Low Ca Conditions Research Articles

Inactivation of theNa-Cl Co-Transporter(NCC) Gene Is Associated With High BMD Through Both Renal and Bone Mechanisms: Analysis of Patients With Gitelman Syndrome andNccNull Mice

Chronic thiazide treatment is associated with high BMD. We report that patients and mice with null mutations in the thiazide-sensitive NaCl cotransporter (NCC) have higher renal tubular Ca reabsorption, higher BMD, and lower bone remodeling than controls, as well as abnormalities in Ca metabolism, mainly caused by Mg depletion. Chronic thiazide treatment decreases urinary Ca excretion (UVCa) and increases BMD. To understand the underlying mechanisms, Ca and bone metabolism were studied in two models of genetic inactivation of the thiazide-sensitive NaCl cotransporter (NCC): patients with Gitelman syndrome (GS) and Ncc knockout (Ncc(-/-)) mice. Ca metabolism was analyzed in GS patients and Ncc(-/-) mice under conditions of low dietary Ca. BMD was measured by DXA in patients and mice, and bone histomorphometry was analyzed in mice. GS patients had low plasma Mg. They exhibited reduced UVCa, but similar serum Ca and GFR as control subjects, suggesting increased renal Ca reabsorption. Blood PTH was lower despite lower serum ionized Ca, and Mg repletion almost corrected both relative hypoparathyroidism and low UVCa. BMD was significantly increased in GS patients at both lumbar (+7%) and femoral (+16%) sites, and osteocalcin was reduced. In Ncc(-/-) mice, serum Ca and GFR were unchanged, but UVCa was reduced and PTH was elevated; Mg repletion largely corrected both abnormalities. Trabecular and cortical BMD were higher than in Ncc(+/+) mice (+4% and +5%, respectively), and despite elevated PTH, were associated with higher cortical thickness and lower endosteal osteoclastic surface. Higher BMD is observed in GS patients and Ncc(-/-) mice. Relative hypoparathyroidism (human) and bone resistance to PTH (mice), mainly caused by Mg depletion, can explain the low bone remodeling and normal/low serum Ca despite increased renal Ca reabsorption.

Interactive effects of Al, Ca and other cations on root elongation of rice cultivars under low pH.

As with other crop species, Al tolerance in rice (Oryza sativa) is widely different among cultivars, and the mechanism for tolerance is unknown. The Ca2+-displacement hypothesis, that is, Al displaces Ca2+ from critical sites in the root apoplast, was predicted to be the essential mechanism for causing Al toxicity in rice cultivars. If displacement of Ca is an essential cause of Al toxicity in rice, Al toxicity may show the same trend as toxicities of elements such as Sr and Ba that are effective in displacing Ca. The interactive effects of Al, Ca, Sr and Ba on root elongation of rice cultivars with different Al tolerances were evaluated in hydroponic culture. Al and Ca accumulation in root tips was also investigated. Not only Al but also Sr and Ba applications inhibited root growth of rice cultivars under low Ca conditions. As expected, rice cultivars more tolerant of Sr and Ba were also tolerant of Al (japonica > indica). Although Mg application did not affect Sr or Ba toxicity, Mg alleviated Al toxicity to the same level as Ca application. In addition, Ca application decreased the Al content in root tips without displacement. These results suggest that Ca does not have a specific, irreplaceable role in Al toxicity, unlike Sr and Ba toxicities. Alleviation of Al toxicity with increasing concentrations of Ca in rice cultivars is due to increased ionic strength, not due to decreased Al activity. The difference in Al tolerance between indica and japonica cultivars disappears under high ionic strength conditions, suggesting that different electrochemical characteristics of root-tip cells are related to the significant difference in Al tolerance under low ionic strength conditions.

A novel anti-anti-activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system.

Expression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells. Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed. Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion. ExsA is an activator of TTSS gene transcription, and ExsD is an anti-activator of ExsA. In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription. Here, we describe the characterization of ExsC as an anti-anti-activator of TTSS expression. Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression. The dependence on exsC for transcription is relieved in the absence of exsD, suggesting that ExsC and ExsD function together to regulate transcription. Consistent with this idea, ExsC interacts with ExsD in bacterial two-hybrid and co-purification assays. We propose a model in which the anti-anti-activator (ExsC) binds to and sequesters the anti-activator (ExsD) under low Ca(2+) conditions, freeing ExsA and allowing for transcription of the TTSS. The P. aeruginosa system represents the first example of an anti-activator/anti-anti-activator pair controlling transcription of a TTSS.

Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca 2+ Involves Luminal Ca 2+ Sensing Sites

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca 2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 μM to 2 mM Ca 2+ (free) and 3 mM ATP (total) on the cytosolic ( cis) side and 20 μM to 20 mM Ca 2+ on the luminal ( trans) side of the channel and with Cs + as the charge carrier. Under conditions of low trans Ca 2+ (20 μM), increasing cis Ca 2+ from 0.1 to 10 μM caused a gradual increase in channel open probability ( P o). Elevating cis Ca 2+ above 100 μM resulted in a gradual decrease in P o. Elevating trans [Ca 2+] enhanced channel activity (EC 50 ≈ 2.5 mM at 1 μM cis Ca 2+) primarily by increasing the frequency of channel openings. The dependency of P o on trans [Ca 2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca 2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N, N-tetraacetic acid. Elevated luminal Ca 2+ enhanced the sensitivity of the channel to activating cytosolic Ca 2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca 2+. Potentiation of P o by increased luminal Ca 2+ occurred irrespective of whether the electrochemical gradient for Ca 2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca 2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca 2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca 2+ are mediated by distinct Ca 2+-sensitive site(s) at the luminal face of the channel or associated protein.

Effect of a low-calcium environment on alkaline phosphatase activity in embryonic rat calvarial bone cells in culture

The effect of a low Ca environment on the alkaline phosphatase (ALP) activity in bone cells was examined. Bone cells were isolated from the calvaria of 18 to 21-day-old fetal rats and cultured in BGJ b or α-minimum essential medium (α-MEM) with calcium concentrations of 1.87 mM (control group), 1.20 mM (middle group) or 0.34 mM (low-Ca group). The control and low-Ca groups grew and reached a confluent state at 10 days of culture and there were no significant differences in the number of cells, total protein, or DNA content between the two groups. The specific ALP activity measured at 10 days of culture of the cells with 0.34, 1.20 and 1.87 mM calcium was 106.3 ± 11.9, 73.3 ± 10.4,and38.8 ± 7.3 unit per mg protein ( n = 9), respectively. When the low-Ca medium was replaced with the control medium during 10 days of culture, the activity decreased to the level of the control group. On the other hand, when control medium was replaced with low-Ca medium, the activity increased to the level of the low-Ca group. After confluence, cell growth in the low-Ca group was poorer than that in the control group with time. The ALP activity markedly increased and the mineralized nodular structures were observed in the control group of α-MEM, but not in the low-Ca group. These findings suggest that stimulation of ALP activity is a compensation mechanism to maintain normal cell functions in a low-Ca condition and that the role of ALP in the low-Ca environment may be different from that on mineralization.

Functional expression of the parathyroid cell calcium receptor in Xenopus oocytes

Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca 2+. To test this hypothesis, Xenopus leavis oocytes were injected with poly(A) +-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca 2+ or other polycations. Cytosolic Ca 2+ concentrations were measured indirectly by recording Cl − currents through the endogenous, cytosolic Ca 2+-activated Cl − channel. Increasing the concentration of extracellular Ca 2+ (from 0.7 to 5 mM) or Mg 2+ (from 0.8 to 10 mM) elicited oscillatory increases in the CT current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg 2+ persisted when extracellular Ca 2+ was reduced to low micromolar levels. La 3+, Gd 3+, or neomycin B also evoked oscillatory increases in the Cl − current in oocytes under conditions of low extracellular Ca 2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca 2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca 2+ receptor protein(s) on parathyroid cells that regulates cellular function.

Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca 2+ (0.1 m M, growth-promoting condition) and physiological Ca 2+ (1.8 m M, differentiation-promoting condition) was investigated. In low Ca 2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca 2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca 2+ was seen at 2.5–5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-β1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca 2+ conditions, exhibiting a K d = 17.3 p M and approximately 690 binding sites/cell under physiological Ca 2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca 2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

A patch‐clamp study of effects of dihydroavermectin on Ascaris muscle

1. Effects of bath-application of the anti-parasitic agent 22,23-dihydroavermectin B1a (DHAVM 20 fM to 0.2 microM) on outside-out and inside-out patches from collagenase-treated muscle membranes of Ascaris suum were examined. 2. DHAVM was tested as a gamma-aminobutyric acid (GABA) agonist by application to outside-out patches. DHAVM failed to open characteristic GABA channels even when high concentrations (20 nM to 0.2 microM) were used. These high concentrations of DHAVM also failed to potentiate opening of channels activated by application of 3-4 microM GABA; instead they acted to depress GABA-activated channel currents by reducing mean conductances and P open. 3. In outside-out patches, low concentrations of DHAVM (1 pM to 100 pM) produced opening of 0-13 channels, the number depending on the patch. The progressive opening of channels gave rise to a 'Staircase' effect. The conductance of these channels was 9-15 pS and open times were long (greater than 100 mS). Ion substitution experiments showed these channels to be permeable to Cl. The channels were not blocked by 20 microM picrotoxin. There was a long delay (greater than 15 s) between DHAVM application and channel opening; this delay and lipophilic nature of DHAVM suggested a site of action in the lipid phase of the membrane. 4. The effect of bath-application of low concentrations of DHAVM on inside-out patches was investigated under low-Ca conditions (to avoid Ca-activated Cl channels). DHAVM (20 fM to 1 nM) did not produce opening of Cl channels but produced opening after a long delay (mean 2.5 minutes) of noisy cation-selective channels which had conductances of 5-30 pS. 5. The actions of DHAVM are discussed.

Isolation of a fraction with Ca 2+ ionophore properties from rat liver mitochondria

Isolation of a small protein with properties of a Ca 2+ ionophore from calf heart mitochondria has recently been reported [ A. Y. Jeng and A. E. Shamoo, 1980, J. Biol. Chem. 255, 6897, 6904]. We have isolated a fraction with similar physical and chemical properties from rat liver mitochondria. In particular, the hepatic preparation is able to bind Ca 2+ with high affinity in such a fashion that the resultant complex is soluble in a hydrophobic phase. It will also transport Ca 2+ through a stirred organic phase (Pressman cell). Interaction of the liver preparation with Ca 2+ is sensitive to inhibitors of mitochondrial Ca 2+ uptake. The hepatic preparation contains both protein and lipid components. The phospholipid components were identified and the behavior of a similar mixture of commercially available phospholipids was compared to that of the ionophore fraction from rat liver mitochondria. All of the Ca 2+ binding properties of the rat liver preparation could be mimicked by the lipids. In a preliminary experiment, reduction of the phospholipid content of the preparation to less than one lipid phosphate per protein molecule (assuming a molecular weight of 3000 by analogy with the calf heart case) resulted in a protein that was unable to bind Ca 2+. We, therefore, suggest that the ability of the preparation to interact with Ca 2+ is due to the constituent phospholipids. Measurements of phospholipid-Ca 2+ interactions in the model systems and under the conditions of low (μ m) Ca 2+ and phospholipid concentration utilized here demonstrated an affinity for Ca 2+ ( K s ~ 1 μm) and a cation selectivity that have not previously been reported.

Leptinotarsin: a presynaptic neurotoxin that stimulates release of acetylcholine.

Leptinotarsin, a toxin found in the hemolymph of the beetle Leptinotarsa haldemani, can stimulate release of acetylcholine from synaptic termini. Leptinotarsin causes an increase in the frequency of miniature end plate potentials (mepps) of the rat phrenic nerve-diaphragm preparation. The increase in the frequency of mepps induced by leptinotarsin is biphasic: about 10% of the total mepps are released in an initial burst that lasts about 90 sec, after which the remaining mepps are released over a period of 10-20 min. Tetrodotoxin has no effect upon the release induced by leptinotarsin, but low-Ca(2+) conditions abolish the first phase. The two phases of release may represent two presynaptic pools of acetylcholine, both of which can be released in quantized form. In a second study, rat brain synaptosomes were incubated with [(3)H]choline and were immobilized on Millipore filters. Leptinotarsin induced release of [(3)H]acetylcholine from this preparation, confirming the release seen by using neurophysiological methods. The ability of leptinotarsin to induce release from either intact nerve terminals or synaptosomes was abolished when the toxin was heated. The releasing activity of leptinotarsin from synaptosomes was also partially dependent upon the presence of Ca(2+) in the perfusing solution. Release from synaptosomes followed first-order kinetics, and was not inhibited by commercial antibodies to black widow spider antigens. The data suggest that leptinotarsin acts as a presynaptic neurotoxin and may be of value as a mechanistic probe in understanding the storage and release of neurotransmitters.

Chemical carcinogenesis studies in mouse epidermal cell cultures.

Studies of tumor induction on mouse skin have provided insight into the basis biology of chemical carcinogenesis, but molecular mechanisms have been more difficult to elucidate. Mouse epidermal cell cultures have proven to be a valuable model for performing mechanistic studies. Previous data have indicated that such cultures proliferate and differentiate in a manner highly analogous to epidermis in vivo. In addition, carcinogen metabolism, DNA repair, and responses to tumor promoters are quite similar in mouse skin in vivo and in vitro. Recent data have extended these observations toward defining the biological characteristics of initiated cells and elucidating the mechanism of action of promoters and antipromoters. When mouse epidermis is cultured under conditions of low extracellular Ca++, proliferation is enhanced and terminal differentiation is inhibited. Addition of Ca++ induces terminal differentiation. If cells are treated with carcinogens under low Ca++ conditions and subsequently switched to standard Ca++, cell colonies which do not terminally differentiate evolve. Such colonies continue to synthesize keratin, are subculturable, and may represent preneoplastic cells. In other experiments, epidermal cells derived from mouse skin treated with carcinogens in vivo also demonstrate prolonged in vitro survival and subculturability while controls have a limited lifespan. Such studies suggest that biological alterations can be detected in epidermal cells exposed to carcinogens well before and the phenotypic expression of neoplasia. Exposure of epidermal cells to phorbol-ester tumor promoters induces ornithine decarboxylase (ODC). This induction is enhanced by corticosteroids and markedly inhibited by retinoids. Ultraviolet light also induces ODC in epidermal cells, but kinetic studies suggest that the early pathway of induction (afferent to the nucleus) is different from that of phorbol esters. The later pathways (efferent from the nucleus-i.e., transcription and translation) appear to be similar. Retinoids have only a minor suppressive effect on ODC induction by UV while corticosteroids enhance UV induction to the same extent as seen with phorbol esters These results suggest that the site of retinoids is in the afferent pathway while steroids act on the efferent pathway.

Simulation of K-contracture curve in smooth muscle (author's transl)

40 mM K-induced isometric contracture of guinea pig taenia coli in the presence of 30 microM dantrolene showed a rapidly rising peak followed by a plateau and then a low sustained tonic contraction. The plateau was delayed by low Ca so that two phasic contractions, fast and slow, were separated from each other. In K-contracture after 15 sec contact with normal Ca following low Ca condition, the fast phasic contraction but not the slow one regained its tension depressed by preceding low Ca, while the slow phasic contraction recovered from its delay. In the presence of 0.2 microM verapamil, K-contracture consisted of the fast phasic contraction without plateau and of the low tonic contraction. The results suggest that K-contracture in the normal state consists of three components, the fast and slow phasic contractions and the tonic contraction, and that dantrolene inhibits a tonic contraction, whereas verapamil inhibits the slow phasic and the tonic contraction. Thus, the contraction curve of each component was tentatively expressed by exponential function, and for simulation of the observed curves a computer was utilized to synthesize the curves using the components of varying parameters. The simulation was successful when based on the above suggestion.

Electrical and adaptive properties of rod photoreceptors in Bufo marinus. I. Effects of altered extracellular Ca2+ levels.

The effects of altering extracellular Ca(2+) levels on the electrical and adaptive properties of toad rods have been examined. The retina was continually superfused in control (1.6 mM Ca(2+)) or test ringer's solutions, and rod electrical activity was recorded intracellularly. Low-calcium ringer's (10(-9)M Ca(2+)) superfused for up to 6 min caused a substantial depolarization of the resting membrane potential, an increase in light-evoked response amplitudes, and a change in the waveform of the light-evoked responses. High Ca(2+) ringer's (3.2 mM) hyperpolarized the cell membrane and decreased response amplitudes. However, under conditions of either low or high Ca(2+) superfusion for up to 6 min, in both dark-adapted and partially light-adapted states, receptor sensitivity was virtually unaffected; i.e., the V-log I curve for the receptor potential was always located on the intensity scale at a position predicted by the prevailing light level, not by Ca(2+) concentration. Thus, we speculate that cytosol Ca(2+) concentration is capable of regulating membrane potential levels and light-evoked response amplitudes, but not the major component of rod sensitivity. Low Ca(2+) ringer's also shortened the period of receptor response saturation after a bright but nonbleaching light flash, hence accelerating the onset of both membrane potential and sensitivity recovery during dark adaptation. Exposure of the retina to low Ca(2+) (10(-9)M) ringer's for long periods (7-15 min) caused dark-adapted rods to lose responsiveness. Response amplitudes gradually decreased, and the rods became desensitized. These severe conditions of low Ca(2+) caused changes in the dark-adapted rod that mimic those observed in rods during light adaptation. We suggest that loss of receptor sensitivity during prolonged exposure to low Ca(2+) ringer's results from a decrease of intracellular (intradisk) stores of Ca(2+); i.e., less Ca(2+) is thereby released per quantum catch.

Calcium and angiotensin tachyphylaxis in rat uterine smooth muscle

Studies were carried out to investigate the relationship between extracellular Ca++ and the ability of a particular smooth muscle to develop tachyphylaxis to angiotensin II (AII). Stimulation of rat uterus by AII was found to be dependent on extracellular Ca++. Placing the tissue in 0 Ca++ completely blocked AII-induced contractions as did the presence of the "Ca++ antagonists" verapamil (10- minus 5M), SKF 525-A (10- MINUS 5M), tetracaine (10- minus 4M), Mn++ (8 times 10- minus 3M), or La-3+ (10- minus 3M). In addition, it is no longer possible to produce tachyphylaxis to AII in deplorazed rat uterus under conditions of pH and Ca++ concentration in which a normally polarized preparation would be unresponsive. Verapamil, on the other hand, was an even more effective antagonist of AII in depolarized preparations (ID50 of 10- minus 8M) than in normal tissues (ID50 of 2.0 times 10- minus 7M). Like the rat uterus, the smooth muscle of the guinea pig ileum also develops tachyphylaxis to AII, and the effect of this peptide was also blocked by 10- minus 5 M verapamil. Rabbit aorta, however, was found to be relatively resistant to both development of tachyphylaxis under conditions of low Ca++ and low pH and also to inhibition by even very high concentrations of verapamil (10- minus 4M). The results of these studies suggest that the Ca++ site involved in the tachyphylactic response to AII may be a physiologically important one in those smooth muscles in which movement of extracellular Ca++ contributes to the inward ion currents during excitation. Verapamil, however, appears to act at a common step in the excitation-contraction sequence in rat uterus. A working model of the interaction of AII with rat uterine smooth muscle is presented.