neurons but absent from the liver , using adenoviruses coding for pv targeted to the cytosol , to selectively buffer ca in this compartment [ 53 , 54 ] .
we found that expression of pv efficiently buffers agonist - induced calcium oscillations in the cytosol and inhibits primary hepatocyte proliferation in vitro as well as in vivo during liver regeneration .  
we found that immediate early gene transcription , early phosphorylation of erk and creb , and hepatocyte progression in the cell cycle after ph were inhibited in rats expressing cytosolic pv .
these data were in line with previous reports describing these pathways and genes as dependant on cytosolic and/or nuclear calcium signalling [ 46 , 5860 ] .
we thus suggested that attenuated [ ca]i oscillations in calcium - buffered hepatocytes resulted in impaired activation of these pathways .
a potential reduction in cam - kinase activation , as previously reported , or reduced erk1/2 activation that we observed in pv - nes expressing hepatocytes may have also contributed to altered creb phosphorylation . since
cam - kinase ii , as well as erk1/2   activity , is sensitive to ca oscillation frequency , an attractive hypothesis would be that cytosolic pv expression , by attenuating agonist - generated ca signals , resulted in impaired phosphorylation of creb .  
cytosolic calcium signalling impacts most likely the early triggering of hepatocyte progression from g0 to g1 and s phases . in line with this view ,
a rise in concentration  in the liver and in the plasma  is observed early after ph for several ca - mobilizing agonists , suggesting these agonists might be involved in initiating the regeneration process . in particular , egf and hgf
elicit cytosolic ca oscillations in hepatocytes , the physiological impact of which has never been specifically addressed .
also , extracellular atp , arginine vasopressin , and noradrenalin , which are comitogenic ca - mobilizing agonists , have been individually reported to contribute to early phases of liver regeneration .
our study thus suggested that buffering hepatocyte calcium signals , potentially generated by these agonists in the minutes after ph , result in delaying hepatocyte cell cycle progression .
there is evidence in the literature for the crucial role of mitochondrial calcium in the regulation of apoptotic processes .
it is well known in particular , that mitochondrial calcium overload can be one of the pathways leading to the swelling of mitochondria and to the rupture of the outer membrane , in turn releasing proapoptotic molecules in the cytosol .
mechanisms for excessive calcium transfer to mitochondria are debated and include mainly interactions between proteins of the bcl2 family and the insp3-r .
anti - apoptotic members appeared as reducing calcium transfer from the er to the mitochondria , whereas proapoptotic factors were reported to enhance this flux . in this context , recent data suggest that mitochondrial ca , as well as cytosolic ca , may be critical for the regulation of liver regeneration after ph in the rat .
the authors suggested that buffering calcium in the mitochondria resulted in a shift in the balance between pro- and antiapoptotic factors , thereby protecting hepatocytes from apoptosis , in vitro in an hepatoma cell line , as well as in vivo in the rat liver after ph .
previous studies have established that growth factors important for liver regeneration such as hgf   and insulin   can differentially affect cytosolic and nuclear calcium in hepatocytes . it has been reported that agonist - induced calcium movements in the nucleus can schematically result from the diffusion of cytosolic calcium to the nucleus and/or from an autonomous insp3 generation and calcium mobilization from local , intra , or perinuclear ca stores [ 64 , 65 ] .
indeed , the nucleus , its envelope and the nucleoplasmic reticulum , has been shown to possess several crucial molecules involved in ca storage , insp3 production , and calcium release , therefore opening the possibility that local nuclear ca signals may occur independently from the cytosol [ 64 , 65 ] . although these two views may coexist in the same cells according to circumstances and cell types , it has been shown in skhep cells that an insp3-sensitive  intranuclear calcium compartment ( i.e. , the  nucleoplasmic reticulum  ) exists .
plc , pip2 , and insp3r have been found in the nucleus , allowing a local insp3 production and providing the machinery necessary to generate autonomous ca signals [ 64 , 65 ] .
we also know that calcium signals in these two compartments  cytosol and nucleus  can have different effects [ 51 , 58 ] .
recent works revealed that buffering calcium in the nucleus , but not in the cytosol , in a hepatoma cell line , resulted in an inhibition of cell proliferation , suggesting that nuclear ca was necessary for centrosome separation and cell progression through early prophase .
gomes et al . further showed that the hgf receptor ( c - met ) can translocate ( upon agonist stimulation ) from the plasma membrane to the nucleus and generate an insp3 production and calcium elevation in the nucleus , independently of cytosolic calcium , in a hepatoma cell line . very similarly
, it was shown by the same group that insulin can induce nuclear calcium signals through a translocation of its receptor to the nucleus , in primary rat hepatocytes .
importantly , the nucleoplasmic reticulum as an intranuclear calcium compartment has not been shown in primary hepatocytes , and some authors claimed that it was not essential for calcium signalling .
important cellular functions are thought to be regulated by nuclear calcium signals , including nuclear pore permeability , transcription factor activity and protein kinase translocation , thereby controlling gene expression . in particular
, the transcriptional activity of creb , nfat , and dream   has been well described as dependent on nuclear ca .
therefore , agonist - induced nuclear ca movements are potentially expected to impact both hepatocyte progression in the cell cycle and more generally cellular homeostasis after ph .
it is important to realize that liver regeneration , seen as a process involving the whole organ  and even the entire organism 
in that view , after ph , ca signalling may also regulate physiological processes unrelated directly to cell cycle control , although they may ultimately interfere with cell cycle progression , not only in hepatocytes but also in other liver cell types ( cholangiocytes , endothelial and kupffer cells ) . whereas we do not have any direct data about the impact of ca signals in nonhepatocytic cells after ph
, we can easily anticipate about ca - dependent physiological processes that may be crucial in hepatocytes after ph . for example
, glucose homeostasis , which regulation is critical for hepatocyte exit from quiescence , may depend on ca signals early after ph .
also , biliary homeostasis , which adaptation after ph is critical for liver regeneration [ 69 , 70 ] , may involve ca - dependent regulation [ 3 , 71 ] .   an integrated full picture of the
 liver calcium signalling  , which is obviously lacking , may improve our knowledge on the interaction network that regulates liver regeneration processes ( figure 1 ) .
 after partial hepatectomy ( ph ) the initial mass of the organ is restored through a complex network of cellular interactions that orchestrate both proliferative and hepatoprotective signalling cascades . among agonists involved in this network many of them drive ca2 + movements . during liver regeneration in the rat , 
 hepatocyte cytosolic ca2 + signalling has been shown on the one hand to be deeply remodelled and on the other hand to enhance progression of hepatocytes through the cell cycle . 
 mechanisms through which cytosolic ca2 + signals impact on hepatocyte cell cycle early after ph are not completely understood , but at least they include regulation of immediate early gene transcription and erk and creb phosphorylation . 
 in addition to cytosolic ca2 + , there is also evidence that mitochondrial ca2 + and also nuclear ca2 + may be critical for the regulation of liver regeneration . 
 finally , ca2 + movements in hepatocytes , and possibly in other liver cells , not only impact hepatocyte progression in the cell cycle but more generally may regulate cellular homeostasis after ph . 
1. Introduction
2. Hepatocyte Ca
3. Intracellular Calcium and Hepatocyte 
4. Nuclear Calcium Signalling and Liver Cell Proliferation