Ca2+ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α2β1 isozyme of Na+/K+-ATPase by ouabain

AimsWe sought to determine the mechanisms of an increase in Ca²⁺ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na⁺/K⁺-ATPase inhibition by ouabain.Main methodsThe caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na⁺ and Ca²⁺ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively.Key findingsWe identified the α₂β₁ and α₁β₁ isozymes of Na⁺/K⁺-ATPase in caveolae vesicles, and only the α₁β₁ isozyme in noncaveolae fraction of the plasma membrane. The α₂-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na⁺/H⁺-exchange inhibitor) and tetrodotoxin (voltage-gated Na⁺-channel inhibitor) pretreatment prevented ouabain induced increase in Na⁺ and Ca²⁺ levels. Ouabain induced increase in Ca²⁺ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na⁺/Ca²⁺-exchange inhibitor) and verapamil (L-type Ca²⁺-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca²⁺ level in the caveolae vesicles, indicating that apart from Na⁺/Ca⁺-exchanger and L-type Ca²⁺-channels, “slip-mode conductance” of Na⁺ channels could also be involved in this scenario.SignificanceInhibition of α₂ isoform of Na⁺/K⁺-ATPase by ouabain plays a crucial role in modulating the Ca²⁺ influx regulatory components in the caveolae microdomain for marked increase in (Ca²⁺)_i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.     

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO^?) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO^? stimulated PKC-α activity and that subsequently increased p³⁸MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p³⁸MAPK inhibitor) eliminated ONOO^? caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO^? induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p³⁸MAPK (p³⁸MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO^? effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO^? was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G_i dependent mechanism. This hypothesis was reinforced by G_iα phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G_i mediated response) to stimulate adenyl cyclase activity upon ONOO^? treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G_i)-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p³⁸MAPK axis dependent phosphorylation of its α-subunit (G_iα) in the pulmonary artery smooth muscle cells.     

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A₂ (PLA₂) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H₂O₂). Exposure of the smooth muscle cells to H₂O₂ dose-dependently stimulates [¹⁴C] AA release, and enhances the cell membrane associated PLA₂ activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H₇ and sphingosine prevent the cell membrane associated PLA₂ activity, and AA release caused by H₂O₂. Treatment of the smooth muscle cells with H₂O₂ stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H₂O₂ caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA₂ and PKC activities correlate linearly. These results suggest that H₂O₂ caused stimulation of the smooth muscle cell membrane associated PLA₂ activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)Abbreviations AA Arachidonic Acid - PLA₂ Phospholipase A₂ - PKC Protein Kinase C - PBS Phosphate Buffered Saline - HBPS Hank's Buffered Physiological Saline - HEPES 4-(2-Hydroxyethyl)-1-Piperazine N-2-Ethanesulfonate - FCS Fetal Calf Serum - ATP Adenosine Triphosphate - H₇ 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine - DMEM Dulbecco's Modified Eagles Medium - TCA Trichloroacetic Acid     

Role of STIM1/Orai1-mediated store-operated Ca²⁺ entry in airway smooth muscle cell proliferation

Hyperplasia of airway smooth muscle cells (ASMCs) is a characteristic change of chronic asthma patients. However, the underlying mechanisms that trigger this process are not yet completely understood. Store-operated Ca(2+) (SOC) entry (SOCE) occurs in response to the intracellular sarcoplasma reticulum (SR)/endoplasmic reticulum (ER) Ca(2+) store depletion. SOCE plays an important role in regulating Ca(2+) signaling and cellular responses of ASMCs. Stromal interaction molecule (STIM)1 has been proposed as an ER/SR Ca(2+) sensor and translocates to the ER underneath the plasma membrane upon depletion of the ER Ca(2+) store, where it interacts with Orai1, the molecular component of SOC channels, and brings about SOCE. STIM1 and Orai1 have been proved to mediate SOCE of ASMCs. In this study, we investigated whether STIM1/Orai1-mediated SOCE is involved in rat ASMC proliferation. We found that SOCE was upregulated during ASMC proliferation accompanied by a mild increase of STIM1 and a significant increase of Orai1 mRNA expression, whereas the proliferation of ASMCs was partially inhibited by the SOC channel blockers SKF-96365, NiCl(2), and BTP-2. Suppressing the mRNA expression of STIM1 or Orai1 with specific short hairpin RNA resulted in the attenuation of SOCE and ASMC proliferation. Moreover, after knockdown of STIM1 or Orai1, the SOC channel blocker SKF-96365 had no inhibitory effect on the proliferation of ASMCs anymore. These results suggested that STIM1/Orai1-mediated SOCE is involved in ASMC proliferation.     

Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy

Pig coronary artery smooth muscle expresses the Na⁺–Ca²⁺-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca²⁺ pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca²⁺ pool. Caveolae may also direct Ca²⁺ traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca²⁺ for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca²⁺ signaling pathways but not others.     

Na+−Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes

Tension generation and Ca²⁺ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na⁺–Ca²⁺ exchanger and the Ca²⁺ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The K_m and V_max of Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [³H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher K_d and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [³H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca²⁺ channel characteristics in isolated sarcolemmal membranes.     

Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells

We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.     

Role of TGF-β1 and TNF-α in IL-1β mediated activation of proMMP-9 in pulmonary artery smooth muscle cells: involvement of an aprotinin sensitive protease

We investigated the role of TGF-β1 and TNF-α in mediating the effect of IL-1β in activating proMMP-9 and proMMP-2, and the involvement of an aprotinin sensitive protease in this scenario in bovine pulmonary artery smooth muscle cells. IL-1β induces TGF-β1 mediated stimulation of 92 kDa proMMP-9 and 72 kDa proMMP-2 mRNA and protein expression; whereas, the elevated level of TNF-α promotes activation of proMMP-9 and proMMP-2. Interestingly, TNF-α induced activation of proMMP-9 appeared to be mediated via a 43 kDa aprotinin sensitive protease. TNF-α inhibited aprotinin and TIMP-1 mRNA and protein expression, which apparently facilitated the proteolytic conversion of proMMP-9 to MMP-9 with the involvement of the aprotinin sensitive protease. The aprotinin sensitive protease did not activate proMMP-2 under IL-1β stimulation, albeit a marked inhibition of TIMP-2 mRNA and protein expression were elicited by TNF-α. Thus, IL-1β induced stimulation of the two progelatinases occurs via different mechanisms.     

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Summary Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na⁺–Ca²⁺ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na⁺–Ca²⁺ exchange. The treatment with dithiothreitol resulted in a decrease inV _max values but did not alter theK _m for Ca²⁺ for the Na²⁺–Ca²⁺ exchange reaction. If Na⁺ replaced K⁺ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na⁺–Ca²⁺ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N-ethylmaleimide, were capable of altering Na⁺–Ca²⁺ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na⁺–Ca²⁺ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na⁺–Ca²⁺ exchange, depending on whether Na⁺ or K⁺ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfurcontaining residues interact with a Na⁺-binding site for Na⁺–Ca²⁺ exchange in cardiac sarcolemmal vesicles.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Role of PKCα-p(38)MAPK-G(i)α axis in NADPH oxidase derived O(2)(·-)-mediated activation of cPLA(2) under U46619 stimulation in pulmonary artery smooth muscle cells

We have recently reported that treatment of bovine pulmonary artery smooth muscle cells with the thromboxane A(2) mimetic, U46619 stimulated NADPH oxidase derived O(2)(·-) level, which subsequently caused marked increase in [Ca(2+)](i)[17]. Herein, we demonstrated that O(2)(·-)-mediated increase in [Ca(2+)](i) stimulates an aprotinin sensitive proteinase activity, which proteolytically activates PKC-α under U46619 treatment to the cells. The activated PKC-α then phosphorylates p(38)MAPK and that subsequently caused G(i)α phosphorylation leading to stimulation of cPLA(2) activity in the cell membrane.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y₁ leads to inhibition of Na⁺,K⁺,Cl⁻ cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y₁ agonists.     

α2 isoform of Na+,K+-ATPase via Na+,Ca2+ exchanger modulates myelin basic protein synthesis in oligodendrocyte lineage cells in vitro

Oligodendrocytes in the CNS myelinate neuronal axons, facilitating rapid propagation of action potentials. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination. In oligodendrocyte lineage cell (OLC) monocultures MBP synthesis starts at DIV4. Ouabain (10 nM), a Na⁺,K⁺-ATPase (NKA) blocker, stimulates MBP synthesis. As OLCs express the α2 isoform of NKA (α2-NKA) that has a high affinity for ouabain, we hypothesized that α2-NKA mediates this effect. Knockdown of α2-NKA with small interfering (si)RNA (α2-siRNA) significantly potentiated MBP synthesis at DIV4 and 5. This effect was completely blocked by KB-R7943 (1 μM), a Na⁺,Ca²⁺ exchanger (NCX) antagonist. α2-NKA ablation increased the frequency of NCX-mediated spontaneous Ca²⁺ transients ([Ca²⁺]t) at DIV4, whereas in control OLC cultures comparable frequency of [Ca²⁺]t was observed at DIV5. At DIV6 almost no [Ca²⁺]t were observed either in control or in α2-siRNA-treated cultures. Immunocytochemical analyses showed that α2-NKA co-localizes with MBP in proximal processes of immature OLCs but is only weakly present in MBP-enriched membrane sheets. Knockdown of α2-NKA in cortical slice cultures did not change MBP levels but reduced co-localization of neurofilament- and MBP-positive compartments. We conclude that α2-NKA activity in OLCs affects NCX-mediated [Ca²⁺]t and the onset of MBP synthesis. We suggest therefore that neuronal activity, presumably in form of local extracellular [K⁺] changes, might locally influence NCX-mediated [Ca²⁺]t in OLC processes thus triggering local MBP synthesis in the vicinity of an active axon.     

A Role for the PKC Signaling System in the Pathophysiology and Treatment of Mood Disorders: Involvement of a Functional Imbalance?

Mood disorders, such as bipolar and major depressive disorders, are frequent, severe, and often disabling neuropsychiatric diseases affecting millions of individuals worldwide. Available mood stabilizers and antidepressants remain unsatisfactory because of their delayed and partial therapeutic efficacy. Therefore, the development of targeted therapies, working more rapidly and being fully effective, is urgently needed. In this context, the protein kinase C (PKC) signaling system, which regulates multiple neuronal processes implicated in mood regulation, can constitute a novel therapeutic target. This paper reviews the currently available knowledge regarding the role of the PKC signaling pathway in the pathophysiology of mood disorders and the therapeutic potential of PKC modulators. Current antidepressants and mood stabilizers have been shown to modulate the PKC pathway, and the inhibition of this intracellular signaling cascade results in antimanic-like properties in animal models. Disrupted PKC activity has been found both in postmortem brains and platelet from patients with mood disorders. Finally, the PKC inhibitor tamoxifen has recently demonstrated potent antimanic properties in several clinical trials. Overall, emerging data from preclinical and clinical research suggest an imbalance of the PKC signaling system in mood disorders. Thus, PKC may be a critical molecular target for the development of innovative therapeutics.     

Binding of kidney nuclear proteins to the 5′-flanking region of the rat gene for Ca2+-binding protein regucalcin: Involvement of Ca2+/calmodulin signaling

Ca²⁺-binding protein regucalcin is expressed in the kidney cortex of rats, as assayed by Northern blot analysis. The existence of kidney nuclear factor which binds to the 5'-flanking region of the rat regucalcin gene was investigated. When nuclear extracts obtained from the kidney cortex of rats were used in gel mobility-shift assays, two protein-DNA complexes were uniquely formed with the DNA fragment containing the 5'-flanking region of the rat regucalcin gene. Competition gel shift experiments indicated the specific binding region of kidney cortex nuclear proteins in the 5-flanking region of the rat regucalcin gene. The two nuclear protein-DNA complexes were formed with the same mobility in rat kidney cortex and liver, which possess detectable amounts of regucalcin mRNA in Northern blot analysis. The binding activities of nuclear factors from kidney cortex to the 5-flanking region of the rat regucalcin gene were inhibited by a single intraperitoneal administration of trifluoperazine, an antagonist of calmodulin, to rats. The present study demonstrates that kidney cortex nuclear proteins specifically bind to the 5-flanking region of the rat regucalcin gene, and that the binding activity may be partly mediated through the Ca²⁺/calmodulin-dependent process.     

Reversible effect of calcium-binding protein regucalcin on the Ca2+-induced inhibition of deoxyuridine 5′-triphosphatase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5′-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca²⁺ up to 5.0 μM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn²⁺, Cd²⁺, Co²⁺, Al³⁺, Mn²⁺ and Ni²⁺ (10 μM) did not have an appreciable effect. The Ca²⁺-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 μM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca²⁺ (10 μM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd²⁺ or Zn²⁺ (10 μM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca²⁺ of various metals, and that the Ca²⁺ effect is reversed by regucalcin.