Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber,Isostichopus badionotus

Summary The physiological properties of mechanical responses and the intracellular localization and translocation of calcium as a pyroantimonate precipitate were studied in the longitudinal retractor muscle (LRM) of a Bermuda sea cucumber. Acetylcholine (ACh)-induced contraction was reduced by lowering the external Ca concentration, and suppressed completely by prolonged soaking in Ca-free solution. The magnitude of ACh-induced contraction was decreased by Mn and La ions. Furthermore, procaine reduced the ACh-induced contraction. The complete removal of Ca and Mg ions from the external medium induced a socalled Ca · Mg-removal contraction. Electron microscopically, numerous subsarcolemmal vesicles were observed in the LRM fibers. In the resting fibers, pyroantimonate precipitates were localized in the subsarcolemmal vesicles and along the inner surface of plasma membrane. While, in the fiber fixed during mechanical activity, the pyroantimonate precipitates were decreased remarkably in the subsarcolemmal vesicles and at the plasma membrane, and diffusely distributed in the myoplasm. Electronprobe X-ray microanalysis showed that the precipitate contains Ca in a significant amount. These results indicate that the contraction of the LRM fibers is caused not only by Ca-influx but also by Ca-release from the intracellular storage sites, such as the subsarcolemmal vesicles and the inner surface of plasma membrane.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Evidence for extracellular localization of activator calcium in dog coronary artery smooth muscle as studied by the pyroantimonate method

Summary Correlated physiological and electron-microscopic studies were made on the source of calcium activating the contractile system (activator calcium) in dog coronary artery smooth muscle fibers. The magnitude of contracture tension induced by 100 mM K⁺ was dependent on external Ca²⁺ concentration and reduced or eliminated by factors known to reduce the Ca²⁺ spike or ca²⁺ influx. Little or no mechanical response was elicited by treatments known to cause release of intracellularly stored calcium. These results indicated that the contractile system is mainly activated by the inward movement of extracellular calcium. In accordance with the physiological experiments, electron-opaque pyroantimonate precipitate containing calcium was found in the lumina of caveolae, but not in any intracellular structures close to the plasma membrane, when the relaxed fibers were fixed in a 1% osmium tetroxide solution containing 2% potassium pyroantimonate. If the contracted fibers were fixed in the same solution, the pyroantimonate precipitate was diffusely distributed in the myoplasm in the form of numerous particles, while the precipitate in the caveolar lumina was scarcely seen. These findings are discussed in connection with the regulation of intracellular Ca²⁺ concentration in dog coronary artery smooth muscle.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

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

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

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.     

Effects of Ca2+ and other divalent cations on K+-evoked force production of slow muscle fibers from Rana esculenta and Rana pipiens

Summary Slow muscle fibers were dissected from cruralis muscles of Rana esculenta and Rana pipiens. Isometric contractures were evoked by application of K⁺-rich Ringer's containing Ca²⁺, Ni²⁺, Co²⁺, Mn²⁺ or Mg²⁺. High (7.2 mmol/liter) external Ca²⁺ concentration raised, 0 Ca²⁺ lowered the K⁺ threshold. Replacing Ca²⁺ by Ni²⁺ or Co²⁺ had an effect similar to that of high Ca²⁺ Ringer's. In Mg²⁺ Ringer's the K⁺ concentration-response curve was flattened. These effects were observed already after short exposure times in both species of slow fibers. When Ca²⁺ was removed for long periods of time the slow fibers of R. esculenta lost their contractile response to application of high K⁺ concentrations much more quickly than those of R. pipiens, while the response to caffeine (20 mmol/liter) was maintained. Upon readmission of Ca²⁺ contractile ability was quickly restored in the slow fibers of both R. esculenta and R. pipiens, but the effects of Ni²⁺ (or Co²⁺, Mn²⁺ and Mg²⁺) were much larger in R. esculenta than in R. pipiens slow fibers. It is concluded that divalent cations have two different sites of action in slow muscle fibers. K⁺ threshold seems to be affected through binding to sites at the membrane surface; these sites bind Ni²⁺ and Co²⁺ more firmly than Ca²⁺. The second site is presumably the voltage sensor in the transverse tubular membrane, which controls force production, and where Ca²⁺ is the most effective species of the divalent cations examined.We are grateful to Mrs. S. Pelvay for technical assistance.     

ATP-dependent Ca2+ transport in wheat root plasma membrane vesicles

Plasma membrane preparations of high purity were obtained from roots of dark-grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. These preparations mainly contained sealed, right-side-out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside-out (cytoplasmic side out) vesicles increased to ca 30%. Inside-out and right-side-out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two-phase system. In this way, highly purified, sealed, inside-out (>60% inside-out) vesicles were isolated and subsequently used for characterization of the Ca²⁺ transport system in the wheat plasma membrane. The capacity for ⁴⁵Ca²⁺ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside-out plasma membrane vesicles) were all enriched in the inside-out vesicle fraction as compared to the right-side-out fraction. This confirms that the ATP-binding site of the ⁴⁵Ca²⁺ transport system, similar to the H⁺-ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The ⁴⁵Ca²⁺ uptake was MgATP-dependent with an apparent K_m for ATP of 0.1 mM and a high affinity for Ca²⁺ [K_m(Ca²⁺/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the ⁴⁵Ca²⁺ uptake seen with ATP. The ⁴⁵Ca²⁺ uptake was stimulated by monovalent cations; K^? and Na⁺ being equally efficient. Vanadate inhibited the ⁴⁵Ca²⁺ accumulation with half-maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K⁺-stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half-maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca²⁺ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP-binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca²⁺ within the cell.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na⁺/Ca²⁺ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca²⁺ reabsorption in the kidney. (1) Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca²⁺ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca²⁺ reabsorption. (2) The vesicle preparation contained high, digitalis-sensitive (Na⁺+K⁺-ATPase activities indicating its origin from the basolateral portion of plasma membrane. (3) The operation of Na⁺/Ca²⁺ antiport device was excluded by the findings that steep Ca²⁺ gradients formed by ATP-dependent Ca²⁺ accumulation in the vesicles were not discharged by extravesicular Na⁺, and did not drive ⁴⁵Ca²⁺ uptake into the vesicles via a Ca²⁺-⁴⁵Ca²⁺ exchange. (4) The ATP-dependent Ca²⁺ uptake into the vesicles became increasingly depressed with time by extravesicular Na⁺. This was not due to an impairment of the Ca²⁺ pump itself, but caused by Na⁺/Ca²⁺ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca²⁺ accumulation in the vesicles. (5) Earlier observations on Na⁺-induced release of Ca²⁺ from vesicles pre-equilibrated with Ca²⁺, seemingly favoring the existence of a Na⁺/Ca²⁺ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na⁺/Ca²⁺ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca²⁺ reabsorption through tubule epithelium essentially depending on the operation of a Na⁺/Ca²⁺ antiport device.     

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch–clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca²⁺-activated K⁺-selective channel K_Ca3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca²⁺] will cause mtK_Ca3.1 opening, thus linking inner membrane K⁺ permeability and transmembrane potential to Ca²⁺ signalling.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.     

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.     

Contractile activation in the retractor unguis muscle of the cockroach Periplaneta americana

1. The K⁺-induced contracture consists of a phasic and a sustained component. Both were eliminated in Ca²⁺-free saline, but the sustained component recovered on the addition of Ca²⁺ to the muscle.2. Procaine mainly inhibited the phasic component. 3. Unlike the sustained component, the phasic component was inhibited by nifedipine in a concentration dependent manner.4. Divalent cations such as Mn²⁺, Co²⁺ and Ni²⁺ markedly increased the sustained component at low concentrations, but decreased it at high concentrations. The cations also modified the phasic component differentially, but to a lesser extent. High concentration abolished the phasic component.5. Ouabain markedly enhanced the sustained component.6. Caffeine contracture was a phasic type. Its duration and amplitude were augmented by pre-soaking the muscle in Na⁺-reduced salines. Immediate pre-treatment with caffeine eliminated the phasic component of the 160mM K⁺-induced contracture.7. These results suggest that a Na-Ca exchange mechanism may play a role in excitation-contraction coupling in insect muscle. Calcium ions flowing into the cell upon membrane depolarization may specifically activate the phasic component by way of a calcium-induced calcium releasing mechanism.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Localization of a Mg2+- or Ca2+-activated ("basic") ATPase in skeletal muscle.

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% ($\text{w}\text{w}$) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Localization of a Mg- or Ca-activated (“basic”) ATPase in skeletal muscle

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.