Effects of prostaglandin D2 on membrane potential in neuroblastoma X glioma hybrid cells as determined with a cyanine dye

A cyanine dye, diS-C₃-(5) was used to determine the effects of prostaglandins on the membrane potential in neuroblastoma X glioma cells (NG 108-15). The largest depolarization was seen with prostaglandin D₂ (ED₅₀ = 1.5 μM), and relative potencies of various prostaglandins (3 μM) were: D₂, 100; I₂, 41; E₁, 17; E₂, 7; and F_2α, 7. 5-Hydroxytryptamine in a dose over 100 μM also depolarized the membrane. The effect of prostaglandin D₂ was observed in a Na⁺-free medium or when Ca²⁺ was replaced by Sr²⁺. The addition of 3 mM ethylene-glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or 5 mM Co²⁺ partially inhibited the effects. These observations suggest that the depolarization of membrane by prostaglandin D₂ may primarily be related to alteration of Ca²⁺ permeability in the cell membrane.     

Activation of Phosphatidylinositol-linked Novel D<Subscript>1</Subscript> Dopamine Receptor Contributes to the Calcium Mobilization in Cultured Rat Prefrontal Cortical Astrocytes

Recent evidences indicate the existence of an atypical D₁ dopamine receptor other than traditional D₁ dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase C_β (PLC_β). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D₁ dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca²⁺]_i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca²⁺]_i. Application of D₁ receptor, but not D₂, α₁ adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca²⁺]_i rise, indicating that activation of the D₁ dopamine receptor was essential for this response. Increase in [Ca²⁺]_i was a two-step process characterized by an initial increase in [Ca²⁺]_i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca²⁺ entry. Furthermore, SKF83959-stimulated increase in [Ca²⁺]_i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D₁ receptor by SKF83959 mediates a dose-dependent mobilization of [Ca²⁺]_i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.     

1α,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1α,25(OH)₂-vitamin D₃ (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca²⁺ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca²⁺ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca²⁺-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca²⁺-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts

Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E₂ via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca²⁺ kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca²⁺ concentration through the activation of B₂ receptors. Removal of extracellular Ca²⁺ attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca²⁺ pump inhibitor, completely inhibited BK-induced increase of intracellular Ca²⁺. These results suggested that bath-applied BK activated store-operated Ca²⁺ channels (SOCCs) following Ca²⁺ store depletion via B₂ receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl₃, LaCl₃ and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca²⁺ influx via B₂ receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.     

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis

Background

Estradiol (E₂) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E₂ can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERα (mERα)-enriched GH₃/B₆/F₁₀ rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E₂ and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects.

Methods

To investigate the activation of different nongenomic pathways, and determine the involvement of mERα, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca²⁺] by Fura-2 fluorescence imaging in cells treated with E₂ or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).

Results

Coumesterol and daidzein increased PRL release similar to E₂ in GH₃/B₆/F₁₀ cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1–10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca²⁺ uptake, though in unique dose-dependent Ca²⁺ response patterns for several aspects of this response. A subclone of GH₃ cells expressing low levels of mERα (GH₃/B₆/D₉) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERα.

Conclusion

Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca²⁺]) via mERα than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E₂ and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.     

Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca²⁺]_i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca²⁺]_i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca²⁺]_i transient in osteoblasts. Fluorescent dyes DiBAC₄(3) and fura-2 AM were respectively used to detect membrane potential and [Ca²⁺]_i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca²⁺]_i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca²⁺]_i transients. Replacing extracellular Na⁺ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca²⁺]_i transients. However, voltage-activated K⁺ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca²⁺ or blocking of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca²⁺]_i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca²⁺ mobilization wherein FSS activated SACs to promote Na⁺ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca²⁺ influx though L-VSCCs switched on [Ca²⁺]_i response in osteoblasts.     

Dependence of membrane potential on Ca2+ transport in cultured cytotrophoblasts of human immature placentas

The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (E_m) and the membrane resistance in a physiological medium were around ?49 mV and 12 MΩ, respectively. The membrane potential was dependent, to a large extent, on the external Ca²⁺ concentration ([Ca²⁺]₀). Deprivation of external Ca²⁺ reduced membrane potential to about ?20 mV, and an increase in [Ca²⁺]₀ caused a hyperpolarization in a saturable manner. The Ca²⁺-dependency of membrane potential was affected remarkably by [K⁺]₀, but not by [Na⁺]₀ or [Cl^?]₀. The intracellular Ca²⁺ injection hyperpolarized the membrane in a Ca²⁺-free medium. A Ca²⁺ channel blocker, verapamil, completely abolished the Ca²⁺-dependent E_m. The Ca²⁺-dependent E_m was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca²⁺-dependent E_m in cultured human cytotrophoblast is caused by a Ca²⁺ influx which, in turn, increases the K⁺ conductance of the cell membrane, presumably due to stimulation of Ca²⁺-activated K⁺ channel.     

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca²⁺ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca²⁺ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode''s solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (E_m), and intracellular Ca²⁺ ([Ca²⁺]_i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]_o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca²⁺ release. However, sugar solutions caused a global increase in [Ca²⁺]_i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in E_m (by 15.04 ± 1.85 mV; n = 8), whereas [Ca²⁺]_i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and E_m was associated with LCR. Both global and localized SR Ca²⁺ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca²⁺ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca²⁺ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in E_m. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.     

G protein‐coupled estrogen receptor‐mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells

G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca²⁺ concentration, [Ca²⁺]_i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca²⁺]_i in RBMVEC. The effect of G‐1 on [Ca²⁺]_i was abolished in Ca²⁺‐free saline or in the presence of a L‐type Ca²⁺ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca²⁺‐activated K⁺ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.     

Effects of prostaglandins e(2) and f(2alpha) on the electrofusion of pea mesophyll protoplasts

The effects of prostaglandins E₂ and F_2α on the electrofusion of pea (Pisum sativum cv Ran 1) mesophyll protoplasts were examined. Prostaglandins E₂ and F_2α influenced electrofusion by lowering the threshold voltage necessary for fusion of dielectrophoretically arranged pairs of protoplasts. The direct current voltage threshold decreased with increasing Ca²⁺ concentration up to 0.1 millimolar CaCl₂ and the effects of prostaglandins E₂ and F_2α were more pronounced when CaCl₂ was present in the medium. Treatment with calcium channel blocker methoxy verapamil did not change the prostaglandin effects, while the addition of ethyleneglycol-bis (β-aminoethyl either)-N,N,N′,N′-tetraacetic acid, which binds free Ca²⁺, increased the threshold voltage. Influence of prostaglandins E₂ and F_2α and Ca²⁺ on the membrane fluidity was investigated by analysis of pyrene fluorescence spectra. The values of the ratio between the maximum fluorescence emission intensities of the excimer and the monomer forms (I_ex/I_mon) indicated that prostaglandins and Ca²⁺ decrease the membrane fluidity. It is proposed that electrically evoked displacement of plasmalemma components takes part in the fusion process (U Zimmermann 1982 Biochim Biophys Acta 694: 227-277). We suggest that prostaglandins E₂ and F_2α facilitate the electrofusion of pea mesophyll protoplasts by changing the fluidity of plasmalemma.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

In vivo activation of cirral movement in Stylonychia by calcium

Summary Motor responses of cirri (= organelles consisting of bundles of cilia) in the protozoan Stylonychia are elicited by positive or negative shifts of the membrane voltage from its resting state. The same responses are evoked at voltages near the Ca²⁺ equilibrium potential (E_Ca) applying extremely positive steps under voltage clamp. Motor responses recorded at large positive voltages approaching E_Ca from the negative side corresponded to cirral activation following physiological depolarization from the resting potential (DCA). The hyperpolarization-induced activation of the cirri (HCA) was documented during step potentials positive to E_Ca, suggesting that the observed HCA of the cirri resulted from an efflux of Ca²⁺ from the ciliary space as compared with DCA, which is related to Ca²⁺ influx. The ciliary responses were graded functions of the rising outward or inward driving force for Ca²⁺. Slopes of reciprocal plots of response latencies near E_Ca as a function of membrane potential indicate a removal of Ca²⁺ during HCA which exceeds the free intraciliary Ca²⁺ content at rest. It is suggested that this excess Ca²⁺ is released from axonemal binding sites.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Terbium binding to axonal membrane vesicles from lobster (Homarus Americanus) peripheral nerve. A probe of calcium binding sites

Tb³⁺, a fluorescent trivalent cation with physicochemical properties similar to Ca²⁺, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb³⁺ can be displaced by other cations in the order: Ca²⁺ > Mg²⁺ = Zn²⁺ > NH₄⁺. The binding of Tb³⁺ to the lower affinity site (K_D(app) = 6.0 μM) is inhibitable by Na⁺, Mg²⁺ and Ca²⁺, whereas the higher affinity site (K_D(app) = 2.2 μM) is only sensitive to Ca²⁺. Using this spectral probe the role of Ca²⁺ in peripheral nerve membrane function can be investigated.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.