Proteinase-activated receptor agonists stimulate the increase in intracellular Ca<Superscript>2+</Superscript> in cardiomyocytes and proliferation of cardiac fibroblasts from chick embryos

We studied activation of cultured cardiomyocytes and cardiac fibroblasts from chick embryos induced by agonists of PAR1 (thrombin and PAR1 peptide agonist) and PAR2 (trypsin, factor Xa, and peptide SLIGRL) by analyzing changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cardiac fibroblast proliferation. Exposure of cardiomyocytes with thrombin induced immediate permanent dose-dependent increase in [Ca²⁺]_i. Ca²⁺ response decreased in a calcium-free medium. Peptide agonists of PAR1 and PAR2 also stimulated the increase in [Ca²⁺]_i in cardiomyocytes. Thrombin induced a short-term increase in [Ca²⁺]_i in cardiac fibroblasts and potentiated cell proliferation. PAR2 agonists trypsin and peptide SLIGRL stimulated proliferation of cardiac fibroblasts. Our results indicate that cardiomyocytes and cardiac fibroblasts from chick embryos have at least two types of PAR (types 1 and 2). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 609–612, December, 2007     

Residual sarcoplasmic reticulum Ca<Superscript>2+</Superscript> concentration after Ca<Superscript>2+</Superscript> release in skeletal myofibers from young adult and old mice

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca²⁺ or significant residual Ca²⁺ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca²⁺ indicators targeted to the SR that exhibit low-Ca²⁺ affinity, a fast biosensor: Ca²⁺ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca²⁺ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca²⁺ with aging. We found CatchER a proper tool to investigate SR Ca²⁺ depletion in young adult and older adult mice, consistently tracking SR luminal Ca²⁺ release in response to brief and repetitive stimulation. We evoked SR Ca²⁺ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca²⁺ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca²⁺ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca²⁺ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.     

Altered intracellular Ca<Superscript>2+</Superscript> regulation in chronic rat heart failure

Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF have not been clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake is required for decreased contractility, we investigated the cytosolic Ca²⁺ transients and SR Ca²⁺ content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca²⁺ transients were markedly reduced in amplitude in HF myocytes (ΔF/F ₀ = 12.3 ± 0.8) compared with control myocytes (ΔF/F ₀ = 17.7 ± 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca²⁺ content (HF: ΔF/F ₀ = 12.4 ± 1.1, control: ΔF/F ₀ = 32.4 ± 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.     

Voltage-gated Ca<Superscript>2+</Superscript> channel mRNAs and T-type Ca<Superscript>2+</Superscript> currents in rat gonadotropin-releasing hormone neurons

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the neuroendocrine regulation of reproduction. We have previously reported that rat GnRH neurons exhibit voltage-gated Ca²⁺ currents. In this study, oligo-cell RT-PCR was carried out to identify subtypes of the α₁ subunit of voltage-gated Ca²⁺ channels in adult rat GnRH neurons. GnRH neurons expressed mRNAs for all five types of voltage-gated Ca²⁺ channels. For T-type Ca²⁺ channels, α_1H was dominantly expressed in GnRH neurons. Electrophysiological analysis in acute slice preparations revealed that GnRH neurons from adult rats exhibited T-type Ca²⁺ currents with fast inactivation kinetics (~20 ms at −30 mV) and a time constant of recovery from inactivation of ~0.6 s. These results indicate that rat GnRH neurons express subtypes of the α₁ subunit for all five types of voltage-gated Ca²⁺ channel, and that α_1H was the dominant subtype in T-type Ca²⁺ channels.     

Long-term presence of angiotensin II type 1 receptor autoantibody reduces aldosterone production by triggering Ca<Superscript>2+</Superscript> overload in H295R cells

Preeclamptic women are reported to have inadequate plasma volume expansion coupled with a suppressed secretion of aldosterone; however, the specific mechanism of preeclampsia remains unclear. We demonstrated that the presence of long-term angiotensin II type 1 receptor autoantibody (AT1-AA) reduces aldosterone production by triggering a Ca²⁺ overload in H295R cells. AT1-AA was discovered in preeclamptic women and reported to activate AT₁R, and consequently elevate intracellular Ca²⁺. We found that AT1-AA significantly prolonged the time of intracellular Ca²⁺ elevation. Besides promoting aldosterone production as a second messenger, Ca²⁺ overload shows a cytotoxic effect. Our data reveals that long-term presence of AT1-AA triggered a Ca²⁺ overload and consequent impairment of aldosterone production, which could be prevented by a PKC inhibitor, Gö 6983, or a calcium channel inhibitor, nifedipine. These findings have clinical significance because AT₁R blockers are not recommended for treatment of preeclampsia due to their potential harm to the fetus. Our findings also emphasize a potential clinical benefit of immunoadsorption or neutralization of AT1-AA in preeclamptic women.     

Nongenomic effect of androgens on Ca<Superscript>2+</Superscript> concentration in human lymphocytes

Testosterone and its high-molecular-weight form (testosterone covalently immobilized on bovine serum albumin) induced a rise of intracellular calcium concentration. The effectiveness of dihydrotestosterone was much lower compared to that of testosterone. Gestagens drospirenone and, to a lesser extent, 17α-acetoxy-3α-butanoyloxy-6-methylpregna-4,6-dien-20-one prevented the testosterone-induced Ca²⁺ entry into cells. Antagonist of intracellular androgen receptors cyproterone acetate had no effect on testosterone-induced variations in calcium concentration. Human lymphocytes can be used as an experimental test system for screening and evaluation of the molecular mechanisms of nongenomic membranotropic effect of androgens and new compounds with antiandrogen activity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 5, pp. 542–544, May, 2007     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

Donepezil suppresses intracellular Ca<Superscript>2+</Superscript> mobilization through the PI3K pathway in rodent microglia

Background

Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer’s disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells.

Methods

We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells.

Results

In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca²⁺ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway.

Conclusions

These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

     

A reappraisal of the Ca<Superscript>2+</Superscript> dependence of fast inactivation of Ca<Superscript>2+</Superscript> release in frog skeletal muscle

Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca²⁺ Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca²⁺ release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca²⁺-free conditions. 2-APB applied extracellularly at 100 μM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between −45 and −35 mV from 7.99 ± 0.73 nC/μF (N = 17) before drug application to 6.27 ± 0.68 nC/μF in the presence of 2-APB. This effect was mostly on the delayed Q_γ component. In fibers treated with the SERCA ATPase inhibitor CPA the Q_γ component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca²⁺ release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 μM. 2-APB also inhibited the L-type Ca²⁺ current (20 ± 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 μM, a typical concentration used for inhibiting SOCE, to fibers held at −80 mV inhibited the charge moved in response to pulses ranging −45 to −30 mV from 7.95 ± 2.59 nC/μF to 3.48 ± 0.9 nC/μF (N = 12). A parallel reduction of Ca²⁺ release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to −100 mV. SKF-96365 also inhibited the L-type Ca²⁺ current (41 ± 8%, N = 4) and increased its rate of inactivation.     

Ca<Superscript>2+</Superscript> regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca²⁺ chelator or inhibitors of Ca²⁺-permeable channels or Ca²⁺-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca²⁺ channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca²⁺-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the γ subunit of epithelial Na⁺ channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 μM amiloride or 10 μM phenamil. Taken together, these findings suggest that [Ca²⁺]_c regulated by endoplasmic Ca²⁺-pump and Ca²⁺-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na⁺ across the basolateral membrane in marginal cells.     

Cold adaptation modulates Ca<Superscript>2+</Superscript> signaling in brown preadipocytes

One-week cold exposure of mice led to a 2-fold increase in the density of α₁-adrenoceptors in brown adipose tissue. The density of α₁-adrenoceptors returned to normal after adaptation to cold for 2 weeks. The reduced Ca²⁺ signaling in stem cells of brown fat activated via β-adrenoceptors and cAMP was transformed into the Ca²⁺-system induced by α₁-adrenoceptors and similar to that in mature brown adipocytes. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 7, pp. 59–62, July, 2004     

cGMP reduces the sarcoplasmic reticulum Ca<Superscript>2+</Superscript> loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca<Superscript>2+</Superscript> by cGMP

Ca²⁺ and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca²⁺ and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca²⁺ storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca²⁺ has not been completely understood. To evaluate this role, intracellular Ca²⁺ concentration ([Ca²⁺]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca²⁺ from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca²⁺]i undershoot. Caffeine-induced Ca²⁺ transient peak and [Ca²⁺]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca²⁺]i undershoot and reduced the subsequent caffeine peak (SR-Ca²⁺ loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca²⁺ ATPase with cyclopiazonic acid inhibited the [Ca²⁺]i undershoot as well as the SR-Ca²⁺ loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca²⁺ loading. Cyclic GMP enhanced [Ca²⁺]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca²⁺ content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca²⁺ loading and the simultaneous release of SR-Ca²⁺. The reduction of SR-Ca²⁺ content induced by cGMP might be a putative mechanism limiting releasable Ca²⁺ in response to a particular stimulus.     

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca²⁺ and voltage-gated potassium (BK_Ca) channels. Here, we explored the effect of LPI on intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels using excised inside-out patches from endothelial cells. The functional expression of IK_Ca was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK_Ca channels with a slope conductance of 39 pS in symmetric K⁺ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK_Ca single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK_Ca single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.     

Prolonged exercise potentiates sarcoplasmic reticulum Ca<Superscript>2+</Superscript> uptake in rat diaphragm

The effects of a single bout of prolonged treadmill exercise [mean=81 (13) min] on sarcoplasmic reticulum (SR) Ca²⁺ release, uptake and ATPase activity were determined in the costal region of rat diaphragm (D) and red gastrocnemius (RG). Glycogen depletion measurements made immediately following exercise suggested that treadmill running substantially recruited the fibers throughout both muscles. SR Ca²⁺ ATPase activity, measured in isolated SR vesicles, decreased in the RG by 33% but remained unchanged in D in response to the exercise bout. This effect in RG was matched by a 37% decline in Ca²⁺ uptake and a 28% depression in Ca²⁺ release when measured in muscle homogenates. Conversely, Ca²⁺ uptake increased between 157% and 263% in the D in the absence of any change in Ca²⁺ release. These data show that the attenuation of SR function that has been consistently observed in limb muscle over the last several decades is absent in diaphragm despite the fact that its fibers appear to experience sufficient activity to deplete their glycogen. In fact, the large increase in Ca²⁺ uptake in D shows that prolonged activity actually potentiates the ability of SR vesicles to sequester Ca²⁺ in the absence of any increase in energy cost. Thus, it appears necessary to re-evaluate the role of exercise in regulating Ca²⁺ sequestration by the SR as different muscles may respond in ways that are dictated by their function. Electronic Publication     

Trifluoperazine protects brain plasma membrane Ca<Superscript>2+</Superscript>-ATPase from oxidative damaging

In the central nervous system (CNS), a number of different pathological processes such as necrosis, Parkinson’s and Alzheimer’s diseases are related to disturbance in calcium homeostasis associated with oxidative stress. Here we compare the susceptibility of rat brain plasma membrane Ca²⁺-ATPase (PMCA) and sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) isoforms to in vitro oxidative stress, and investigate a putative role of trifluoperazine (TFP), an antipsychotic drug that is also a powerful inhibitor of Ca²⁺-transporter proteins, in protecting these enzymes. It is shown that, in rat brain, PMCA is very sensitive to the damage induced by preincubation with Fe²⁺-ascorbate, or Fe²⁺-ascorbate plus H₂O_2, while SERCA is resistant. Inhibition of PMCA activity promoted by Fe²⁺/ascorbate medium is fully prevented by the presence of μM concentrations of either butylated hydroxytoluene (BHT) or TFP, but only partially protected, or reversed, by dithiothreitol (DTT), pointing to some protein cysteine(s) as one of the main targets for a lipid peroxidation-dependent damaging mechanism. However, when 0.5–1 mM H₂O₂ is added together with Fe²⁺/ascorbate, both BHT and TFP only partially prevent ATPase activity inhibition, and DTT does not confer any protection, suggesting two possible additional mechanisms involving both lipid peroxidation and direct damage to PMCA at amino acid residues other than cysteines. A possible use of μmolar concentrations of TFP as a direct antioxidant protector for PMCA under oxidative stress conditions is discussed.     

Osteoblasts modulate Ca<Superscript>2+</Superscript> signaling in bone-metastatic prostate and breast cancer cells

Metastatic prostate and breast cancers display a predilection for the skeleton. The high incidence of skeletal metastasis may be a reflection of favorable reciprocal interactions between the bone microenvironment and disseminated cancer cells. Here we show that bone-metastatic PC3-ML prostate cancer cells and MDA-231 breast cancer cells—when co-cultured with human osteoblasts—down-regulate the increase in cytosolic free calcium (Ca²⁺) induced by agonist stimulation. This osteoblast promoted alteration of Ca²⁺ signaling develops and reverts in a time-dependent manner. Most importantly, the Ca²⁺ responses of cancer cells lacking bone metastatic potential are not affected by osteoblasts. The limited increase in cytosolic Ca²⁺ observed in bone-metastatic cells does not result from depleted intracellular Ca²⁺ stores but rather a decreased entry of Ca²⁺ from the extracellular space. Interestingly, the inhibition of histone deacetylase in cancer cells replicates the changes in Ca²⁺ signaling induced by osteoblasts, suggesting the participation of epigenetic mechanisms. Finally, cancer cells harvested from skeletal metastases induced in mice showed Ca²⁺ responses identical to cells co-cultured with osteoblasts. However, Ca²⁺ signaling in cancer cells recovered from metastases to soft-tissues was not affected, emphasizing the role of the bone microenvironment in regulating the functional behavior of bone-metastatic cells. We propose that osteoblasts protect selected malignant phenotypes from cell death caused by an excessive increase in cytosolic Ca²⁺, thereby facilitating their progression into macroscopic skeletal metastases.     

Excitation contraction uncoupling by high intracellular [Ca<Superscript>2+</Superscript>] in frog skeletal muscle: a voltage clamp study

Raising the intracellular [Ca²⁺] ([Ca²⁺]_i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca²⁺]_i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca²⁺ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca²⁺]_i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca²⁺]_i intracellular solution, permeabilization of the plasma membrane by a Ca²⁺ ionophore) all of which produced impairment of excitation–contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/μF meanwhile the Ca²⁺ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 μM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca²⁺]_i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca²⁺.     

Effect of Ca<Superscript>2+</Superscript> gradient on the structure of sarcoplasmic reticulum membranes

The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca²⁺ gradient corresponding to the status of Ca²⁺ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca²⁺-ATPase, ryanodine receptor, and lipids). The transmembrane Ca²⁺ gradient modulates the protein-lipid interactions and structural characteristics of the membrane. The proposed model can be used for studies of the effects of pharmacologically active substances and endogenous regulators. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 517–521, November, 2007