Calcium signals have distinct organization and underlying mechanisms in different astrocytic compartments thus allowing complex spatiotemporal signaling. Signals mediated by fluctuations in cytosolic Na+ are instrumental for coordination of Na+ dependent astrocytic transporters with tissue state and homeostatic demands. Astroglial ionic excitability may also involve K+, H+, and Cl−. The cyclic AMP signalling system is, in comparison to ions, much slower in targeting astroglial effector mechanisms. This evidence review summarizes the concept of astroglial intracellular excitability.Classic physiology divides all neural cells into excitable neurons and nonexcitable neuroglia. Neuroglial cells, chiefly responsible for homeostasis and defense of the nervous tissue, coordinate their complex homeostatic responses with neuronal activity. This coordination reflects a specific form of glial excitability mediated by complex changes in intracellular concentration of ions and second messengers organized in both space and time. Astrocytes are equipped with multiple molecular cascades, which are central for regulating homeostasis of neurotransmitters, ionostasis, synaptic connectivity, and metabolic support of the central nervous system. Astrocytes are further provisioned with multiple receptors for neurotransmitters and neurohormones, which upon activation trigger intracellular signals mediated by Ca This was an inert tissue, forming a filling or basic substance that surrounds and covers all components of the organism being in a way a predecessor of the connective tissue of Rudolf Virchow.The concept of physiological excitability and the definition of excitable and nonexcitable tissues was formulated by Albrecht von Haller, who exposed different organs or their parts to injury, by squeezing and stinging, by sprinkling with cold, hot, or corrosive substances or by electrocuting. Analyzing responses to such interrogations, von Haller proposed to classify all organs into sensible (cerebellulas (minute brains) that are connected by nerve fibers, which receive sensations from or project motor impulses into the peripheral organs. Swedenborg described these structures as the substrates for brain function. He wrote “From each cortical gland proceeds a single nerve fiber; this is carried down into the body, in order that it may take hold of some part of a sensation or produce some action.”globules” or “kugeln” both denoting spheres) from the microscopic observations were made by Christian Gottfried EhrenbergAlthough the cellular theory had not yet been established by the time of von Haller’s work the notion of the cell as an elementary living entity has been considered; the term being invented by Robert Hook in the 1650s.Kugeln der Belegungsmassen) and nerve fibers (Primitivfasern), whereas the passive substance was defined as intermediate substance (Zellgewebescheide) made from fibers and threads. The ideas about specific brain connective tissue were further developed by Carl von Rokitansky,bona fide connective tissue, glial cells have been visualized and identified by many neuroanatomists These considerations changed the role of glia from being regarded as simply a connective tissue to an active counterpart of neurons in executing brain functions.A pupil and assistant of Purkinje, Gabriel Gustav Valentin was the first to contemplate two types of nervous elements, which he called nervous masses. One of these masses representing “the creative, active, higher principle” while the second “the receiving and guiding, passive, lower principle.”,,The advent of electrophysiology and intracellular recordings led to a detailed characterization of electrical excitability of nerves, muscle, and neurons. The very first observation of experimentally evoked muscle contractions were made in the 1660s by Jan Swammerdam, who designed the classic frog neuromuscular preparation.in vivo, in organotypic cultures, in isolated optic nerve preparations from amphibians or in the isolated ganglionic chain of the leech+ accumulation in the extracellular space.Some 150 years later, the seminal discoveries of Hodgkin and Huxley provided the first quantitative description of the ionic conductance changes underlying the action potential,,in vivo expression of these receptors was tightly regulated by the neurochemical environment: the neuroglial receptor pattern is tailored to neurotransmitters operating in a particular brain region.2+indicators, it turned out that chemical or mechanical stimulation of glia almost invariably triggered complex cytoplasmic Ca2+ signals, which, in a form of Ca2+ waves, could propagate over long distances through the gap-junction connected glial syncytium. Thus, the concept of calcium excitability of neuroglia was developed.+ fluxes that generate cytoplasmic Na+ signals as well as with highly organized changes in cytosolic second messengers such as InsP3 and cAMP; the former being linked to Ca2+ signaling whereas the latter being connected with numerous intracellular enzymatic cascades and influenced by Ca2+. Consequently, a coherent concept of intracellular astroglial excitability is in need of definition.When the technique for making purified glial cell cultures was developedAppearance of the central nervous system (CNS), which emerged early in evolution, was accompanied by division of neural cells into neuron, which represent executive arm responsible for sensory input, information processing, and initiation of peripheral responses and homeostatic and defensive neuroglia. Neuroglial cells of the brain and the spinal cord are classified into macroglia  and microglia, which are scions of fetal macrophages invading the CNS early in the development. Neurons are universally considered as the only excitable cells of the nervous system; they generate fast action potentials, which are conducted over large distances and initiate neurotransmitter release responsible for synaptic connectivity. Nonetheless, fast signaling (in addition to relatively slow ones) does also occur in glial cells, which respond to physiological stimulation with transient fluctuations in their ionic content; these ionic signals are the substrate for rapid stimulus-induced glial excitability.l-serine.,,Astrocytes are the principal homeostatic cells of the CNS, which constantly adapt operation of elaborated homeostatic molecular cascades to neuronal activity and brain state. Astrocytes control CNS homeostasis at many levels. First and foremost astrocytes are responsible for CNS ionostasis—the ionic composition of the interstitial fluid, which are tightly associated with changes in brain state, such as sleep and arousal.i), which regulate astroglial physiological activity. Similarly to neurons, astrocytes, are activated in response to sensory stimulation; numerous experiments in vivo in anesthetized and awake animals have demonstrated synchronous cytosolic [Ca2+]i transients engulfing groups of astrocytes in the sensory cortex.,2+ signals originating from endoplasmic reticulum (ER) Ca2+ release and/or from Ca2+ entry across the plasmalemma,+-dependent transporters, generating a massive Na+ influx, which triggers cytosolic Na+ signals.,,Sensing the neural tissue environment involves the stimulation of astroglial membrane receptors. Activation of these receptors does not trigger regenerative transmembrane depolarization, instead it produces changes in intracellular ion activity reflecting changes in free ion concentration , which represents the prime neuronal plexus localized in the brain stem; projections of the LC neurons synchronously release NA in various brain and spinal cord regions. In the adult human brain, the LC consists of only around 50 000 neuronsAstrocytes are major targets of NA in the CNS; mature astrocytes express adrenoceptors of both α and β varieties while the density of adrenoceptors in astrocytic processes seems to be significantly higher than in neurons.Maintenance of cellular ionic homeostasis is one of the most fundamental conditions for life; all living organisms on planet Earth are keeping the ionic composition of cytosol and organelles under tight control at the expense of considerable energy. Ionic gradients between the extracellular space and the cytosol are driving ion fluxes. These ionic fluxes originate from opening of ion channels following for example an environmental stress in unicellular organisms or due to release of chemical messengers in multicellular ones. Thus, from the very beginning of life, ions evolved as dynamic intracellular signalers coupling extrinsic challenges to intracellular processes. Conceptually, living cells are constantly balancing the preservation of their ionic composition with generation of ionic fluctuations organized in space and time. To achieve this steady-state, evolution selected transport systems moving ions along and against concentration gradients. In essence, all changes in the cytosolic concentration of any ion, di- or monovalent, can regulate/modulate various cellular events, and hence may act as second messengers in biological systems. Ionic signalingis shaped by dynamic interactions of diffusion  and primary or secondary ion transport , which requires energy. All these molecular cascades are in operation in astrocytes.2+ concentration acts as a ubiquitous physiological signal, operating in most  cells and tissues. Changes in the Ca2+ concentration in various cellular compartments trigger or regulate a wide variety of cellular processes. The Ca2+ homoeostatic and signaling system involves relatively few molecular elements  which, by operating in concert, shape Ca2+ signals in the cytosol and in the organelles while at the same time preventing life-endangering Ca2+ overloads. Changes in [Ca2+]i are sensed by numerous Ca2+-binding proteins, which translate Ca2+ signals into cellular activity.It is universally acknowledged that an increase in the Ca2+ signaling is characterized by a complex spatiotemporal organization, which reflects the elaborate astrocyte architecture. Furthermore, different types of astrocytes seemingly have distinct [Ca2+]i dynamics with idiosyncratic underlying mechanisms. The morphological compartments of protoplasmic astrocytes (which are probably the most studied class of astroglia) are represented by (i) soma; (ii) main processes also known as branches; (iii) secondary to tertiary processes designated as branchlets; (iv) peripheral parenchymal and perisynaptic processes known as leaflets; and (v) perivascular processes, which terminate with end feet plastering blood vessels  domains of elevated [Ca2+]i. Focal [Ca2+]i transients can either be spontaneous, with no association with neuronal activity,2+ signals can result from neuronal activity and stimulation of astroglial receptors.2+ signals in the peripheral processes of protoplasmic astrocytes are shorter in duration than in the soma,2+ influx through Ca2+ permeable ionotropic receptors,,+/Ca2+ exchanger (NCX).,2+ events in branchlets and branches can be amplified by Ca2+ released from the ER and mitochondria. The higher surface-to-volume ratio of branchlets allows larger plasma membrane Ca2+ influx and hence larger [Ca2+]i fluctuations.2+]i fluctuations more frequently reach the threshold for Ca2+-induced Ca2+ release through InsP3 receptors. Hence, loss of fine astrocytic branchlets in pathological conditions such as epilepsy can be linked to reduced astrocytic Ca2+ activity.Numerous lines of evidence have demonstrated that Ca2+ signals in protoplasmic astrocytes, as well as [Ca2+]i transients in the primary processes are larger in amplitude and slower, are often synchronized with neighboring astrocytes .,3 receptor type II  eliminates Ca2+ signals in the soma and in the primary processes, leaving [Ca2+]i dynamics in branchlets and leaflets very much undisturbed or only partially suppressed.Somatic Cam the ER 53,85,86 2+ signalling  does not operate in all types of astrocytes. For example, in Bergmann glial cells  Ca2+ signalling microdomains are associated with specific morphological structures—the appendages. These appendages emanate from the primary radially oriented processes of the Bergmann glial cells; each appendage contains mitochondria and projects leaflets that contact 50–70 synapses formed by axons of granular neurons  and InsP3 receptor-mediated Ca2+ release, with subsequent activation of SOCE.,2+]i-induced Ca2+ release was shown to substantially contribute to α1-adrenoceptor-mediated Ca2+ signals2+]i dynamics in the peripheral fine branchlets of cortical mouse astrocytes  were reported to originate from mitochondrial Ca2+ release through the flickering mitochondrial permeability transition pore.2+ signals in branchlets (which possess ER) may involve combination of InsP3-induced Ca2+ release and plasmalemmal Ca2+ entry. Spatial restriction of [Ca2+]i increases could result from local mitochondria, which act as powerful Ca2+ buffers2+]i increases in astroglial processes.2+ signals can be associated with plasmalemma-ER junctions that have been described in cultured primary astrocytes; these junctions are rich in InsP3 receptors, SERCA pumps, and NCX being thus a substrate for focal Ca2+ signaling.2+ signaling, which most likely changes depending on the physiological context, astrocyte morphology, age, and environmental settings.This type of segregated Ca neurons . Activat2+]i dynamics in vivo, in awake animals, remain rather limited. It seems that sensory stimulation triggers large and global  [Ca2+]i elevations controlled mainly by noradrenergic stimulation of α1 adrenoceptors.,,2+]i increase in response to transcranial direct current stimulation.2+ signals evoked by acetylcholine release from projections of the nucleus basalis of Meynert; these [Ca2+]i responses are mediated through muscarinic ACh receptors and involve InsP3-induced Ca2+ release.,in vivo setting communicate through propagating Ca2+ waves, which were characterized in detail in vitro and in brain slices,,Data on astroglial i fluctuations remain largely unanswered questions. Similarly to other cells, astroglial Ca2+ signals regulate gene expression and provide for excitation-metabolic coupling; formation of [Ca2+]i microdomains in astroglial branchlets immobilize mitochondria thus securing local metabolic support2+ signals may trigger astrocyte morphological plasticity.,2+ signalling is implicated in secretion, both exocytotic and nonvesicular.,,2+ signals.,3-mediated Ca2+ signalling does not affect increases in local blood flow in response to sensory stimulation.2+]i transients occurring in the end feet shortly before vasodilatation.,2+-dependent slow tonic regulation of the vascular tone through continuous release of prostaglandins.2+ signals were also reported to undergo changes in sleep, while suppression of astroglial [Ca2+]i dynamics by knocking out the InsP3 receptor type II affects slow-wave sleep with an increase in the number of micro-arousals, abnormal brain rhythms, and an increased frequency of slow-wave sleep state transitions and sleep spindles.How Ca2+ signaling controls reactive astrogliosis, the archetypal astrocytic defensive program. Challenge of astrocytes with damage-associated molecular patterns, such as ATP or β-amyloid triggers [Ca2+]i rises,2+ signaling by genetic ablation of InsP3 receptorsIn a pathological context, Ca+]i are of particular significance for astrocytic homeostatic function, because the absolute majority of plasmalemmal transporters engaged in maintaining various aspects of molecular homeostasis are driven by transmembrane Na+ gradient. These transporters not only utilize Na+ gradients; their operation produces Na+ fluxes thus transporters being simultaneously the sensors and modifiers of [Na+]i. The resting [Na+]i in astrocytes lies in the range of 15–20 mM,,,+]i, which may last for tens of seconds.,,,,+]i transients were shown to spread in the form of propagating [Na+]i waves through individual cells  led to the concept of astroglial Na+ signaling.,Changes in [Na2+]i and [Na+]i dynamics in astrocytes, the underlying mechanisms are quite distinct. Astrocytic Na+ signals rely on plasmalemmal Na+ movements only, as no Na+ storing structures exist in the cells  or Na+-dependent bicarbonate symporter (NBCe1). The NKA in astrocytes incorporates the α2 subunit; which is not expressed in neurons (which possess α 1 and α 3 subunits). As a result, the affinity of astroglial NKA to K+ is substantially lower than in neurons. The EC50 for K+ for astroglial NKA composed from α2/β1 subunits is ∼3.6 mM, while EC50 for K+ in neuronal NKA  varies between 0.25 and 0.65 mM.+] (∼3–3.5 mM) the neuronal NKA K+ binding sites are fully saturated; whereas for astrocytic NKA half of the K+ binding sites remain unoccupied. Consequently, an increase in interstitial [K+] activates astroglial NKA, which is the main mechanism for extracellular K+ sensing and buffering. Neuronal NKA is activated solely by an increase in [Na+]i. The NKA-dependent transport of K+ and Na+ in astrocytes maintains ion gradients critical for operation of homoeostatic transporters; in essence, the NKA acts as the master regulator of astroglial homoeostatic physiology. Increases in NKA transport, which accompany neuronal activity (to buffer K+ or to expel excessive Na+ entering the cell in the course of glutamate uptake) are also linked to astroglial l-lactate production and hence are central for the operation of astrocyte-to-neurone-lactate shuttle (ANLS).,Extrusion of Na+ from astrocytes is mediated solely by Na+/K+ ATPase (NKA). Inhibition of NKA in cultured astroglia by ouabain or by removal of extracellular K+ results in an increase in [Na+]+ signaling is represented by the NCX; astrocytes express all three subtypes of this exchanger . These subtypes are quite similar from the functional point of view, exchanging Na+ and Ca2+ with a 3:1 stoichiometry.,+]i or small depolarizations turn NCX into the reverse mode when it brings Ca2+ into the cell in exchange for Na+; in this mode, the NCX generates a [Ca2+]i rise while accelerating recovery to resting [Na+]i.,,2+]i rises the NCX is forced into the forward mode in which it assists the recovery of [Ca2+]i transients while producing Na+ influx.2+ and Na+ signaling.The second key player of Na2+]i dynamics, astrocytic Na+ signals may be confined to microdomains. Such local subcellular [Na+]i transients have been characterized in Bergmann glia and in hippocampal protoplasmic astrocytes.,+ buffers/binding sites, which may account for the localization of [Na+]i rises. The Na+ transporters in the plasmalemma endowed with Na+ binding sites may act as some sort of highly localized and relatively immobile Na+ buffers. Alternatively, Na+ (and other cations) may be trapped in tiny leaflets by binding sites associated with the inner side of the plasma membrane.+ signals can propagate from cell to cell by diffusion through gap junctions; the speed of these waves in [Na+]i may reach 100–150 mm/s.,Similar to i is represented by the SLC membrane transporters, which fulfill an astroglial homoeostatic function. Changes in [Na+]i may affect not only the efficacy of transports but also change their operational direction. The reversal is well documented for NCX (see above) and can also occur to some other transporters, such as, for example, GABA or glycine transporters, which have been shown to reverse in physiological settings following an increase in [Na+]i.+]i may translate to various biochemical and cellular responses though action on enzymes; in an astroglial context Na+ regulates glutamine synthetase thus affecting availability of glutamine for the glutamate (GABA)-glutamine shuttle.+ ions are also known to modulate or open various types of ion channels, such as, for example, Na+-dependent K+ channels or Kir4.1 inward rectifying K+ channels.,+ signaling therefore was proposed as a mechanism for rapid tuning of astroglial homeostatic cascades to neuronal activity.There are surprisingly large varieties of Na−. Changes in [Cl−]i regulate plasmalemmal channels  serine/threonine protein kinases.,−]i contribute to the regulation of several fundamental cellular processes such as cell differentiation and death.,Chloride, the major inorganic anion in the living tissues, is a likely contributor to astroglial ionic excitability. There are multiple indications for signaling role of intracellular Cl− is the central ion for mediating inhibitory currents in neural cells, and hence fluctuations in [Cl−] in the interstitial fluid are of paramount importance for balancing neurotransmission. Experiments on cultured astrocytes demonstrated that astrocytes maintain high [Cl−]i ranging between 20 and 50 mM, which corresponds to ECl = −35 mV.in situ in acute brain slices. In Bergmann glial cells of the cerebellum Cl− imaging indeed revealed high [Cl−]i of around 50 mM in newborn and 35 mM in mature mice.−]i at 3–4 mM.−]iin vivo is of pressing importance; as it may reveal either regional or state-dependent differences.At the same time, Cl− homeostasis depends on Cl− diffusion through several sets of anion channels that include (i) GABAA and glycine receptors; (ii) inwardly rectifying chloride channels ClC-1, -2, and -3; (iii) Ca2+-dependent Cl− channels; (iv) anion channels of the Bestrophin (Best) family and by (v) volume-regulated anion channels VRAC or SWELL1.− efflux or influx depending on the [Cl−]i; at the same time molecular mechanism(s) for Cl− accumulation into astrocytes remains to be identified. The only known Cl− accumulating transporter, Na+/K+/Cl− co-transporter NKCC1/SLC12A1, has been frequently identified in astrocytes in culture, however, whether NKCC1 operates in situ or in vivo remains controversial.,− fluxes.,− signaling in astrocytes are yet to be fully characterized; the role of astrocytes as a source for Cl− to maintain inhibitory transmission has been proposedAstrocytic Cl+-rich Primordial Ocean. Surprisingly, the cytoplasm of most cells has high K+ and low Na+ concentrations. Several hypotheses explaining this phenomenon have been developed. For example, protocells could have emerged in the K+ enriched vents at the bottom of the ocean; or they may have appeared in the inland basins molded from K+ rich clay and filled with rainwater.+ plays a vital role in cellular life. High [K+]i is required for protein synthesis and sets the cell membrane potential, while K+ efflux repolarizes the cell membrane following action potentials, excitatory postsynaptic potentials, and dendritic spikes in neurons.Life on Earth is believed to have emerged around four billion years ago in a Na+ fluxes across astroglial membranes. Astrocytes remove excess K+ at the peak of neuronal activity and then return K+ back to restore neuronal ionic gradients; in pathology, astrocytes are capable of redistributing K+ through the syncytial networks. Notably, most of the K+ removed by astrocytes from the synaptic cleft during neuronal activity comes from K+ efflux through ionotropic glutamate receptors, predominantly of NMDA type.+ into astrocytes is mainly mediated by NKA (discussed in the previous section), while K+ efflux is mediated by inwardly rectifying K+ channels.+ microdomains in perisynaptic astroglial processes, but whether these domains exist remains to be experimentally seen. The mechanisms of formation of K+ microdomains are similarly unknown. Recently, the role of intramembrane negative charges preventing K+ diffusion+]i have a signaling role and directly modulate cellular functions similarly needs to be tested. One testable possibility is that K+-mediated depolarization can affect voltage-dependent steps of glutamate transporter cycle, hence, affecting glutamate uptake.Hence, neuronal activity is associated with substantial K+]i. This phenomenon is known as “depolarization-induced alkalinization” and results in accumulation of H+ in the extracellular space.+/+ is represented by phosphofructokinase; activation of the latter is perceived as a key step in stimulation of astrocyte-neuronal lactate shuttle.Neuronal activity is accompanied by a transient decrease in astroglial i.Given the relatively complex design of cAMP nanosensor, it is not surprising that the first single-cell measurements of [cAMP]i, consistent with the action of β-adrenergic receptors.i was characterized by a monoexponential rise to a plateau with a time-constant of ∼15 s, much slower than the agonist-induced increases in [Ca2+]i in astrocytes.i represents the balance between the production of cAMP by adenylyl cyclases (AC) and its enzymatic degradation by phosphodiesterases.i were recorded and were considered to be due to an interaction with Ca2+ signaling,Stimulation of astrocytes with adrenaline at 29 nM induced a half-maximal increase in [cAMP]2+ signaling operate in different time domains in astrocytes, there is an interaction between these pathways.i is tonic, lasting several minutes, the swift changes in [Ca2+]i are phasic, often exhibiting oscillations . Glycogen-derived l-lactate exits astrocytes through monocarboxylate transporters (MCTs) 1 and 4 and/or yet unidentified ion channelsl-lactate can also act as an extracellular signal where it binds to l-lactate metabotropic receptors or to yet unknown receptors.Astroglial glycogen represents an energy reserve, which is used during increased activity to support many CNS functions, including memory formation and consolidation.2+- and/or cAMP-pathways, such as ARs and purinoreceptors. Upon stimulation of LC neurons, NA is released, with subsequent activation of metabotropic adrenoceptors and increases in astrocytic [Ca2+]i and [cAMP]i.,2+ and cAMP as second messengers to the regulation of aerobic glycolysis and glycogenolysis in astrocytes remains unclear and even controversial. It is thought that aerobic glycolysis and glycogenolysis are primarily elevated through the cAMP-dependent pathway in astrocytes,,2+ signals might also be involved.Aerobic glycolysis together with glycogenolysis is regulated in astrocytes by a variety of receptors on the surface of astrocytes that are linked to intracellular Ca2+ and cAMP regulates many cellular processes affecting the function of astrocytes and neighboring neurons in health and disease. This intracellular excitability provides regulatory clues in distinct space and time domains, which underlies the capacity of adapting to dynamic and life-long changes that occur during the function of the CNS in health and disease.In conclusion, astroglial noradrenergic signaling, involving CaAstrocytes are an indispensable part of the nervous tissue, which together with neurons and other neural cells produce a cellular fabric responsible for brain function. Homoeostatic cascades in the astrocytes, which support the most fundamental functional properties of the CNS, are tightly correlated with neuronal activity and tissue demands. This coordination is a function of astroglial excitability mediated through spatiotemporal fluctuations of intracellular ions and second messengers.A.S. work was supported by Russian Science Foundation (grant number 20-14-00241); R.Z. is supported by grants from the Slovenian Research Agency , CIPKEBIP, COST Nanonet, COST Mouse Ageing, and COST CM1207 – GLISTEN.None declared.


Recently, it was reported that calcineurin is activated in a variety of cancers. Given that abnormalities in calcineurin signaling can lead to malignant growth and cancer, the calcineurin signaling pathway could be a potential target for cancer treatment. For example, NFAT, a typical substrate of calcineurin, activates the genes that promote cell proliferation. Furthermore, cyclin D1 and estrogen receptors are dephosphorylated and stabilized by calcineurin, leading to cell proliferation. In this review, we focus on the cell proliferative functions and regulatory mechanisms of calcineurin and summarize the various substrates of calcineurin. We also describe recent advances regarding dysregulation of the calcineurin activity in cancer cells. We hope that this review will provide new insights into the potential role of calcineurin in cancer development.Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by regulating the phosphorylation states of several targets. Intracellular Ca While the extracellular Ca2+ concentration is 11.5 mM, the steady-state intracellular Ca2+ concentration is kept very low at several tens of nanomoles. It is well known that the concentration of Ca2+ varies greatly in different cellular compartments, for example, intracellular calcium, 100 nM; endoplasmic reticulum (ER), 0.5–1 mM; and mitochondria, 100–200 nM [Intracellular calcium ions , which cleaves phosphatidylinositol 4,5-biphosphate (PIP2) to produce diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsP3). InsP3 then binds to the InsP3 receptor (InsP3R) and stimulates the release of Ca2+ from intracellular stores, such as ER [2+ [2+ release via InsP3R is regulated by complex feedback mechanisms [The binding of a hormone or growth factor to a G protein coupled receptor (GPR) or a tyrosine kinase receptor (RTK) initiates the Cahate InsP. InsP3 t2+ concentration trigger signal transduction, which regulates a wide range of cellular events such as gene expression, cell cycle, cell motility, autophagy, and apoptosis [2+ diffuse through the cell and have effects on the distal sites [2+ increases in the mitochondria and causes the release of cytochrome c and subsequently triggers apoptosis [2+, the dysregulation of calcium homeostasis can impair cell function and trigger cell death, and by doing so, may contribute to heart disease, cancer, and mental disorders. As a result, Ca2+ signals must be tightly regulated during the cell cycle. Indeed, previous studies have characterized Ca2+ transients during the cell cycle [2+ in rat fibroblasts and found that complex changes occurred in calcium levels during the cell cycle [2+ were the lowest at the beginning of the G1 phase but were subsequently increased at the G1/S border.Changes in Capoptosis ,5. Localal sites . Long-tepoptosis . Due to ll cycle ,9. For ell cycle . They sh2+ oscillations are narrow in the sense of periodic rise and fall, while Ca2+ concentration alterations are broad in the sense of simple Ca2+ concentration changes. To facilitate different cellular processes, intracellular downstream effectors decode the frequency, amplitude, and duration of intracellular Ca2+ oscillations [2+ binding sites. CaM is involved in the regulation of cell proliferation and cell cycle through CaM-dependent phosphorylation/dephosphorylation events [2+ binds to CaM, the Ca2+/CaM complex can interact with and control target proteins such as serine/threonine phosphatase calcineurin (CaN) and the family of Ca2+/CaM-dependent protein kinases (CaMK).Callations . The mosn events . When Ca2+/CaM regulates cell cycle progression, especially through the activation state of the cyclin-dependent kinase (CDK) complex, is of great interest, and therefore has been studied intensively. It has been shown that although all eukaryotic cells require Ca2+ signaling for cell proliferation, some transformed cells and tumor cell lines are less dependent on Ca2+ for cell growth [2+-mediated signaling pathways, therefore, play important roles in tumorigenesis, tumor progression, metastasis, and invasion [The molecular mechanisms through which Cal growth ,14. Ca2+invasion . In thisCell division is precisely regulated by a series of CDKs, whose activity is dependent on the binding to different cell cycle-specific cyclins . Cyclins1 phase is the retinoblastoma protein (Rb). In the early G1 phase, CDK4/6-cyclin D initiates the phosphorylation of Rb, and, subsequently, CDK2-cyclin E phosphorylates Rb [1/S boundary and to commence the S phase. CDK2-cyclin A plays an important role in S phase progression through the phosphorylation of proteins involved in DNA replication [2 phase [2 phase, CDK1-cyclin B is activated, allowing for the entry of cells into mitosis [2/M transition, CDK1-cyclin A activity is necessary for prophase initiation [One of the critical targets of the CDK-cyclin complex in the Glates Rb ,18,19,20lates Rb ,22. Thislication ,24. As c[2 phase ,26. In t mitosis . During itiation .2+ is required at several different points during the cell cycle. Cells are most sensitive to the depletion of extracellular Ca2+ at early G1 and near the G1/S boundary [1, Ca2+ regulates the expression of immediate-early genes, such as FOS, JUN, and MYC. Ca2+ is also required for Rb phosphorylation at the G1/S boundary. Spontaneous Ca2+ oscillations at the G1/S phase transition have been described in synchronized immortalized cell lines. These Ca2+ oscillations are accompanied by DNA replication [In mammalian cells, Caboundary ,29. In elication .2+ levels increase. Ca2+ regulates certain targets directly and others indirectly through Ca2+-binding proteins, such as CaM. Ca2+/CaM activates various pathways involved in the regulation of cellular processes, such as secretion, cell motility, ion homeostasis, and gene transcription. CaM also modulates a large number of intracellular enzymes, including protein kinases, protein phosphatases, phosphodiesterases, adenylyl cyclases, and ion channels [Once hormone receptors are activated, intracellular Cachannels .1, specifically the G1/S transition. Consistent with this notion, CaM antagonists block cell cycle progression in early or late G1 phases [2+, CaM undergoes a major conformational change, and forms a Ca2+/CaM complex to interact with a variety of target proteins such as cellular kinases [2+/CaM signaling are the CaMKs [2 phases, is also activated by the CaN/NFAT pathway [1 to S phase [1/S [2/M transition [CaM is required for cell cycle progression through G1 phases . Further1 phases . Upon bi kinases . The beshe CaMKs ,36. CaN he CaMKs  and CDK4he CaMKs . The samhe CaMKs . In conthe CaMKs . In addi pathway . CaMK re S phase ,43, and  S phase . In the  S phase . As a rease [1/S ,47,48 oransition , with deansition ,51. Not ansition ,52,53.2+-binding regulatory subunit, calcineurin B (CnB). In mammals, three independent genes, PPP3CA, PPP3CB, and PPP3CC, encode CnAα, CnAβ, and CnAγ, respectively. CnAα and CnAβ exhibit ubiquitous expression patterns, whereas the CnAγ expression is restricted to the testis and brain [PPP3R1 and PPP3R2, respectively). The CnB1 protein is expressed ubiquitously, whereas the CnB2 protein is specifically expressed in the testes. CnA contains an amino-terminal catalytic domain followed by a CnB-binding domain, a CaM-binding domain, and an autoinhibitory domain. CaN also contains a nuclear localization signal (NLS) in the catalytic domain and a nuclear export signal (NES) in the carboxyl terminus. The autoinhibitory domain of CnA blocks the catalytic site and the NLS [2+-binding motifs and an amino-terminal myristylation site. CaN is activated by the increased intracellular Ca2+ concentration in the cell and plays essential roles in multiple signaling processes [2+/CaM to the regulatory domain leads to the attenuation of autoinhibition, followed by dramatic enzymatic activation. Of note, although there are multiple kinases that are regulated by CaM, and CaN is the only phosphatase directly regulated by Ca2+/CaM.CaN, also known as protein phosphatase 2 B (PP2B), is a serine/threonine protein phosphatase that is conserved in all eukaryotes ,55,56,57nd brain ,59,60,61 the NLS . CnB conrocesses . The bin2+ concentrations, and a small portion of CaN can translocate to the nucleus and interact with the target substrates [As mentioned above, CaN is widely distributed in various mammalian tissues and is particularly abundant in neural tissues . Howeverbstrates .Several proteins have been reported to inhibit CaN, including AKAP79 (A-kinase anchoring protein-79) , PMCA2  transcription factors ,103. Fou S phase ,117. NFApromoter . In addipromoter  or inducpromoter . Thus, NGiven the diverse expression and function of each NFAT isoform, its dysregulation is known to be associated with tumorigenesis, Alzheimer’s disease, and the development of autoimmune and inflammatory diseases. A novel therapeutic strategy for treating NFAT-related diseases is to develop new ways to selectively regulate specific NFAT isoforms .Approximately 600 proteins with conserved sequences that bind to CaN have been identified . Many of2+ increase caused by mitochondrial depolarization. Activated CaN dephosphorylates DRP1, which then relocates to the mitochondria and promotes mitochondrial fission [Key proteins associated with tumor development such as myocyte enhancer factor 2 (MEF2), kinase suppressor of ras 2 (KSR2), DAXX, c-Jun, and nuclear factor I (NFI), are known CaN substrates ,136,138. fission . In neur fission . CaN-dep fission , while d fission .1/S progression mediated by the degradation of cyclin D1. FK506 also decreases the protein level of CDK4 via a yet unknown mechanism. Taken together, CaN activates CDK4-cyclin D1 through multiple mechanisms to promote cell cycle progression [Recently, we found that CaN inhibits cyclin D1 degradation by dephosphorylation of the Thr286 residue  Figure a. Treatmgression ,38. The gression . It is agression . This ingression  , a Ca2+-selective ion channel, is highly expressed in prostate cancer and is a prognostic marker [2+ to enter the cell, which in turn promotes NFAT activation [The mechanisms that drive CaN activation in cancer are not well understood. In some cancers, CaN hyperactivation has been associated with mutations. For example, the EL4 murine T-cell lymphoma cell line expresses a mutant form of CaN, in which the aspartic acid at position 477 is mutated to asparagine and the negative regulation of the phosphatase activity by the autoinhibitory domain is impaired by this mutation . In contc marker ,170. Inctivation . It is ativation ,173,174.tivation . Moreovetivation . Recentltivation , which iHighly activated CaN is supposed to activate its substrates, including NFAT, to promote proliferation. Similar to CaN, its target NFAT is also constitutively activated or overexpressed in numerous cancers and may contribute to cancer development and progression . ConversOwing to the high frequency of CaN/NFAT activation in cancer and the contribution of these molecules in cancer progression, the CaN/NFAT pathway could be a potential therapeutic target. Indeed, the anticancer effects of CaN inhibitors have been studied extensively in the past. For instance, cyclosporine A or FK506 induced apoptosis and rapid tumor clearance, resulting in the regression of leukemia . Cyclosp2+ channels/transporters/pumps can provide promising potential for the treatment of cancer patients. [2+ concentrations alters the expression levels of the genes involved in cell proliferation, resulting in an antitumor effect [2+ reduces the growth rate of tumor cells without affecting the cells in normal tissues [2+ levels.There is growing acceptance that targeting dysregulated Caatients. . Bufferir effect . Moreove tissues . Further2+ is involved in tumor growth and alters the expression of the genes involved in cell proliferation. Furthermore, previous studies have suggested that the modulation of nuclear Ca2+ signaling may be effective in cancer therapy. Activation of CaN and its downstream dephosphorylation has been identified as a mechanism by which nuclear Ca2+ regulates cell proliferation and cell cycle progression. As discussed above, dephosphorylation of proteins by CaN plays an important role in tumor formation and progression. Therefore, in the future, it will be necessary to identify the molecular mechanism of CaN activation and the substrates that promote cancer cell growth. Targeting the interactions of activated CaN with specific substrates in cancer cells, without affecting the normal immune function of CaN, may effectively inhibit the growth of cancer cells, leading to the establishment of new tumor-specific therapies.Nuclear Ca


We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca Cl- accunctions . Subsequar cells . Acetylcar cells . ACh ress (IP3R) . The ER ecretion . To stimpical PM . As Cl- ecretion .2+ signal have been described in preparations of enzymatically isolated preparations of salivary gland acinar cells or lobules     to allow5–8/min) . At high2+ signaling have provided a wealth of information to greatly inform our models of salivary secretion, there are general caveats which apply to these data. Primarily, the cells are studied out of a physiological context with no extracellular matrix, basement membrane and with probable disruption of cellular junctions as a result of the enzymatic isolation. A further complication is that isolated cells are typically exposed for a period of time to a constant concentration of agonist which in many cases is a non-hydrolysable analog, as opposed to the neural delivery of transmitter occurring in a pulsatile fashion that is subject in vivo to enzymatic degradation. Whether these factors influence the spatiotemporal characteristics of Ca2+ signals in salivary glands is unknown. Additionally, since only indirect measurements of fluid secretion inferred from volume changes can be made in isolated acinar cell preparations  microscopy. Notably, stimulation of the endogenous neural input to the gland induces Ca2+ signals with major spatiotemporal properties which are distinct from those observed in isolated acinar cells. Specifically, following moderate stimulation, apically localized Ca2+ signals are generated which did not propagate globally. Furthermore, the apical signals consist of rapid oscillations. At stimulation intensities optimum for secretion, a significant apical-basal standing Ca2+ gradient was also established. These striking disparities suggest a modification of our previous model is necessary HzeGt(ROSA)26Sor/MwarJ) (tm3(cre/ERT2)SkzBhlha15/J) (To generate mice expressing a fluorescent Cae/MwarJ)  floxed b2)Skz/J) . The STO2)Skz/J) . The exp2)Skz/J) . Bipolar2)Skz/J) .2+] in acinar cells  as described in Materials and methods (each image a three frame average to result in acquisition at 10 fps). Under basal conditions, prior to stimulation, the cells were quiescent . Electriar cells . Followiar cells , while mncreased . During ncreased . The maxereafter , probablereafter .2+] , but saliva secretion progressively increased following 5–10 Hz nerve stimulation  . Higher 2+] . At mode2+ release and subsequent Ca2+ influx  and restimulated 20–70 mins later. No significant effect was observed on the magnitude of the initial peak, presumably because sufficient refilling of the ER Ca2+ store had occurred during exposure to blocker. However, the plateau measured following 5 min of stimulation was significantly attenuated in drug injected animals vs. vehicle , we observed both the recruitment of responding cells and an increase in the amplitude of the [Ca2+] response , while the increase in Ca2+ signal following increased stimulation in less responsive regions was proportionately smaller   . Therefo2+] observed, we systemically administered a cholinesterase inhibitor to block the clearance of ACh and effectively increase the local [ACh]. Physostigmine (0.1 mg/kg body weight IP) enhanced the peak amplitude of the Ca2+ signals following stimulation , but became more prominent at 3 Hz with shorter latency, an increase in the number of responding grids and peak amplitude  and following 5 Hz stimulation   . At 10 Hanimals) . While t2+ signals evoked by neural stimulation, we generated standard deviation (SD) images from the stimulated portion of the image series. This analysis revealed that at all physiological stimulus strengths the increase in [Ca2+]i was highly heterogeneous, with the largest increases in signal occurring in the apical aspects of acinar clusters, directly below the apical PM . The apical-basal gradient was further analyzed by plotting the amplitude of the maximum fluorescent change across the line profile which further reinforces that at each stimulus intensity the signal rapidly decays away from the apical initiation sites to the nucleus and is ~25% of the apical signal in the extreme base of the cell . In total, these data demonstrate that in contrast to experiments performed in in vitro preparations, physiological stimulation results in prominent, apical Ca2+ signals which do not propagate and globalize to result in a homogeneous [Ca2+]i throughout the cell, such that steep apical-basal gradients are established.To further investigate this spatial heterogenicity, we performed MP line scans following stimulation at 3–10 Hz stimulation where the scanned line was orientated to interrogate changes in fluorescence originating in the apical region extending through the nucleus to the base of an individual cell. 2+ signals, Python software scripts were written in the Jupyter lab notebook environment to automate the designation of apical ROIs from masks generated from average or SD images. These apical ROIs were then subsequently used to quantitate the magnitude and frequency of signals within these regions . 2+ signal, the relationship between secretion and the frequency of Ca2+ oscillation was less strong  by wide field imaging at 37°C. Individual acinar cells failed to respond to 5 nM CCh; however, at 25 nM CCh single cells within an acinus responded by evoking repetitive Ca2+ transients . This led to periodic increases of [Ca2+] in the basal regions of the cell and hence activation of basal Ca2+ activated K+ (KCa) channels. Activation of the KCa channels is critical for maintenance of the electrochemical potential driving the efflux of Cl- and thus secretion. However, the spatiotemporal information from the current in vivo data is obviously not compatible with these older models. We have therefore constructed a new model, as shown in 2+ released from the apical ER, from IP3R that are situated in close proximity to the TMEM16A  give identical qualitative results. As VPLC increases, the [Ca2+] oscillation frequency increases and the latency decreases. At high stimulation levels, the [Ca2+] oscillation frequency is approximately 0.5–1 Hz, and the oscillations are superimposed on an increasing baseline, which is due to a decreasing cell volume as a result of saliva secretion. These features all agree well with experimental data. One aspect of the data not reproduced by the model is the appearance of very low frequency baseline [Ca2+] spikes at the lowest stimulation frequencies. These baseline spikes are highly likely to be due to stochastic properties of the IP3R, as described by Falcke and others  which has neither apical KCa nor a propagated [Ca2+] wave. Note that the old model with no apical KCa is quantitatively different from the new model with no apical KCa. Fluid secretion in the new model with no apical KCa is slightly greater than that in the old model with no KCa, and is shown in panel F. These simulations show that absence of both a propagated [Ca2+] wave and apical KCa channels significantly decreases saliva secretion. The associated [Ca2+] traces, averaged from the apical and basal regions, as well as from the cytoplasmic region of the cell (representing a domain between these regions), are shown in 2+] is between 25% and 35% of apical [Ca2+], as observed experimentally, while in the model of 2+] is a significantly greater fraction of apical [Ca2+]. The [Ca2+] traces for the model with neither apical KCa nor a propagated wave are very similar to those shown in 2+] is illustrated by the model line scan shown in 2+ is largely restricted to the apical region of the cell. The experimental data show more spatial inhomogeneity (due to the presence of the nucleus and the mitochondria), features which are not included in the model.2+] wave, diffusion of Ca2+ from the apical region will still activate a fraction of the KCa channels. This is illustrated in 2+ from the apical region can activate those basal KCa channels which are situated close to the apical membrane, and this is sufficient to cause significant fluid flow. Furthermore, consistently with previous theoretical work  and KCa1.1 (‘BK’) together with Na/K ATPases are present in the apical PM of salivary acinar cells  were crossed with male heterogenous Mist1CreERT2  to generate Mist1+/– x GCaMP6f+/– transgenic mice. Tamoxifen (0.25 mg/g body weight) dissolved in corn oil was administered to mice at least 6 weeks old of either gender, by oral gavage once a day for 3 consecutive days. Imaging was carried out 1–4 weeks after the last tamoxifen administration. All animal procedures were approved by University Committee on Animal Resources (UCAR-2001-214E).Adult female GCaMP6f2+ signals.The animal was anesthetized with Ketamine  and Xylazine . The animal was restrained with the ventral side up, and the animal’s body temperature was maintained at 37°C by a heat pad during surgery and imaging. A submandibular gland was exposed by making an incision in the skin on the ventral side of the neck. Connective tissue around the gland was teased away using forceps to allow the gland to be raised away from the body but remaining attached by a duct/nerve/blood vessels bundle. A pair of tungsten wires  was inserted to the bundle as stimulation electrodes. The lifted gland was placed on a 10 x 13 mm custom build small holder situated directly above the neck so that the position of the gland was not influenced by movement as a result of breathing. The gland was held by a cover glass to flatten the surface and to keep tissue moist with Hanks salt solution between the holder and the cover glass . A 25x w0 = 0%).For line scan imaging, an acinar cell cluster that arranged in the image plane such that that apical and basal sides of the acinar cell could be clearly identified and the imaging plane depth crossed near the center of the cell. A line was drawn from the apical end, passing through a nucleus, to the basal end of an acinar cell. The scanning speed was 1–1.6 ms per line for 10 s duration. The apical, cytosolic, and basal region were defined as 3 µm wide line within each image, and fluorescent intensity before stimulation for each region served as baseline intensity , bovine serum albumin (0.5%), and Collagenase Type II . Cells were washed twice in HBSS with 0.5% BSA and resuspended in a HBSS solution containing 0.5% BSA and 0.02% trypsin inhibitor. Cells were then resuspended in imaging buffer  and seeded onto a coverslip to allow attachment of cells. Cells were then perfused with imaging buffer and stimulated with agonist. Ca2+ imaging was performed using an inverted epifluorescence Nikon microscope with a 40 X oil immersion objective (NA=1.3) equipped with an environmental chamber allowing control of experimental temperature. Cells were excited at 488 nm from a monochromator, and emission was monitored at 530 nm. Images were captured every 500 ms with an exposure of 10 ms and 4 × four binning using a digital camera driven by TILL Photonics software. Image acquisition was performed using TILLvisION software and data was exported to Microsoft excel and analyzed in Prism (Graph Pad).SMG acinar cells were enzymatically isolated from 2- to 4-month-old, Mist10), and %ΔF/F0 ((F– F0)/ F0 x 100) was calculated using the Image calculator function, so that the converted 32-bit image series represents [Ca2+] changes over time expressed as %ΔF/F0 in 8x8 alley of grids in the XY dimension .Amplitude, latency, and percent of responding areas in fluorescence fields were analyzed using ImageJ software (NIH). Standard Deviation (SD) Images were generated from the image time series by using the Stacks Z project function in image J. XY drift during the imaging, when present, was corrected by applying the Descriptor-based series registration plugin (Stephan Preibisch). The imaging field was divided into 8 by eight grids to yield 64 regions of interest (ROIs) of dimension 32x32 µm square. The average intensity of each ROI grid was generated for each frame. The average of first 100 frames prior to stimulation served as baseline fluorescence . The new software toolset consists of an assembly of Python (https://www.python.org) scripts deployed in a collection of Jupyter Lab (https://jupyter.org) ‘notebooks’. The notebooks were designed to automate repetitive analysis steps and give flexibility to interactively explore and analyze the image data sets and makes use of image processing algorithms in the scikit-image (https://scikit-image.org) package. ROI were identified by generating a difference image whereby the average image prior to stimulation was subtracted from the average image during the period of stimulation on a pixel-by-pixel basis (https://www.scipy.org) package were employed. The script employs a sequence of signal resampling, low-pass zero phase filtering, high-pass zero phase filtering and a generic peak detector followed by a mapping back into the original response data. Detected peaks for each stimulation frequency were plotted in the notebook as black dots overlaid on region summary plots as shown in A computer based, automated region-of-interest (ROI) detection method that specifically targets cellular regions with pronounced changes was developed since no off-the-shelf tool met our particular requirements to efficiently process the data  can diffuse a short distance to activate those KCa channels situated on the basolateral membrane close to the apical membrane. This is why the model exhibits substantial fluid flow even when there are no apical KCa channels; in the absence of apical KCa channels only a small proportion of the basolateral KCa channels will be activated by Ca2+ diffusing from the apical region, but that is sufficient to allow for some fluid flow.The model has two interconnected modules; a Caral data . All ionthe cell  and all 2+ oscillation module is based on a closed-cell Class I model in which oscillations arise via sequential activation and inactivation of the IP3R. [Ca2+] buffering is assumed to be fast and linear, and the IP3R model is taken from 2+ obeys a reaction-diffusion equation in the cell interior, but the effective diffusion coefficient of Ca2+ is small (due to buffering) which allows for localized increases in [Ca2+] which are not propagated throughout the cell. Because the IP3R are situated 50–100 nm from the ClCa , release of Ca2+ through the IP3R is modeled as a boundary flux term, which avoids the necessity for a high-resolution finite element mesh in the apical region, which would greatly slow the calculations. The diffusion coefficient of IP3 is two orders of magnitude greater than that of Ca2+, and thus IP3 is effectively spatially homogeneous throughout the cell.The CaIn the new model, all parameters and equations remain unchanged from those in the old model  with the2+ flux through RyR (VRyR) was set to 0. This is the simplest way of removing RyR from the model.The maximum Ca+] altered to compensate. The apical/basolateral KCa current ratio was varied over a range of values , but for most computations the model assumes that the KCa channels have the same density on the apical and basolateral membranes. We note that the KCa current is also determined by the [Ca2+] immediately adjacent to the membrane, and thus the spatial distribution of Ca2+ will determine the exact apical/basolateral KCa current ratio. It is only when [Ca2+] is spatially homogeneous that this ratio will equal the apical/basolateral surface area ratio.A KCa channel, with the same conductance as the basal KCa channel, was introduced into the apical membrane, with the equations for cytosolic and lumenal i gradients, and how increasing stimulation frequency changes the Ca2+ response? More data are needed to justify the choice of model parameter values.3. The computational model does not adequately reproduce the main features of the Ca2+ signals in vitro is not the case for all exocrine acinar cells. The authors should acknowledge that local apical responses have been seen in other acinar cells (e.g. pancreatic acinar cells) in vitro, upon stimulation with physiological concentrations of hormones, low concentrations of neurotransmitter, or directly by IP3.4. The absence of local apical Ca2+ signaling in vivo and in vitro. While the use of GCaMP6f is a good way to rule out effects of different indicators , other factors are likely to contribute and can be easily tested:5. The current data do not allow a fair comparison of the spatiotemporal aspects of Ca(a) The in vivo experiments were conducted at 37 deg C while the in vitro imaging seems to have been done at room temperature (temperature was not indicated in the Methods). Cooler temperature is likely to affect oscillation frequency as well as the rate of pumping or sequestration which could affect propagation.(b) The concentrations of ACh used in vitro may not relate to the neural stimulation frequencies in vivo. The 100 nM response in vitro is quite large, so it may not be fair to compare this with low- frequency stimulation in vivo where signals are confined to the apical pole. Perhaps more localized signals would result from lower ACh concentrations.6. The method for standard deviation (SD) image processing should be described. This approach highlights areas that oscillate, but may de-emphasize areas of tonic elevation which would also be expected to drive secretion. At 5 Hz stimulation it shows that oscillations are confined mostly to the apical region , but at 10 Hz the cells don't seem to oscillate , so it is not clear why SD images are presented here. It would be more informative to plot a cross-section of GCaMP6 signal from apical to basal before and during stimulation.Reviewer #1:This work offers a new look at the calcium signals that drive secretion in salivary gland cells. Based on studies of isolated cells in vitro, the common view was that calcium signals originating in the apical pole of the cell propagate to the basal pole, so that chloride channels allow chloride ions to exit across the apical membrane while K channels in the basal membrane are opened to maintain the electrical driving force for chloride exit. Through an elegant use of 2-photon microscopy to monitor the spatial spread of calcium signals in intact glands in vivo, the authors describe a different pattern, in which the signals do not propagate but are much more confined to the apical pole of the cell, and they show that secretion occurs even without significant spread of calcium to the basal pole. These results are significant as they challenge current mechanistic models for secretion, and they imply a functional role for K channels in the apical membrane, which until now have been commonly considered to only function in the basolateral membrane.2+ and therefore are sufficient to support secretion. A modified computational model intended to support the need for apical KCa channels does not offer strong support, as it does not faithfully reproduce several of the experimentally observed Ca2+ responses. Finally, the differences between the spatiotemporal aspects of Ca2+ signaling in vivo and in vitro are difficult to assess as critical conditions such as temperature and agonist concentration were not matched in the two experimental scenarios.However, the paper has several shortcomings that need to be addressed to better support a need for revising the current secretion model. It is not clear whether KCa channels in proximal regions of the basolateral membrane are activated by the apically confined CaReviewer #2:2+ signals and salivary secretion in vivo in a mouse model which expressed the Ca2+ indicator protein, GCamp6F, in salivary glands. Previously reported studies measured fluid secretion in vivo by injecting the animals with pilocarpine and collecting the oral secretion or by using protocols where the gland is perfused. However, cytosolic [Ca2+] changes, which are correlated with, and required for, salivary fluid secretion have been predominantly measured in isolated acinar or cell cluster preparations and some in tissue slices. By using intravital multiphoton (MP) microscopy together with stimulation of the neural input to the gland, the authors were able to measure the spatiotemporal properties of Ca2+ signals within the acinar cells and collect saliva that is secreted into the oral cavity. Thus, this study is an important breakthrough in this field as it reveals the exact [Ca2+] changes associated with salivary gland fluid secretion.In this study Dr. Yule and his colleagues describe very impressive and painstaking work in which they have monitored for the first time Ca2+ signals detected in vivo are markedly different from those previously reported using isolated acinar cells (by this group and others) which demonstrated that Ca2+ signals are initiated apically but propagate as a wave globally across the cell without generation of Ca2+ gradients. The present findings show that following moderate stimulation, apically localized oscillatory Ca2+ signals are generated that do not propagate globally and cause minimal secretion. However, at stimulation intensities optimum for secretion, there is a spread of Ca2+ across the cell although with an apical-basal standing Ca2+ gradient. Based on these differences the authors have proposed a revised model which suggests that the apical region contains all the machinery needed for saliva secretion and that Ca2+ released predominately in the apical region, via IP3R, can regulate the function of locally situated ion channels without requirement for a propagated wave of increased [Ca2+] across the cell. As I have noted above the data described in this manuscript provide new understanding of the physiological response of salivary gland to neuronal stimulation and how fine-tuning of the spatiotemporal properties of the Ca2+ signals regulate fluid secretion.This study shows that major spatiotemporal properties of the Ca2+ across the cell and almost 3-fold higher secretion relative to that at 5Hz. While increase in % responding cells at higher stimulus strengths could result in higher total secretion, possible contribution of Ca2+ influx pathways in the spread of Ca2+ in individual acinar cells, e.g. via Orai1 that is localized in the apical region of the cell, cannot be ruled out. Also not clear is whether the spread enhances secretion. Thus, when stimulus is low, secretion is minimal and supported only by release and oscillatory apical [Ca2+]. At higher stimulus strengths there might be an additional Ca2+ influx component which is activated very early after stimulation, likely during IP3-mediated Ca2+ release. Since the basolateral membrane extends all the way to the tight junction, is it possible that several components shown basally localized in the model could actually be quite close to the apical membrane region, near the tight junctions.The main question I have is regarding stimulus strengths that induce optimal levels of secretion (10 and 30Hz) where there is propagation of Ca2+ signals".Thus, it is important to distinguish between initial stimulation of secretion vs regulation of sustained secretion. For example; the last sentence in the abstract clearly, and correctly, states "salivary secretion can be efficiently stimulated by apically localized CaReviewer #3:This paper describes a nice set of experiments where the authors have measured calcium responses in salivary acinar cells from living animals and, importantly, correlated these with measures of salivary secretion. The work is very well performed and the data interesting and definitely an advance in the field. I cannot think of another study that combines, so well, cellular calcium responses with genuine physiological output.StrengthsIntravital images are very good and the combination with fluid secretion gives a nice correlation between calcium responses and physiological output – something which is impossible in vitro.Good initial analysis of the images and convincing data where the calcium responses are further analysed.The conclusions that local calcium responses can drive physiological responses is interesting in the context of salivary glands.WeaknessesThe authors claim that local responses are not seen in vitro is dependent on taking a very narrow view of exocrine cells. Local responses are readily seen in other exocrine cells, like pancreatic acinar cells. These local responses can be driven directly by IP3, low concentrations of neurotransmitter, or physiological concentrations of hormones. Furthermore, these responses drive enzyme secretion. Therefore, although these local responses have not been measured intravitally, it is highly likely that they are the native response in the pancreas.The new model put forward is conjecture. It is built entirely around the observations of localised calcium responses but has no supporting data on changes in membrane potential or activation of specific channels.The in vitro GCaMP data is not convincing. GCaMP and Fura have different affinities for calcium and so this is a serious potential confounder in their experiments. For the in vitro work, the isolated cells are clearly fragmented and the cell chosen is at the edge of the cell cluster. It would be expected that the cells will completely lose their polarisation and structural factors that might limit the calcium response.While I like the analysis, I think that much of it is unnecessary. The authors, for example, show that regions that respond well at low stimulation also respond well at high levels of stimulation. That's fine, but the authors then do not explore what the basis of these differences are.2+ signals in vivo necessitate a new model for salivary fluid secretion" for further consideration by eLife. Your revised article has been evaluated by Kenton Swartz (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "The characteristics of intracellular CaThank you for the revised manuscript with the additional data and clarifications. Your responses have addressed most of the major concerns of the reviewers. The only remaining issue is whether KCa channels in the apical membrane are required for secretion. I think this is central to the significance of the paper. The study shows beautifully how Ca signals are mostly apical and do not propagate to the basal membrane in vivo, but the most interesting implication is that it necessitates having KCa channels near or in the apical membrane. To broaden the appeal of your paper to more than exocrine gland physiologists it will be important to establish this point clearly.We understand that an experimental approach  to establish the role of apical KCa channels is not feasible. Thus, the model offers the best opportunity to test this idea. The model needs a fuller description, not including all the details and equations, but rather a description of the salient features that will allow the reader to grasp how it works, and what assumptions it makes. The comments below summarize the uncertainties in the model's operation that need to be addressed:1. How are apical, cytoplasmic, and basal regions defined in the model, and how do they correspond to the same regions in the experimental data? Throughout the paper there are references to "apical PM" or "apical region." The apical PM is the membrane bounded by tight junctions, but what does "apical region" mean – does this include lateral membrane between the apical PM and the nucleus? The model cartoon in Figure 13 does not explain the distinction between apical and basolateral. Likewise, on p. 12: "We emphasise that, in the model, KCa channels and Na/K- ATPases are also present in the basal membrane, and thus the secretory machinery is not restricted to the apical membrane." Is "basal" here referring to lateral membrane as well? I imagine that some of this vagueness is because the anatomy is complex and the localization of channels is not precisely known, but this needs to be clarified, particularly for non-expert readers.+ current ratio was determined by the apical/basal surface area ratio." After Ca2+ is released from the ER, is it assumed to diffuse to the lateral membrane where it activates KCa channels?  Or is only the basal Ca2+ signal being used to determine KCa current? The data in Figure 10 show that there is significant Ca2+ elevation in the cytoplasmic region below the apical membrane even at low stimulation frequencies, which could activate channels in the lateral membrane. What spatial distribution of KCa channels is assumed in the model? These things need to be clearly and simply explained.2. How is the KCa current simulated? This is a crucial part of the model, but it is not clear how this was done. The only description is (p. 25) "The apical/basal K3. The model shows that with no apical KCa channels  you still get ~80% of the maximal response . This is almost maximal and implies only a modest effect of true apical PM channels. Figure 14F Y axis should be plotted from 0 rather than 60, in order to give a truer impression of the effect of KCa channel localization. There is also some confusion here: Figure 14B shows only 50% of max response if you remove apical KCa channels – but I assume that this is because the total number of channels was not constant in this case, resulting in fewer KCa channels than in Figure 14F. If so, that is not a fair comparison.4. On p. 17 there is a reference to Supp. Figure 4, but that figure was not included.5. I would suggest some rewording of the title, as it seems too focused on what was done, rather than the most significant result. Perhaps you can revise it to focus on localized calcium signaling that engages apical K channels in vivo. Essential revisions:2+ signals associated with secretion in vivo and demonstrating a consequent need for a revised model of apical K channel activation to maintain the driving force for Cl-efflux across the apical membrane and fluid secretion.The major significance of this work rests on the novel pattern of Ca2+ activates KCa channels in the apical membrane is based on the observation of low levels of secretion (<5 Hz stimulation) associated with apically localized Ca oscillations. However, these signals are not strictly localized to the apical membrane and would likely activate KCa channels in the basolateral membrane that are close to the tight junctions. In addition, a large increase in secretion is seen for stimuli (5-10 Hz) that propagate Ca to the basal pole, consistent with previous models incorporating activation of basal KCa channels. The contribution of KCa channels in the apical membrane needs to be demonstrated experimentally. This could be addressed by applying blockers to the apical surface, although this may be difficult. Overall, to justify the model, a need for apical KCa channels or evidence of their contribution to secretion, needs to be shown.1. Is a new model needed to describe secretion in vivo? The argument for a new model in which CaWhile a direct demonstration that apical KCa channels participate in the secretory mechanism would be ideal, we believe that the experiments proposed  is not practically feasible. Specifically, given the architecture of an acinus, it is not possible to effectively isolate the apical plasma membrane (~1-5 % of PM in the center of a group of cells) to apply a pharmacological agent locally with any degree of certainty without it acting basolaterally. In addition, it is difficult to imagine how this could be performed in every apical domain in the gland to assess its effect on fluid secretion into the oral cavity following neural stimulation. Further, this problem is not obviously amenable to any straightforward genetic approach. Our previous data has demonstrated the presence of apical KCa channels in salivary gland acinar cells and we provide a more complete discussion of evidence for their functional importance.2+ signals and secretion. At the least the contributions of Orai1 channels should be discussed, taking into account previously published work.2. The apical Ca signals are ascribed solely to release through IP3R, but Orai1 channels, known to be expressed in these cells, are likely to contribute as well. Preferably, Orai1 inhibitors applied systemically (as was done for the cholinesterase inhibitor) should be tested for effects on Ca2+ signal following extended stimulation with physiological stimulus strengths is blunted by the Orai antagonist. The initial peak is largely unaffected, consistent with the Ca2+ response to sustained stimulation being mediated by Ca2+ release and subsequent Ca2+ influx.We have performed new in vivo experiments using the Orai channel blocker, GSK7975A, which show that the sustained phase of the Ca2+ response. It shows a large initial spike upon stimulation  that is not seen experimentally . It also oscillates at a much lower frequency  than measured in vivo (0.5-1 Hz). In addition, the model needs to be tested under more conditions to confirm that it can account for the experimental data; e.g., does it predict the intracellular [Ca2+]i gradients, and how increasing stimulation frequency changes the Ca2+ response? More data are needed to justify the choice of model parameter values.3. The computational model does not adequately reproduce the main features of the Ca2+ signals observed experimentally. A more detailed discussion of the model and predictions is also now provided, specifically to explicitly state that basolateral KCa channels in the original (and present) model are present throughout the PM to regions directly adjacent to tight junctions. We now test the model responses under more conditions. We show that increasing stimulation increases the frequency of oscillations, and decreases the latency , in agreement with data. We also compare model and experimental line scans and show that the [Ca2+] gradient through the cell in the model is qualitatively similar to that observed experimentally. We test model behaviour for lower values of the IP3 diffusion coefficient, and show that the behaviour remains unchanged . We simulated the model with periodic stimulation of PLC activity, to mimic the effects of periodic neural stimulation, and showed that the [Ca2+] traces and secretion remain essentially unchanged . Again, because of the similarity with the results already shown here we do not include these results explicitly, we just state the result. We now give a more detailed discussion of where the model parameter values are derived. Since these parameter values have been developed over many years, and have already been presented in multiple papers, we do not give full details again in the present submission. For example, the parameters that govern our model of the IPR were determined by fitting the model to single-channel data, and this work is described in a series of papers between 2009 and 2019 (Refs 1-7 below). However, any interested reader is now told more precisely where further details about parameter values can be found.The model has been modified and the computational aspects of these studies have been expanded to test more conditions. The modified model now also more faithfully reflects the characteristics of the Ca1. Gin, E., L.E. Wagner, D.I. Yule, and J. Sneyd, Inositol trisphosphate receptor and ion channel models based on single-channel data. Chaos, 2009. 19(3): p. 037104.2. Gin, E., M. Falcke, L.E. Wagner, 2nd, D.I. Yule, and J. Sneyd, A kinetic model of the inositol trisphosphate receptor based on single-channel data. Biophys J, 2009. 96(10): p. 4053-62.3. Gin, E., M. Falcke, L.E. Wagner, D.I. Yule, and J. Sneyd, Markov chain Monte Carlo fitting of single-channel data from inositol trisphosphate receptors. J Theor Biol, 2009. 257(3): p. 460-74.4. Siekmann, I., L.E. Wagner, 2nd, D. Yule, E.J. Crampin, and J. Sneyd, A kinetic model for type I and II IP3R accounting for mode changes. Biophys J, 2012. 103(4): p. 658-68.5. Siekmann, I., L.E. Wagner, D. Yule, E.J. Crampin, and J. Sneyd, A Park/Drive Model for the Inositol-Trisphosphate Receptor (IPR). Biophysical Journal, 2012. 102(3): p. 110a-110a.6. Cao, P., X. Tan, G. Donovan, M.J. Sanderson, and J. Sneyd, A deterministic model predicts the properties of stochastic calcium oscillations in airway smooth muscle cells. PLoS Comput Biol, 2014. 10(8): p. e1003783.7. Cao, P., G. Donovan, M. Falcke, and J. Sneyd, A stochastic model of calcium puffs based on single-channel data. Biophys J, 2013. 105(5): p. 1133-42.2+ signals in vitro is not the case for all exocrine acinar cells. The authors should acknowledge that local apical responses have been seen in other acinar cells (e.g. pancreatic acinar cells) in vitro, upon stimulation with physiological concentrations of hormones, low concentrations of neurotransmitter, or directly by IP3.4. The absence of local apical Ca2+ signals in acutely isolated pancreatic acinar cells is now included.A discussion of the numerous reports of apically localized Ca2+ signaling in vivo and in vitro. While the use of GCaMP6f is a good way to rule out effects of different indicators , other factors are likely to contribute and can be easily tested:5. The current data do not allow a fair comparison of the spatiotemporal aspects of Ca(a) The in vivo experiments were conducted at 37 deg C while the in vitro imaging seems to have been done at room temperature (temperature was not indicated in the Methods). Cooler temperature is likely to affect oscillation frequency as well as the rate of pumping or sequestration which could affect propagation.(b) The concentrations of ACh used in vitro may not relate to the neural stimulation frequencies in vivo. The 100 nM response in vitro is quite large, so it may not be fair to compare this with low- frequency stimulation in vivo where signals are confined to the apical pole. Perhaps more localized signals would result from lower ACh concentrations.oC, but the Ca2+ are still invariably global at physiological temperature.We have now provided new in vitro data that demonstrates that acini are more sensitive to agonist stimulation and oscillations are more rapid at 376. The method for standard deviation (SD) image processing should be described. This approach highlights areas that oscillate, but may de-emphasize areas of tonic elevation which would also be expected to drive secretion. At 5 Hz stimulation it shows that oscillations are confined mostly to the apical region , but at 10 Hz the cells don't seem to oscillate , so it is not clear why SD images are presented here. It would be more informative to plot a cross-section of GCaMP6 signal from apical to basal before and during stimulation.2+ signal is ~20-30% of that in the apical domain.We describe the method for SD image generation. While we contend that this method is an effective means of visualizing the sub-cellular gradients , we now provide plots of multiple ROI across the acinus to better visualize the gradients . We also provide a new figure where line scans have been performed across an acinus (figure 10). Both these approaches demonstrate that the magnitude of the basal CaReviewer #1:2+ and therefore are sufficient to support secretion.[…] The paper has several shortcomings that need to be addressed to better support a need for revising the current secretion model. It is not clear whether KCa channels in proximal regions of the basolateral membrane are activated by the apically confined Ca2+. This fact has been explicitly stated in the new version of the manuscript. The effect of this localization can be seen in the blue curve in new Figure 14B, which shows how, in the old model modified to have no propagated apical-to-basal Ca2+ wave, there is still an increase in secretion upon stimulation. However, this increase is only about 50% of the saliva secretion attained if apical KCa channels are included, as in the new version of the model .We appreciate the opportunity to clarify this point. In the model the basolateral membrane extends all the way to the tight junction, and thus a fraction of the basal KCa channels are close enough to the apical membrane to be activated by Ca2+ responses.A modified computational model intended to support the need for apical KCa channels does not offer strong support, as it does not faithfully reproduce several of the experimentally observed Ca2+]. In the new version of the model this dependence on [Ca2+] has been removed (as described on new page 24). This change has removed the artifact of the large initial spike, but has made no difference to the conclusions drawn from the model results. The model simulations now closely resemble the data, in that they are oscillations (with the correct frequency of around 0.5 to 1 Hz at high stimulation) superimposed on an increasing baseline as seen in new figure 14 A and discussed on page 12.The large initial peak in the first version of the model has now been corrected by making a slight change to the way in which we model the activity of PLC. In the first version of the model PLC activity was a function of  The main question I have is regarding stimulus strengths that induce optimal levels of secretion (10 and 30Hz) where there is propagation of Ca2+ influx pathways play a major role in maintaining the Ca2+ signal, and thus secretion, especially during prolonged stimulation. We now include data which shows that systemic administration of an Orai channel blocker GSK7579A markedly inhibits in vivo Ca2+ signals to physiologically optimum nervous stimuli. Notably, but only after extended stimulation (~5 min) and has little impact on the initial phase of the Ca2+ rise. These data are included as new figure 4.This is an important point because it is highly likely that Ca2+]. At higher stimulus strengths there might be an additional Ca2+ influx component which is activated very early after stimulation, likely during IP3-mediated Ca2+ release. Since the basolateral membrane extends all the way to the tight junction, is it possible that several components shown basally localized in the model could actually be quite close to the apical membrane region, near the tight junctions.Also not clear is whether the spread enhances secretion. Thus, when stimulus is low, secretion is minimal and supported only by release and oscillatory apical  The authors claim that local responses are not seen in vitro is dependent on taking a very narrow view of exocrine cells. Local responses are readily seen in other exocrine cells, like pancreatic acinar cells. These local responses can be driven directly by IP3, low concentrations of neurotransmitter, or physiological concentrations of hormones. Furthermore, these responses drive enzyme secretion. Therefore, although these local responses have not been measured intravitally, it is highly likely that they are the native response in the pancreas.2+ signals occur at low agonist concentrations and exposure to IP3. Additional references have been added to the text to reflect this. A discussion point is that the majority of these studies were performed in whole-cell patch clamped isolated cells, where cytoplasmic buffering is imposed by the internal pipette solution and which itself could influence the spatial characteristics of the Ca2+ signals and thus it would still be informative to observe Ca2+ signals stimulated in vivo in the exocrine pancreas.The Authors acknowledge that there is a rich literature in isolated pancreatic acinar cells demonstrating that local CaThe new model put forward is conjecture. It is built entirely around the observations of localised calcium responses but has no supporting data on changes in membrane potential or activation of specific channels.2+ signal in vivo, a previous study  exhaustively demonstrated that there were apical KCa3.1 and KCa1.1 in the apical PM of salivary acinar cells, which were activated by local uncaging of Ca2+. A more extensive discussion of this previous data has been added to the text in the revised manuscript.The model has been developed over a number of years and is faithfully built on experimental data. More details are given regarding the model parameters in the revised paper, which have been derived experimentally and published over many years. While it is correct that the present study did not monitor any effectors of the local CaThe in vitro GCaMP data is not convincing. GCaMP and Fura have different affinities for calcium and so this is a serious potential confounder in their experiments. For the in vitro work, the isolated cells are clearly fragmented and the cell chosen is at the edge of the cell cluster. It would be expected that the cells will completely lose their polarisation and structural factors that might limit the calcium response.2+ signal in vitro would strengthen our case that there is a real difference between in vivo and isolated cells. However, we stress that in 20 years working on Ca2+ signaling in salivary gland acinar cells, stimulated by secretagogue or IP3 directly, we have never observed an apical confined Ca2+ rise. We now include additional data, from experiments performed at 37oC and over a wider range of secretagogue concentrations.We agree that a more thorough characterization of the attributes of the CaWhile I like the analysis, I think that much of it is unnecessary. The authors, for example, show that regions that respond well at low stimulation also respond well at high levels of stimulation. That's fine, but the authors then do not explore what the basis of these differences are.2+ signals in salivary acinar cells for the first time is important per se. The response heterogeneity observed is a feature which can only really be meaningful when studied in vivo, given that any variability in response in isolated cells could be due to the isolation procedure. Exploring further the underlying mechanisms will be the subject of future study.The Authors contend that this analysis establishing the “baseline” characteristics of in vivo CaThank you for the revised manuscript with the additional data and clarifications. Your responses have addressed most of the major concerns of the reviewers. The only remaining issue is whether KCa channels in the apical membrane are required for secretion. I think this is central to the significance of the paper. The study shows beautifully how Ca signals are mostly apical and do not propagate to the basal membrane in vivo, but the most interesting implication is that it necessitates having KCa channels near or in the apical membrane. To broaden the appeal of your paper to more than exocrine gland physiologists it will be important to establish this point clearly.We understand that an experimental approach  to establish the role of apical KCa channels is not feasible. Thus, the model offers the best opportunity to test this idea. The model needs a fuller description, not including all the details and equations, but rather a description of the salient features that will allow the reader to grasp how it works, and what assumptions it makes. The comments below summarize the uncertainties in the model's operation that need to be addressed:The Authors wish to thank the Reviewers and Editor for their constructive comments. In response we have made changes to the text and modified figures. We believe this has further clarified our description of the architecture of the acini as it relates to the experimental and computational approaches with the overarching goal of making the paper more accessible to a wider readership. Specifically, this includes a more extensive description of the model, including an additional supplemental figure and additional results of the model simulations. We provide a point-by-point response to the comments provided below.1. How are apical, cytoplasmic, and basal regions defined in the model, and how do they correspond to the same regions in the experimental data? Throughout the paper there are references to "apical PM" or "apical region." The apical PM is the membrane bounded by tight junctions, but what does "apical region" mean – does this include lateral membrane between the apical PM and the nucleus? The model cartoon in Figure 13 does not explain the distinction between apical and basolateral. Likewise, on p. 12: "We emphasise that, in the model, KCa channels and Na/K- ATPases are also present in the basal membrane, and thus the secretory machinery is not restricted to the apical membrane." Is "basal" here referring to lateral membrane as well? I imagine that some of this vagueness is because the anatomy is complex and the localization of channels is not precisely known, but this needs to be clarified, particularly for non-expert readers.2+ signal. It is possible this volume encompasses some lateral membrane in the region defined as “cytoplasmic”, however this does not impact the secretion model because of how this Ca2+ release is modelled (see below).In the introduction, we now provide a paragraph describing the general architecture of exocrine acinar cells, including making it clear that basal and lateral membranes are functionally identical. The cartoon in Figure 13 has been amended to illustrate this architecture. In an effort to define the “apical region” we also highlight published data demonstrating that the apical ER and apical plasma membrane are intimately associated and can be considered a region of privileged communication. In the experimental figures, the apical domain is defined as in the line scan data, where the apical domain is considered to be 3 μm from the apical PM. The line scan and positioning of ROIs in experimental data is designed primarily to illustrate the spatial heterogeneity of the Ca2+ flux is not exactly a boundary flux, but comes through IPR which we know are situated very close (within approximately 50 nm) of the TMEM channels, which are certainly on the apical membrane. However, this simplifying assumption appears to introduce no significant quantitative differences; we know this because we have also run simulations in which the IPR Ca2+ flux occurs in an apical “region” consisting of all the grid points within 3  microns of the apical membrane, and the model simulations show no significant quantitative differences in any of these cases. We now explain this in more detail on new pages 24 and 25.In the model the apical, basal and lateral membranes of the model are clearly defined by anatomical reconstructions from Z stacks in which the TMEM channels and the Na/K ATPases are labelled. This procedure is discussed in detail in previous papers, but here, for clarity, we now include a three-dimensional plot of the cell  on which all the computations in this paper were performed. The acinar membranes are labelled in red and the basolateral membranes in blue. Thus, the reader can now obtain a much clearer intuitive understanding of the model domain, without having to read our previous papers. Although there is no such clear-cut experimentally-based definition of apical or basal “regions”, this is unimportant for the model, in which the concept of apical and basal “regions” plays essentially no role. This is because the IPR flux is modelled as a boundary flux through the apical membrane, while PLC activity is restricted to the basal membrane. Thus, the spatial positioning of these two critical Ca pathways is determined directly (and reasonably precisely) by experimental data. Of course, this requires some simplifying assumptions in the model; for example, the IPR CaAlthough the model does technically make a distinction between the basal membranes and the lateral membranes  this distinction is of no importance in2+ released from the apical “region”  can diffuse to neighboring basolateral surface triangles and activate KCa channels on the basolateral surface. This is why the model exhibits substantial fluid flow even when there are no KCa channels in the apical membrane.the current simulations, which (without exception) assume that all the ion channels and transporters  are uniformly distributed across both the basal and lateral membranes. This means that Ca+ current ratio was determined by the apical/basal surface area ratio." After Ca2+ is released from the ER, is it assumed to diffuse to the lateral membrane where it activates KCa channels?  Or is only the basal Ca2+ signal being used to determine KCa current? The data in Figure 10 show that there is significant Ca2+ elevation in the cytoplasmic region below the apical membrane even at low stimulation frequencies, which could activate channels in the lateral membrane. What spatial distribution of KCa channels is assumed in the model? These things need to be clearly and simply explained.2. How is the KCa current simulated? This is a crucial part of the model, but it is not clear how this was done. The only description is (p. 25) "The apical/basal K2+] in the tetrahedron that contains that triangle. Thus Ca2+ released from the apical region can diffuse a short distance to the neighboring basolateral membrane to activate KCa channels there.We now explain in much more detail (new page 25) exactly how activation of the KCa channels works in the model. Briefly , the channels on each surface triangle are affected only by the average [Ca3. The model shows that with no apical KCa channels  you still get ~80% of the maximal response . This is almost maximal and implies only a modest effect of true apical PM channels. Figure 14F Y axis should be plotted from 0 rather than 60, in order to give a truer impression of the effect of KCa channel localization. There is also some confusion here: Figure 14B shows only 50% of max response if you remove apical KCa channels – but I assume that this is because the total number of channels was not constant in this case, resulting in fewer KCa channels than in Figure 14F. If so, that is not a fair comparison.necessary for fluid flow, appear to be required for optimal fluid flow, hence the change in the title for the manuscript. We have modified the figure as requested by the reviewer and added simulations for additional levels of stimulus strengths.In the previous resubmission we acknowledged that the model predicts that the absence of apical KCa channels reduces salivary secretion by ~25%. We contend that this is not insignificant. Nevertheless, there is indeed only a partial effect from the presence of apical KCa channels, as the model is capable of significant fluid secretion in their absence. We now emphasize more strongly throughout that the model predicts that apical KCa channels, although not old model, not the new one, which explains the quantitative differences with Figure 14F. The curves in Figure 14 B are designed to show how the old model is poor at generating fluid flow in the absence of apical KCa, and thus a new model is needed. Of course, we note that even the old model can generate some fluid flow even in the absence of apical KCa. In both models it is simply a matter of how much fluid flow there is, not whether fluid flow exists at all.We also now explain at greater length  that the curve with no apical KCa in Figure 14B corresponds to simulations from the 4. On p. 17 there is a reference to Supp. Figure 4, but that figure was not included.Supp Figure 4-  is now included.5. I would suggest some rewording of the title, as it seems too focused on what was done, rather than the most significant result. Perhaps you can revise it to focus on localized calcium signaling that engages apical K channels in vivo.2+ signals promote optimal salivary gland fluid secretion”, which we feel better reflects the data.We have amended the title to “Highly Localized intracellular Ca