Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium,and by other divalent ions

Summary Direct inhibitory effects of Ca²⁺ and other ions on the epithelial Na⁺ channels were investigated by measuring the amiloride-blockable²²Na⁺ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol. 81:643–666, 1983) we found that the presence of micromolar concentrations of Ca²⁺ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive²²Na⁺ uptake could not be blocked by Ca²⁺ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba²⁺, Sr²⁺, or VO²⁺, but not by Mg²⁺. The Ca²⁺ inhibition constant was a strong function of pH, and varied from 0.04 m at pH 7.8 to >10 m at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of²²Na⁺ uptake in vesicles that contained 0.5 m Ca²⁺. This Ca²⁺ activity produced a maximal inhibition of²²Na⁺ uptake at pH7.4 but had no effect at pH7.0. The tracer fluxes measured in the absence of Ca²⁺ were pH independent over this range. The data is compatible with the model that Ca²⁺ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca²⁺ from binding to this site but does not by itself inhibit transport. The fact that the apical Na⁺ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca²⁺, rather than, or in addition to changes in the cytoplasmic level of free Ca²⁺.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers

Summary Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] _o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] _o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is 20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca²⁺ artificial seawater (ASW) or in Ca²⁺-free ASW decreases approximately linearly with increasing logarithm of [K] _o . The slope of the plot is slightly steeper for fibers bathed in Ca²⁺-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK _o depends on the external Na⁺ and Ca²⁺ concentrations, as well as on external pH, but is independent of external Mg²⁺ concentration. External application of 10^–4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca²⁺ by injection of 250mm EGTA before exposure of the fiber to 100mm K _o leads to 60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K _o by 50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na₂ before elevation of [K] _o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K _o for time periods ranging from 30 sec to 10 min causes a small (60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K _o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca²⁺ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Influence of isolation media on synaptosomal properties: Intracellular pH,pCa, and Ca2+ uptake

Preparations of synaptosomes isolated in sucrose or in Na⁺-rich media were compared with respect to internal pH (pH₁), internal Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and⁴⁵Ca²⁺ uptake due to K⁺ depolarization and Na⁺/Ca²⁺ exchange. We found that synaptosomes isolated in sucrose media have a pH_i of 6.77±0.04 and a [Ca²⁺]_i of about 260 nM, whereas synaptosomes isolated in Na⁺-rich ionic media have a pH_i of 6.96±0.07 and a [Ca²⁺]_i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca²⁺ only by voltage sensitive calcium channels (VSCC'S) when K⁺-depolarized, while the Na⁺-rich synaptosomes take up⁴⁵Ca²⁺ both by VSCC'S and by Na⁺/Ca²⁺ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca²⁺ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca²⁺ uptake by VSCC'S, but not by Na⁺/Ca²⁺ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca²⁺ flux through channels and through Na⁺/Ca²⁺ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca²⁺ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF 2,7-Biscarboxyethyl-5(6)-carboxyfluorescein - BCECF/AM acetoxymethyl ester of BCECF - [Ca²⁺]_i Internal free calcium ion concentration - CBZ-DMB 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil - DMB 2, 4-dimethylbenzamil - DMSO dimethyl sulfoxide - Indo-1/AM acetoxymethyl ester of Indo-1 - MES 2-|N-Morpholino|ethanesulfonic acid - NMG N-methyl-D-glucamine - pH_i internal pH - TPP⁺ tetraphenylphosphonium - _p plasma membrane potential     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm ⁴⁵Ca²⁺-containing buffer results in a small ( two fold) increase in [Ca²⁺] _i , a significant decrease in osmotic fragility, and a decrease in intracellular K⁺ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na⁺]. This decrease in [K⁺] _i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K⁺ to 125mm prevents the A23187-induced changes in osmotic fragility, K⁺ content and cell volume, but does not prevent the ionophore-induced uptake of⁴⁵Ca²⁺. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in⁴⁵Ca²⁺ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K⁺ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in⁴⁵Ca²⁺ uptake and concomitant change in K⁺ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K⁺] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca²⁺ until Ca²⁺ uptake is increased substantially at the higher concentrations of A23187.     

GTPγS-induced calcium transients and exocytosis in human neutrophils

Exocytosis and intracellular free calcium ([Ca²⁺]_in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5-O(3-thiotriphosphate) (GTPS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of 40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca²⁺]_in increases from 150 nM to 600 nM with a sigmoidal time course. The peak concentration is reached within 30 s but the main increase occurs during 3s. [Ca²⁺]_in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca²⁺]_in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons

Summary We studied the effects of lanthanum (La³⁺) on the release of ³H-norepinephrine(³H-NE), intracellular Ca²⁺ concentration, and voltage clamped Ca²⁺ and K⁺ currents in cultured sympathetic neurons. La³⁺ (0.1 to 10 m) produced concentration-dependent inhibition of depolarization induced Ca²⁺ influx and ³H-NE release. La³⁺ was more potent and more efficacious in blocking ³H-NE release than the Ca²⁺-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 m, La³⁺ produced a complete block of the electrically stimulated rise in intracellular free Ca²⁺ ([Ca²⁺] _i ) in the cell body and the growth cone. The stimulation-evoked release of ³H-NE was also completely blocked by 3 m La³⁺. However, 3 m La³⁺ produced only a partial block of voltage clamped Ca²⁺ current (I _Ca). Following La³⁺ (10 m) treatment ³H-NE release could be evoked by high K⁺ stimulation of neurons which were refractory to electrical stimulation. La³⁺ (1 m) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K⁺ current (I _A ) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated ³H-NE release is a result of the unique ability of La³⁺ to activate a stabilizing, outward K⁺ current at the same concentration that it blocks inward Ca²⁺ current.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.