Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Essential role of STIM1 in the development of cardiomyocyte hypertrophy

Store-operated Ca²⁺ entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Characterization of the 1,25-dihydroxycholecalciferol-stimulated calcium influx pathway in CaCo-2 cells

The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃)-induced increase in intracellular Ca²⁺ ([Ca²⁺] _i ) in individual CaCo-2 cells. In the presence of 2mm Ca²⁺, 1,25(OH)₂D₃-induced a rapid transient rise in [Ca²⁺] _i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca²⁺-free buffer, this hormone still induced a transient rise in [Ca²⁺] _i , although of lower magnitude, but [Ca²⁺] _i then subsequently fell to baseline. In addition, 1,25(OH)₂D₃ also rapidly induced⁴⁵Ca uptake by these cells, indicating that the sustained rise in [Ca²⁺] _i was due to Ca²⁺ entry. In Mn²⁺-containing solutions, 1,25(OH)₂D₃ increased the rate of Mn²⁺ influx which was temporally preceded by an increase in [Ca²⁺] _i . The sustained rise in [Ca²⁺] _i was inhibited in the presence of external La³⁺ (0.5mm). 1,25(OH)₂D₃ did not increase Ba²⁺ entry into the cells. Moreover, neither high external K⁺ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca²⁺ channel agonist, alone or in combination, were found to increase [Ca²⁺] _i , 1,25(OH)₂D₃ did, however, increase intracellular Na⁺ in the absence, but not in the presence of 2mm Ca²⁺, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca²⁺ channels. 1,25(OH)₂D₃ does appear to activate a La³⁺-inhibitable, cation influx pathway in CaCo-2 cells.     

Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry

Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.     

Characterization of cardiomyocyte excitation-contraction coupling in the FVB/N-C57BL/6 intercrossed "chocolate" brown mice

Mice are extensively used for gene modification research and isolated cardiomyocytes are essential for evaluation of cardiac function without interference from non-myocyte contribution. This study was designed to characterize cardiomyocyte excitation-contraction coupling in FVB/N-C57BL/6 intercrossed brown mice. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix softedge system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca(2+) rise and decay rate. Resting cell length was longer in age- and gender-matched C57BL/6 and brown mice compared to FVB strain. PS and +/- dL/dt were significantly lower in brown mice compared to FVB/N and C57BL/6 groups. TPS was shortened in C57BL/6 mice and TR(90) was prolonged in brown mice compared to other groups. Resting intracellular Ca(2+) level and single exponential intracellular Ca(2+) decay constant were comparable among all three mouse lines. Rise in intracellular Ca(2+) in response to electrical stimulus was higher in C57BL/6 mouse myocytes whereas bi-exponential intracellular Ca(2+) decay was faster in brown mice. Myocytes from all three groups exhibited similar fashion of reduction in PS in response to increased stimulus frequency. These data suggest that inherent differences in cardiomyocyte excitation-contraction coupling exist between strains, which may warrant caution when comparing data from these mouse lines.     

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+).     

Direct interaction and functional coupling between voltage-gated CaV1.3 Ca channel and GABAB receptor subunit 2

Although Ca_V1.2 and Ca_V1.3 are two subtypes of L-type Ca²⁺ channels expressed in the CNS, functions of Ca_V1.3 have not been well elucidated compared to Ca_V1.2. Here, we found that Ca_V1.3-NT associates with GABA_BR2-CT using yeast two-hybrid, GST pull-down and co-immunoprecipitation assays. We also demonstrated co-localization of Ca_V1.3 and GABA_BR2 in HEK293 cells and cultured hippocampal neurons. Whole-cell patch-clamp and Ca²⁺-imaging experiments revealed that activation of GABA_BR increases Ca_V1.3 currents and intracellular Ca²⁺ via Ca_V1.3, but not Ca_V1.2. These results show a physical and functional interaction between Ca_V1.3 and GABA_BR, suggesting the potential pivotal roles of Ca_V1.3 in the CNS.

Structured summary

MINT-7975667: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by two hybrid (MI:0018)MINT-7975740: Cav1.3 (uniprotkb:P27732) and GABABR2 (uniprotkb:O75899) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7966007, MINT-7966016: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by anti bait coimmunoprecipitation (MI:0006)MINT-7975712, MINT-7975691: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by pull down (MI:0096)MINT-7966026: GABABR2 (uniprotkb:O88871) and Cav1.3 (uniprotkb:P27732) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Basal and ethanol-induced cardiac contractile response in lean and obese zucker rat hearts

Obesity plays a pivotal role in metabolic and cardiovascular diseases. Certain types of obesity may be related to alcohol ingestion, which itself leads to impaired cardiac function. This study analyzed basal and ethanol-induced cardiac contractile response using left-ventricular papillary muscles and myocytes from lean and obese Zucker rats. Contractile properties analyzed include: peak tension development (PTD), peak shortening amplitude (PS), time to PTD/PS (TPT/TPS), time to 90% relaxation/relengthening (RT(90)/TR(90)) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT and +/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes and fluorescence decay time (FDT). In papillary muscles from obese rats, the baseline TPT and RT(90) were significantly prolonged accompanied with low to normal PTD and +/-VT compared to those in lean rats. Muscles from obese hearts also exhibited reduced responsiveness to postrest potentiation, increase in extracellular Ca(2+) concentration, and norepinephrine. By contrast, in isolated myocytes, obesity reduced PS associated with a significant prolonged TR(90), normal TPS and +/-dL/dt. Intracellular Ca(2+) recording revealed decreased resting Ca(2+) levels and prolonged FDT. Acute ethanol exposure (80-640 mg/dl) caused comparable concentration-dependent inhibitions of PTD/PS and DeltaFFI, associated with reduced +/-VT in both groups. Collectively, these results suggest altered cardiac contractile function and unchanged ethanol-induced depression in obesity.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Altered Ca2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca(2+) transients and Ca(2+) sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca(2+)](i) amplitude and an increased time constant for decay (tau), both of which correlated with a reduced Ca(2+) content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca(2+) sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability (P(o)) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca(2+) sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca(2+) homeostasis observed in senescent myocytes.     

Intracellular Ca2+ in pancreatic acinar cells: Regulation and role in stimulation of enzyme secretion

Evidence for a primary role for intracellular Ca²⁺ in the stimulation of pancreatic enzyme secretion is reviewed. Measurements of cytoplasmic free Ca²⁺ concentration have allowed direct demonstration of its importance in triggering enzyme secretion and defined the concentration range over which membrane Ca²⁺ pumps must work to regulate intracellular Ca²⁺. Current evidence suggests a key role for the Ca²⁺ Mg-ATPase of rough endoplasmic reticulum in regulating intracellular Ca²⁺ and accumulating a Ca²⁺ store which is released by the action of inositol-l,4,5 trisphosphate following stimulation of secretion.Abbreviations Used EGTA (ethylene dioxy) diethylene-dinitrilotetraacetic acid - BAPTA 1,2-bis (2-aminophenoxy) ethane NNN,N-tetracetic acid - InsP₃ inositol trisphosphate - Ins-1,4,5P₃ and Ins-1,3,4P₃ isomers of inositol trisphosphate with the position of phosphate groups assigned - Ins-1,3,4,5P₄ inositol tetrakisphosphate     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

(Ca2+ + Mg2+) ATPase activity in kidney basolateral membrane in diabetes: role of atrial natriuretic peptide

Summary The (Ca²⁺ + Mg²⁺) ATPase which serves as a Ca²⁺ pump in the kidney basolateral membranes is essential to the maintenance of an intracellular Ca²⁺ concentration optimal for kidney function. Since atrial natriuretic peptide (ANP) is known to participate in the Ca²⁺ homeostasis mechanism, altered levels of ANP in diabetes may vary the pump activity and consequently the kidney function. In order to examine the modulatory role of ANP on (Ca²⁺ + Mg²⁺) ATPase in short- (6 weeks) and long-term (6 months) diabetes, rats were injected with streptozotocin (65 mg/kg body wt, i.v.). At 6 weeks, the plasma ANP was decreased whereas, ANP-receptor binding in the kidney basolateral membrane was increased. In contrast, there was an increased plasma ANP and decreased ANP receptor binding at 6 months. Insulin treatment to diabetic animals normalized these parameters. The (Ca²⁺ + Mg²⁺) ATPase activity was unchanged both at 6 weeks and 6 months. Our results demonstrate that the unchanged Ca²⁺ pump activity in short-term and long-term diabetes serves to maintain the Ca²⁺ homeostasis in the kidney cells and thus may maintain the hyperfiltration state in diabetes. Unaltered (Ca²⁺ + Mg²⁺) ATPase is achieved by the initial up-regulation and subsequent down-regulation of the ANP receptors.