The bulk of Ca2+ released to the myoplasm is free in the sarcoplasmic reticulum and does not unbind from calsequestrin

Calsequestrin (CS) is the major Ca2+ binding protein contained in the lumen of sarcoplasmic reticulum (SR). Ca2+ binding properties and tissue concentration of CS of frog skeletal muscle were measured. At equilibrium, maximal Ca2+ binding capacity of purified CS was about 1.2 mumol Ca2+/mg protein. Apparent Kds for Ca2+ were around 50 microM in the absence of salts, around 0.9 mM in the presence of 100 mM KCl, and around 1.1 mM under 'physiological' conditions. Quantitation of CS in homogenates was accomplished by three methods (Stains-all staining, immunoblotting and 45Ca ligand overlay). Frog muscle contained about 0.5 mg of CS/g wet weight, that is 6.1 mM CS inside the SR. At rest the in situ free [Ca2+] of SR was calculated to be 3.6 mM, and, thus, CS is largely saturated with Ca2+. Moreover, computer simulations of Ca2+ release indicated that about 75% of Ca2+ released during a twitch is free in the SR and does not unbind from CS.     

Heterogeneity of microsomal Ca2+ stores in chicken Purkinje neurons.

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca2+ stores, i.e. the Ca2+ pump, the receptors for inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca2+ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP3 receptor, while the Ca2+ pump was present in both intermediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immunogold labeling of ultrathin cryosections with anti-CS and anti-IP3 receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP3 receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP3 receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP3 and the rest possibly sensitive to Ry.     

Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.     

P400 protein is one of the major substrates for Ca2+/calmodulin-dependent protein kinase II in the postsynaptic density-enriched fraction isolated from rat cerebral cortex, hippocampus and cerebellum.

Concanavalin A-binding glycoprotein with 250 K M(r) found in the postsynaptic density (PSD)-enriched preparation (or synaptic cytoskeleton) from rat cerebellum was identified with P400 protein from the physicochemical properties and enrichment in the cerebellum. Proteins homologous to the cerebellar 250 K M(r) protein occurred in the PSD-enriched preparations from rat cerebral cortex and from hippocampus, although the contents in the preparations were very low. The 250 K M(r) proteins in the PSD-enriched preparations from cerebellum and from cerebrum were highly phosphorylated by Ca2+/calmodulin (CaM)-dependent protein kinase II. The protein of synaptic plasma membrane (SPM) and PSD-enriched fractions prepared from cerebral cortex were not phosphorylated by the cAMP-dependent protein kinase endogenous to the fractions, whereas the protein from cerebellum was done in SPM and PSD-enriched fractions. The facts suggest that P400 or P400-like protein is closely associated with Ca2+/CaM-dependent protein kinase II in the PSD-enriched preparations, especially in the preparation from cerebral cortex. Phosphorylation of the protein by Ca2+/CaM-dependent protein kinase II may play an important role in the postsynaptic function in both cerebellum and at least in some areas of cerebrum.     

Calciosome, a sarcoplasmic reticulum-like organelle involved in intracellular Ca2+-handling by non-muscle cells: studies in human neutrophils and HL-60 cells

Calciosomes are intracellular organelles in HL-60 cells, neutrophils and various other cell types, characterized by their content of a Ca2+-binding protein that is biochemically and immunologically similar to calsequestrin (CS) from muscle cells. In subcellular fractionation studies the CS-like protein copurifies with functional markers of the inositol 1,4,5-trisphosphate (IP3) releasable Ca2+-store. These markers (ATP-dependent Ca2+-uptake and IP3-induced Ca2+-release) show a subcellular distribution which is clearly distinct from the endoplasmic reticulum and other organelles. In morphological studies, antibodies against rabbit skeletal muscle CS protein specifically stained hitherto unrecognized vesicles with a diameter between 50 and 250 nm. Thus both, biochemical and morphological studies indicate that the calsequestrin containing intracellular Ca2+-store, now referred to as the calciosome, is distinct from other known organelles such as endoplasmic reticulum. Calciosomes are likely to play an important role in intracellular Ca2+-homeostasis. They are possibly the intracellular target of inositol 1,4,5-trisphosphate and thus the source of Ca2+ that is redistributed into the cytosol following surface receptor activation in non-muscle cells.     

Glycosylation site for chondroitin sulfate on the neural part-time proteoglycan, neuroglycan C

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate (CS) proteoglycan that is expressed predominantly in the central nervous system (CNS). NGC dramatically changed its structure from a proteoglycan to a nonproteoglycan form with cerebellar development, whereas a small portion of NGC molecules existed in a nonproteoglycan form in the other areas of the mature CNS, suggesting that the CS glycosylation of NGC is developmentally regulated in the whole CNS. As primary cultured neurons and astrocytes from cerebral cortices expressed NGC in a proteoglycan form and in a nonproteoglycan form, respectively, CS glycosylation seems to be regulated differently depending on cell type. To investigate the glycosylation process, cell lines expressing a proteoglycan form of NGC would be favorable experimental models. When a mouse NGC cDNA was transfected into COS 1, PC12D, and Neuro 2a cells, only Neuro 2a cells, a mouse neuroblastoma cell line, expressed NGC bearing CS chains. In PC12D cells, although three intrinsic CS proteoglycans were detected, exogenously expressed NGC did not bear any short CS chains just like NGC in the mature cerebellum. This suggests that the addition of CS chains to the NGC core protein is regulated in a manner different from that of other CS proteoglycans. As the first step in investigating the CS glycosylation mechanism using Neuro 2a cells, we determined the CS attachment site as Ser-123 on the NGC core protein by site-directed mutagenesis. The CS glycosylation was not necessary for intracellular trafficking of NGC to the cell surface at least in Neuro 2a cells.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

Regional expression and subcellular localization of the voltage-gated calcium channel β subunits in the developing mouse brain

J. Neurochem. (2012) 122, 1095-1107. ABSTRACT: Ca(2+) channel β subunits determine the maturation, biophysical properties and cell surface expression of high voltage-activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Ca(v) β subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Ca(v) β(1) , Ca(v) β(3) and Ca(v) β(4) protein levels increased with age, although there were marked region- and developmental stage-specific differences in their expression. Ca(v) β(1) was predominantly expressed in the strata oriens and radiatum of the hippocampus, and only weakly in the cerebellum. The Ca(v) β(3) subunit was mainly expressed in the strata radiatum and lucidum of the hippocampus and in the molecular layer of the cerebellum. During development, Ca(v) β(3) protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the hippocampus increased with age throughout all subfields. Ca(v) β(4) protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Ca(v) β(4) was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Ca(v) β(1) and Ca(v) β(3) were predominantly located post-synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Ca(v) β(4) subunits were detected in the pre-synaptic and post-synaptic compartments of both regions, albeit more strongly at post-synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Ca(v) β subunits, and their possible role in pre- and post-synaptic transmission.     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

Toward the elucidation of the mechanism of attachment and entry of malaria sporozoites into cells: synthetic polypeptides from the circumsporozoite protein of Plasmodium falciparum bind Ca2+ and interact with model phospholipid membranes

Through the joint use of CD, Fourier transform ir (FTIR), and attenuated total reflectance FTIR we have found that synthetic polypeptide models of the Plasmodium falciparum circumsporozoite (CS) protein repeat domain bind calcium ions in helicogenic environments. Ca(2+)-(NANP)n complexes (n greater than or equal to 20) interact vectorially with model phospholipid membranes orienting their polypeptide axes preferentially along those of the lipid acyl chains. It is proposed that the P. falciparum CS protein central region, rather than acting as a molecular lure helping the parasite to evade host immune control, plays, as a specific Ca2+ macroligand, a critical functional role during attachment, invasion, and development of the malaria parasite in the hepatic cell.     

Stimulation of excitatory amino acid release from adult mouse brain glia subcellular particles by high mobility group box 1 protein

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [(3)H]d-aspartate form gliosomes in a concentration-dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [(3)H]d-aspartate was independent of modifications of cytosolic Ca(2+) , but it was blocked by dl-threo-beta-benzyloxyaspartate (dl-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca(2+)-independent and dl-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca(2+). Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca(2+). These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.     

The cortico-basal ganglia-thalamocortical circuit with synaptic plasticity. I. Modification rules for excitatory and inhibitory synapses in the striatum

It is pointed out that Ca(2+)-dependent modification rules for NMDA-dependent (NMDA-independent) synaptic plasticity in the striatum are similar to those in the neocortex and hippocampus (cerebellum). A unitary postsynaptic mechanism of synaptic modification is proposed. It is based on the assumption that, in diverse central nervous system structures, long-term potentiation/depression (LTP/LTD) of excitatory transmission (depression/potentiation of inhibitory transmission, LTDi/LTPi) is the result of an increasing/decreasing the number of phosphorylated AMPA and NMDA (GABA(A)) receptors. According to the suggested mechanism, Ca(2+)/calmodulin-dependent protein kinase II and protein kinase C, whose activity is positively correlated with Ca(2+) enlargement, together with cAMP-dependent protein kinase A (cGMP-dependent protein kinase G, whose activity is negatively correlated with Ca(2+) rise) mainly phosphorylate ionotropic striatal receptors, if NMDA channels are opened (closed). Therefore, the positive/negative post-tetanic Ca(2+) shift in relation to a previous Ca(2+) rise must cause NMDA-dependent LTP+LTDi/LTD+LTPi or NMDA-independent LTD+LTPi/LTP+LTDi. Dopamine D(1)/D(2) or adenosine A(2A)/A(1) receptor activation must facilitate LTP+LTDi/LTD+LTPi due to an augmenting/lowering PKA activity. Activation of muscarinic M(1)/M(4) receptors must enhance LTP+LTDi/LTD+LTPi as a consequence of an increase/decrease in the activity of protein kinase C/A. The proposed mechanism is in agreement with known experimental data.     

Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.     

Evidence that spinach leaves express calreticulin but not calsequestrin.

The presence of either calreticulin (CR) or calsequestrin (CS-like proteins in spinach (Spinacia oleracea L.) leaves has been previously described. Here we report the purification from spinach leaves of two highly acidic (isoelectric point 5.2) Ca(2+)-binding proteins of 56 and 54 kD by means of DEAE-cellulose chromatography followed by phenyl-Sepharose chromatography in the presence of Zn(2+) (i.e., under experimental conditions that allowed the purification of CR from human liver). On the other hand, we failed to identify any protein sharing with animal CS the ability to bind to phenyl-Sepharose in the absence of Ca(2+). Based on the N-terminal amino acid sequence, the 56- and 54-kD spinach Ca(2+)-binding proteins were identified as two distinct isoforms of CR. Therefore, we conclude that CR, and not CS, is expressed in spinach leaves. The 56-kD spinach CR isoform was found to be glycosylated, as judged by ligand blot techniques with concanavalin A and affinity chromatography with concanavalin A-Sepharose. Furthermore, the 56-kD CR was found to differ from rabbit liver CR in amino acid sequence, peptide mapping after partial digestion with Staphylococcus aureus V8 protease, pH-dependent shift of electrophoretic mobility, and immunological cross-reactivity with an antiserum raised to spinach CR, indicating a low degree of structural homology with animal CRs.     

[3H]N-Methylscopolamine binding to heart atrium and four brain regions from the mallard (Anas platyrhynchos)

1. Binding characteristics of membrane preparations from four brain regions and heart (HT) right artria from mallard ducks were documented using the muscarinic ligand [³H]N-methylscopolamine([³H]NMS).2. Brain regions used were: cerebellum (CL), medulla/pons (MP), corpus striatum (CS), and hippocampus (HI). Cholinesterase (ChE) activity from these same tissues was also measured.3. High affinity binding sites were identified in all tissue preparations, and were shown to be saturable and linear. Maximal number of [³H]NMS binding sites (Bmax) (fmoles [³H]NMS/mg protein) were: CL = 90.6, MP = 104.1, HI = 276.0, CS = 229.3, HT = 65.52. Dissociation constants (K_D) (nM [³H]NMS) for the same tissues were: CL = 0.204, MP = 0.293, HI = 0.347, CS = 0.163, HT = 0.447.     

A functional dermatan sulfate epitope containing iduronate(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate) disaccharide in the mouse brain: demonstration using a novel monoclonal antibody raised against dermatan sulfate of ascidian Ascidia nigra

Oversulfated chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid structures bind growth factors, promote the neurite outgrowth of hippocampal neurons in vitro, and have been implicated in the development of the brain. To investigate the expression of functional oversulfated DS structures in the brain, a novel monoclonal antibody (mAb), 2A12, was generated against DS (An-DS) from ascidian Ascidia nigra, which contains a unique iD disaccharide unit, iduronic acid (2-O-sulfate)alpha1-->3GalNAc(6-O-sulfate), as a predominant disaccharide. mAb 2A12 specifically reacted with the immunogen, and recognized iD-enriched decasaccharides as minimal structures. The 2A12 epitope was specifically observed in the hippocampus and cerebellum of the mouse brain on postnatal day 7, and the expression in the cerebellum disappeared in the adult brain, suggesting a spatiotemporally regulated expression of this epitope. Embryonic hippocampal neurons were immunopositive for 2A12, and the addition of the antibody to the culture medium significantly reduced the neurite growth of hippocampal neurons. In addition, two minimum 2A12-reactive decasaccharide sequences with multiple consecutive iD units were isolated from the An-DS chains, which exhibited stronger inhibitory activity against the binding of various growth factors and neurotrophic factors to immobilized embryonic pig brain CS/DS chains (E-CS/DS) than the intact E-CS/DS, suggesting that the 2A12 epitope at the neuronal surface acts as a receptor or co-receptor for these molecules. Thus, we have selected a unique antibody that recognizes iD-enriched oversulfated DS structures, which are implicated in the development of the hippocampus and cerebellum in the central nervous system. The antibody will also be applicable for investigating structural alterations in CS/DS in aging and pathological conditions.     

Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum.

A brain-specific multifunctional calmodulin-dependent protein kinase, calmodulin-dependent protein kinase IV, which exhibited characteristic properties quite different from those of calmodulin-dependent protein kinase II, was purified approximately 230-fold from rat cerebellum. The purified preparation gave two protein bands with molecular weights of 63,000 (alpha) and 66,000 (beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both of which showed protein kinase activity as examined by the activity gel method. The molecular weight of the enzyme was estimated as about 67,000 from sedimentation coefficient (3.2 S) and Stokes radius (50 A), indicating a monomeric structure of the enzyme. The enzyme phosphorylated smooth muscle myosin light chain, synapsin I, microtubule-associated protein 2, tau protein, myelin basic protein, histone H1, and tyrosine hydroxylase in a Ca2+/calmodulin dependent manner, suggesting that the enzyme is a multifunctional calmodulin-dependent protein kinase capable of phosphorylating a large number of substrates. A synthetic peptide, Lys-Ser-Asp-Gly-Gly-Val-Lys-Lys-Arg-Lys-Ser-Ser-Ser-Ser, was found to be a specific substrate for this kinase and, using this peptide as substrate, the distribution of the enzyme activity in various rat tissues was examined. The activity was found in cerebral cortex, brain stem, and cerebellum, most abundantly in cerebellum, but other tissues tested, including liver, spleen, kidney, lung, heart, skeletal muscle, and adrenal gland showed very little activity.     

Purification and characterization of a human NO synthase.

A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L-citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system.     

Molecular cloning of stanniocalcin 1 and its extracorpuscular regulation by salinity and Ca2+ in the Japanese flounder

Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca2+ ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1-25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca2+ increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca2+ level.