Free Ca Levels Research Articles

Formation of a Complex of the Catalytic Subunit of Pyruvate Dehydrogenase Phosphatase Isoform 1 (PDP1c) and the L2 Domain Forms a Ca2+ Binding Site and Captures PDP1c as a Monomer

Pyruvate dehydrogenase phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca(2+)-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca(2+) was evaluated in activity and L2 binding studies. An increase in the Mg(2+) concentration decreased the Ca(2+) concentration required for half-maximal activation of PDP1 from 3 to 1 microM, but this parameter was unchanged at 3 microM with PDP1c. Near 1 microM Ca(2+), tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg(2+). With just Ca(2+) included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c.Ca(2+).L2 complex is supported by micromolar Ca(2+) concentrations and becomes sensitive to the Mg(2+) level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 +/- 2.5 microM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K(d) of approximately 0.3 microM. Isothermal titration calorimetry and (45)Ca(2+) binding studies failed to detect binding of Ca(2+) (<100 microM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca(2+), and complex formation hinders the tendency of PDP1c to form a dimer.

Inhibition of Growth Hormone Secretion in Mild Primary Hyperparathyroidism

Introduction: Impairment in growth hormone (GH) secretion has been reported to occur in primary hyperparathyroidism (PHP) with strikingly elevated (>150 pg/ml) plasma PTH and free Ca levels. Patients with these characteristics are relatively few, whereas the great majority of patients with biochemically diagnosed PHP are asymptomatic and show borderline or slightly elevated plasma PTH and Ca levels. We wondered whether also patients in these latter conditions show a defective GH secretory pattern. Methods: In order to answer this question, 8 female subjects (mean age ± SE: 44 ± 1.3 years) were selected at the time of a checkup examination from a larger population of persons in fairly good clinical condition. Inclusion criteria were plasma PTH values slightly above the normal range (up to 50% higher than the maximum limit) with free Ca levels in the upper normal range or slightly higher (experimental group). Normal values in our laboratory are ionized calcium: 1.22–1.42 mmol/ml and plasma PTH: 12–72 pg/ml. A group of 15 age-matched healthy women with plasma PTH and Ca levels in the middle normal range and significantly lower than values found in the experimental group was also selected and used as control. Experimental and control groups were tested with arginine [0.5 mg/kg body weight (BW)] infused intravenously over 30 min and arginine plus GH-releasing hormone (GHRH; 1 µg/kg BW in an intravenous bolus injection). The GH responses to these challenging stimulations were compared between groups. Results: Basal serum GH values were similar in all subjects. Both arginine and arginine plus GHRH induced a significant GH rise in both groups; however, the GH responses were significantly lower in the experimental than in the control group. Mean GH peak was 27.7 and 14.6 times higher than baseline after arginine and 57.5 and 26.6 times higher than baseline after arginine plus GHRH in the control and experimental group, respectively. No significant correlation was observed between PTH or Ca levels and the GH responses to challenging stimuli in any group. Conclusion: These data show that impairment in GH secretion is associated with slightly elevated levels of PTH in the presence of serum Ca values in the upper normal range. GH responses to stimulations were reduced by about 50% in our hyperparathyroid subjects. A long-time duration of this relatively small decline of GH secretory activity may be supposed to contribute to age-related catabolic processes in a large number of patients with mild primary hyperparathyroidism.

Histamine reduces gap junctional communication of human tonsil high endothelial cells in culture

The regulation of gap junctional communication by histamine was studied in primary cultures of human tonsil high endothelial cells (HUTECs). We evaluated intercellular communication, levels, state of phosphorylation, and cellular distribution of gap junction protein subunits, mainly connexin (Cx)43. Histamine induced a time-dependent reduction in dye coupling (Lucifer yellow) associated with reduction in connexin43 localized at cell–cell appositions (immunofluorescence), without changes in levels and phosphorylation state of connexin43 (immunoblots). These effects were prevented with chlorpheniramine, an H 1 receptor blocker; indomethacin, a cyclooxygenase blocker; or GF109203X, a protein kinase C inhibitor. Treatment with phorbol myristate acetate, a protein kinase C activator, and 4bromo (4Br)-A23187, a calcium ionophore, mimicked the histamine-induced effects on dye coupling. 8Bromo-cAMP doubled the dye coupling extent and prevented the histamine-induced reduction in incidence of dye coupling. After 24-h histamine treatment, known to desensitize H 1 receptors, reapplication of histamine increased cell coupling in a way prevented by ranitidine, an H 2 receptor blocker. Thus, activation of H 1 and H 2 receptors, which increase intracellular levels of free Ca 2+ and cAMP, respectively, may affect gap junctional communication in opposite ways. Stabilization of actin filaments with phalloidine diminished but did not totally prevent histamine-induced cell shape changes and reduction in dye coupling. Hence, the histamine-induced reduction in gap junctional communication between HUTEC is mediated by cytoskeleton-dependent and -independent mechanisms and might contribute to modulate endothelial function in lymphoid tissue.

Prefibrillar Amyloid Protein Aggregates Share Common Features of Cytotoxicity

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

Elicitation of reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a Ca2+-dependent process involving simultaneous activation of NADPH oxidase and cytochrome oxidase genes.

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals.

Expression and proteolytic activity of calpain in lens epithelial cells of oxidative cataract.

To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca(2+), the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H(2)O(2)-induced cataract. Rat lenses were cultured in vitro and cataract was induced by H(2)O(2). The level of intracellular free Ca(2+) was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate. There were significant differences of the level of intracellular free Ca(2+) (P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity of calpain (P=0.001, 0.000, 0.000) between H(2)O(2)-induced and control group at 6, 12 and 24 h, respectively. H(2)O(2) can increase intracellular free Ca(2+), then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

Ca 2+ dependent k + efflux in hypertension: an 39k-nmr spectroscopic study

P-387 Key Words: Calcium, Potassium,

Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors: IMPLICATIONS FOR PHOTORECEPTOR GUANYLYL CYCLASE (RetGC) REGULATION IN MAMMALIAN PHOTORECEPTORS

Guanylyl cyclase-activating proteins (GCAP) are EF-hand Ca(2+)-binding proteins that activate photoreceptor guanylyl cyclase (RetGC) in the absence of Ca(2+) and inhibit RetGC in a Ca(2+)-sensitive manner. The reported data for the RetGC inhibition by Ca(2+)/GCAPs in vitro are in disagreement with the free Ca(2+) levels found in mammalian photoreceptors (Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., and Fain, G. L. (2002) J. Physiol. (Lond.) 542, 843-854). We have found that binding of Mg(2+) dramatically affects both Ca(2+)-dependent conformational changes in GCAP-1 and Ca(2+) sensitivity of RetGC regulation by GCAP-1 and GCAP-2. Lowering free Mg(2+) concentrations ([Mg](f)) from 5.0 mm to 0.5 mm decreases the free Ca(2+) concentration required for half-maximal inhibition of RetGC ([Ca]((1/2))) by recombinant GCAP-1 and GCAP-2 from 1.3 and 0.2 microm to 0.16 and 0.03 microm, respectively. A similar effect of Mg(2+) on Ca(2+) sensitivity of RetGC by endogenous GCAPs was observed in mouse retina. Analysis of the [Ca]((1/2)) changes as a function of [Mg](f) in mouse retina shows that the [Ca]((1/2)) becomes consistent with the range of 23-250 nm free Ca(2+) found in mouse photoreceptors only if the [Mg](f) in the photoreceptors is near 1 mm. Our data demonstrate that GCAPs are Ca(2+)/Mg(2+) sensor proteins. While Ca(2+) binding is essential for cyclase activation and inhibition, Mg(2+) binding to GCAPs is critical for setting the actual dynamic range of RetGC regulation by GCAPs at physiological levels of free Ca(2+).

PEROXIDATION OF MEMBRANE LIPID AND CALCIUM DISTRIBUTION IN GRAPE MESOPHYLL CELLS DURING CROSS ADAPTATION TO TEMPERATURE STRESSES

Changes in the membrane protective system and Ca 2+ homeostasis in young grape (Vitis vinifera) plants during the enhancement of chill resistance with heat acclimation and heat resistance with cold acclimation were examined to explore the mechanism of cross adaptation to temperature stress. MDA content in heat (cold) acclimated leaves by cold (heat) stress was less than that in the non-acclimated control plants which showed that heat (cold) acclimation could alleviate the damages of cell membrane caused by cold (heat) stress. Higher leaf levels of glutathione (GSH) and ascorbate acid (AsA) also were found with pre-cold (heat) acclimation under heat (cold) stress than in the non-acclimated control plants. Cold and heat stresses have been shown to cause transient elevated levels of cytosolic free Ca 2+, but there is little evidence showing that Ca 2+ homeostasis was maintained in cross adaptation, a crucial factor for confirmation of the existence of cross adaptation and the elucidation of the mechanisms of cross adaptation. Ca 2+-homeostasis was demonstrated by examining Ca 2+ distribution using the cytochemical method of the calcium antimonite precipitation. The results revealed calcium was distributed mainly within the vacuole and intercellular spaces at the optimum growth temperature. When the plants were treated by either heat or cold acclimation for 3 h, calcium levels in the cytoplasm increased dramatically. Meanwhile, deposits in the vacuole concentrated in the tonoplast and deposits in the intercellular space decreased after heat acclimation whereas, after cold acclimation, the deposits in vacuole decreased significantly but did not change in the intercellular space. These results indicate that heat acclimation mostly pumped Ca 2+ out of intercellular spaces whereas cold acclimation pumped Ca 2+ out of vacuoles. These results revealed two physiological phenomena: on one hand, heat (cold) acclimation increased Ca 2+ in cytoplasm to increase the integrity of membranes under cold (heat) stress; on the other hand, increased Ca 2+ in the cytoplasm induced the expression of GSH and AsA genes increasing GSH and AsA levels. When non-acclimated plants were stressed at 44 ℃ or -3 ℃ for 3 h, compared to control plants, more Ca 2+ particles were located in the inner side of the plasma membrane, fewer Ca 2+ particles were in the intercellular spaces and vacuole, the plasma membrane and chloroplast showed signs of damage, and chloroplast ultrastructure was destroyed. However, when heat acclimated plants were exposed to chilling stress at -3 ℃ for 3 h and cold-acclimated plants were exposed to heat stress at 44 ℃ for 3 h, more Ca 2+ particles were found in the intercellular spaces and in vacuoles and few Ca 2+ particles were on the inner side of the plasma membrane. Cell membranes and chloroplasts were not destroyed. These results indicate that too much intracelluar Ca 2+ can be pumped off in heat-acclimated cells under cold stress or cold-acclimated cells under heat stress but cannot be pumped off in non-acclimated cells, suggesting that calcium homeostasis is very important in grape mesophyll cells during cross adaptation to temperature stress.

Inner mitochondrial membrane potential (DeltaPsim), cytoplasmic ATP content and free Ca2+ levels in metaphase II mouse oocytes.

The relative magnitude of the inner mitochondrial membrane potential (DeltaPsim) has been suggested to indicate the competence of mammalian gametes and early embryos. This study examined the response of cultured somatic cells and mouse oocytes to inhibitors and conditions that affect DeltaPsim or metabolism, or both, and measured treatment-specific changes in ATP and cytoplasmic free Ca(2+). During and after treatment, relative DeltaPsim, free Ca(2+), and ATP levels and cortical granule density were determined. Comparable responses of somatic cells and metaphase II mouse oocytes to experimental manipulations that affect DeltaPsim and metabolism were observed and reversible loss of DeltaPsim was associated with increased intracellular free Ca(2+), which in certain instances resulted in parthenogenetic activation. The findings support a mitochondrial basis for pericortical J-aggregate fluorescence but not for a direct association between high DeltaPsim and metabolism. The results extend previous findings indicating that high-polarized (high DeltaPsim, JC-1 J-aggregate-forming) mitochondria occur in pericortical domains in mouse and human oocytes and early preimplantation stage embryos and support the notion that this spatial distribution may be related to localized ionic and metabolic regulation.

Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin.

Calmodulin (CaM)-regulated plasma membrane Ca(2+)-ATPase (PMCA) is critical for the regulation of free intracellular Ca(2+) levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H(2)O(2) was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H(2)O(2) (25-100 microM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H(2)O(2)-exposed PMCA was disrupted by treatment with H(2)O(2). Rates of inactivation by H(2)O(2) of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H(2)O(2) did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H(2)O(2) protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H(2)O(2) quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H(2)O(2) enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H(2)O(2) led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H(2)O(2) exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H(2)O(2), it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.

Activation of calcium signaling by hepatitis B virus-X protein in liver cells.

Hepatitis B virus x gene product (HBx) is known to be a transactivator of transcriptional elements that regulate the expression of a variety of genes associated with the growth, differentiation, survival and the apoptosis of cells. However, the exact mechanism of the activation and inhibition of cellular events by HBx remains uncertain. The present study was designed to measure the effect of HBx, on the signal transduction pathways associated with intracellular Ca(2+) mobilization following HBx transfection in the stable Chang liver cells (CHL-X). Enhanced cell proliferation by HBx in CHL-X was confirmed by MTT assay and by the immunodetection of PCNA. The transactivation of AP-1 by HBx induced in CHL-X was inhibited by cyclosporin A (CsA), a mitochondrial Ca(2+) channel blocker and by BAPTA-AM, a cytosolic Ca(2+) blocker. Activation of the SAPK/JNK signaling pathway by HBx was evidenced by the increased phosphorylations of c-Jun (Ser63) and of JNK (Thr183/Tyr185). Increased phospho-Erk/Erk and phospho-Raf1/Raf in HBx-induced CHL-X indicated that HBx might stimulate the MAPK pathway. PI3K activity and cytosolic free Ca(2+) levels were elevated in HBx-induced CHL-X. These results imply that HBx transactivates both JNK and MAPK signal transduction pathways in association with the mobilization of cytosolic Ca(2+).

Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeutic potential of calpain inhibitors for prevention of neurodegeneration.

Calpain is a Ca(2+)-activated proteolytic enzyme involved in neurodegeneration in a variety of injuries and diseases of the central nervous system (CNS). Many calpain homologs have been discovered. Depending on the tissue distribution, calpains are broadly classified as ubiquitous and tissue-specific. Ubiquitous calpain isoforms, -calpain and m-calpain, are abundantly expressed in the CNS. Calpastatin, an endogenous protein inhibitor, regulates the activity of ubiquitous calpain. Overactivation of calpain may degrade calpastatin, limiting its regulatory efficiency. Molecular structures of calpain and calpastatin have been deduced from cDNA cloning. The precise physiological function of calpain remains elusive. However, experimental evidence strongly suggests an important role for calpain in causing neurodegeneration in various injuries and diseases of the CNS. The increase in intracellular free Ca(2+) levels in the course of injuries and diseases in the CNS causes overactivation of calpain, promoting degradation of key cytoskeletal and membrane proteins. Cleavage of these key proteins by calpain is an irreversible process that perturbs the integrity and stability of CNS cells, leading to programmed cell death or apoptosis. Calpain in conjunction with caspases can cause apoptosis of the CNS cells. An aberrant Ca(2+) homeostasis inevitably activates calpain, which plays a crucial role in the pathophysiology of the CNS injuries and diseases. Therefore, calpain is a potential therapeutic target to prevent neurodegeneration. To this end, various cell-permeable calpain inhibitors have been synthesized for pharmacological inhibition of calpain activity. Some calpain inhibitors have shown significant neuroprotection in animal models of the CNS injuries and diseases, indicating their therapeutic potential.

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity.

The CACNA2D2 gene, a new subunit of the Ca(2+)-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca(2+) signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca(2+) contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca(2+) were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca(2+) contents.

A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes.

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K(+) homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca(2+)-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K(+). Membrane potentials for half-maximal open probability (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P(o)=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca(2+)]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 microM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 ms, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26+/-0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLFs and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K(+) recycling dynamics in the mammalian cochlea.

Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.

When boar spermatozoa are incubated in a medium designed for in vitro fertilization, many of them become agglutinated at the acrosomes. We previously reported that bicarbonate and cyclic adenosine 3',5'-monophosphate (cAMP) promote agglutination. The aim of the present study is to examine the role of cytoplasmic free Ca(2+) in boar sperm agglutination induced by a cell-permeable cAMP analogue. Spermatozoa were collected from five mature boars, washed, and resuspended in a modified Krebs-Ringer-Hepes solution lacking calcium chloride. The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 minutes and were then used to determine the percentages of head-to-head agglutinated spermatozoa. Percentages of head-to-head agglutinated spermatozoa in the samples rose significantly after incubation, from 28% to 61%-62%, after adding to the medium a cell-permeable, phosphodiesterase-resistant cAMP analogue (cBiMPS, 10 microM) or an adenylyl cyclase stimulator (sodium bicarbonate, 5 mM) plus a cell-permeable phosphodiesterase inhibitor (IBMX, 25 microM). However, the promoting effects of these reagents were blocked when spermatozoa were pretreated with a cell-permeable Ca(2+) chelator (BAPTA-AM, 25 microM), whereas the same pretreatment with a cell-impermeable Ca(2+) chelator (BAPTA, 25 microM) had almost no influence on sperm agglutination. Adding thapsigargin, a potential Ca(2+)-ATPase inhibitor, to the medium raised the percentages of agglutinated spermatozoa in a concentration-dependent manner for concentrations up to 4 microM. When 4 microM thapsigargin and 10 microM cBiMPS were examined for their effects on free Ca(2+) levels in sperm heads by using a cell-permeable Ca(2+) indicator (fluo-3/AM), the samples incubated with both or either of these reagents contained many head-to-head agglutinated cells that exhibited intense fluorescence in the heads. In control samples incubated without these reagents by contrast, most spermatozoa were free (unagglutinated) cells and characterized by almost no or only slight fluorescence in the heads. Moreover, morphological observation of Giemsa-stained preparations revealed that most agglutinated spermatozoa possessed darkly stained acrosomes, which distinguished them from acrosomereacted spermatozoa. This indicated that the sperm agglutination was not a result of the acrosome reaction. Furthermore, with indirect immunofluorescence of Ca(2+)-ATPases, the mouse monoclonal antibody to this enzyme demonstrated high affinity to the acrosomes of permeabilized spermatozoa. Based on these results, we conclude that cytoplasmic free Ca(2+) is involved in sperm head-to-head agglutination induced by a cAMP analogue.

Structure and Ca2+ regulation of frog photoreceptor guanylate cyclase, ROS-GC1.

Rod outer segment membrane guanylate cyclase (ROS-GC) is a critical component of the vertebrate phototransduction machinery. In response to photoillumination, it senses a decline in free Ca(2+) levels from 500 to below 100 nM, becomes activated, and replenishes the depleted cyclic GMP pool to restore the dark state of the photoreceptor cell. It exists in two forms, ROS-GC1 and ROS-GC2. In outer segments, ROS-GCs sense fluctuations in Ca(2+) via two Ca(2+)-binding proteins, which have been termed GCAP1 and GCAP2. In the present study we report on the cloning of two ROS-GCs from the frog retinal cDNA library. These cyclases are the structural and functional counterparts of the mammalian ROS-GC1 and ROS-GC2. There is, however, an important difference between the regulation of mammalian and frog ROS-GC1: In contrast to the mammalian, the frog form does not require the myristoylated form of GCAP1 for its Ca(2+)-dependent modulation. This feature is not dependent upon the ability of frog GCAP1 to bind Ca(2+) because unmyristoylated GCAP1 mutants which do not bind Ca(2+), activate frog ROS-GC1. The findings establish frog as a suitable phototransduction model and show a facet of frog ROS-GC signaling, which is not shared by the mammalian form.

Direct measurement of free Ca 2+ shows different regulation of Ca 2+ between the periplasm and the cytosol of Escherichia coli

As in eukaryotes, bacterial free Ca 2+ can play an important role as an intracellular signal. However, because free Ca 2+ is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca 2+ in the periplasmic space of living Escherichia coli. Periplasmic free Ca 2+ was measured by targeting the Ca 2+-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca 2+ was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca 2+, resulting in the inner membrane being exposed to free Ca 2+ concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca 2+ as predicted by the Donnan potential. With micromolar concentrations of external free Ca 2+, the periplasm concentrated free Ca 2+ some three to sixfold with respect to the external medium. A Ca 2+ gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca 2+ being some one to threefold higher. At millimolar levels of external free Ca 2+, a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca 2+ in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca 2+ in this compartment, where it may constitute a sink for regulation of Ca 2+-dependent processes in the cytoplasm.

Examining the role of mitochondrial respiration in vanilloid-induced apoptosis.

The vanilloids capsaicin and resiniferatoxin are natural products that contain a vanillyl moiety (4-hydroxy-3-methoxybenzyl). Both vanilloids can induce apoptosis in certain cell types by a mechanism that has not been fully elucidated but may involve plasma membrane or mitochondrial targets. We investigated the role of mitochondrial respiration in vanilloid-induced apoptosis. Cytofluorometric analysis was used to evaluate the effects of vanilloids on apoptosis, Ca(2+) mobilization, hydroperoxide generation, and DNA content in cells from two human cutaneous squamous cell carcinoma (SCC) cell lines (parental cells) and in their respiration-deficient clones. Oxygen consumption by the cells was determined polarographically. The majority of the parental SCC cells underwent apoptosis after a 12-hour exposure to 100 micro M capsaicin or 10 micro M resiniferatoxin. The induction of apoptosis was associated with the mitochondrial permeability transition (i.e., an increase in the permeability of the inner mitochondrial membrane associated with the opening of a nonspecific pore). Exposure of parental cells to either vanilloid was not associated with an increase in intracellular free Ca(2+) levels but was associated with a rapid increase in hydroperoxide generation and a decrease in oxygen consumption. After vanilloid treatment, the respiration-deficient clones generated less hydroperoxide and were resistant to the mitochondrial permeability transition and the induction of apoptosis. Moreover, vanilloid treatment inhibited cell proliferation in the respiration-deficient clones by promoting G(1) arrest. Vanilloid-induced apoptosis in the parental SCC cells appears to involve the inhibition of mitochondrial respiration. The apoptogenic effects promoted by vanilloid treatment in parental SCC cells, as well as the antiproliferative effects observed in their respiration-deficient clones, suggest that vanilloids may be useful for preventing or treating skin cancers or other hyperproliferative skin disorders.

Differential effects of mu-opioid receptor ligands on Ca(2+) signaling.

Activation of mu-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca(2+) levels (Ca(2+)i). The first Ca(2+)i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca(2+) (Ca(2+)e). The second phase peaking at approximately 15 s (peak 2) was independent of Ca(2+)e and thus represents Ca(2+) release from intracellular stores. A decrease in temperature from 37 to 25 degrees C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca(2+)e-dependent third response phase is thought to represent capacitative Ca(2+)e influx evoked after release of Ca(2+) from internal stores. Agonists and antagonists of two major classes of opioid ligands, oxymorphinans (morphine and naloxone) and oripavines (etorphine and diprenorphine), had differential effects on Ca(2+) currents. Although morphine activated both phases with equal potency, etorphine was 20-fold less potent at stimulating peak 1 over peak 2. Similarly, the antagonists, naloxone and diprenorphine, blocked the Ca(2+) response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca(2+)e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca(2+) release). However, diprenorphine potently inhibited peak 1 as well when added to the cells before morphine, indicating limited or slow access of diprenorphine to these morphine binding sites. The existence of multiple, functionally distinct binding site conformations could account for these findings. In conclusion, different opioid ligands can differentially affect Ca(2+) response patterns resulting from MOR activation.