Muscle Contraction during Hyperpolarizing Currents in the Crab

Isolated muscle fibers from the motor legs of the crab Trichodactilus dilocarcinus were submitted to strong hyperpolarizing currents of varied intensities which produced tension during the current pulse. Threshold for tension was obtained with intensities of about 0.2 x 10^–5 A, changing E_m to ca. –150 mV (starting from a resting potential ofca. –80 mV). At the closure of the anodic square pulse, a second phase of tension usually appeared superimposed upon the one obtained during hyperpolarization. The first phase of tension increased with the increase of Ca⁺⁺ concentration in the bath. Sr⁺⁺ produced the same type of mechanical output as Ca⁺⁺. When added to the normal Ca⁺⁺ concentration, Ba⁺⁺ and Mn⁺⁺ in low concentrations (up to 21.5 mM) also increased the tension of this phase, but at higher concentrations they blocked both phases while Mg⁺⁺ did not alter the tension. Of all the divalent cations employed, only Sr⁺⁺ is capable of developing tension as a substitute for Ca⁺⁺ in the external media. Procaine administered in a dosage (5 x 10^–3 W/V)which would suppress the contracture due to caffeine (10 mM), did not modify the tension developed during the hyperpolarization. The preceding data indicate that the Ca⁺⁺ required for tension during hyperpolarization comes from sites which would differ from those usually postulated for tension due to depolarization in the muscle fibers of other crustaceans (American crayfish). Furthermore, the external source of Ca⁺⁺ appears to be one mainly implicated in the induction of tension due to inward current pulses.     

Galvanotaxis of human granulocytes: electric field jump studies

The static and dynamic responses of human granulocytes to an electric field were investigated. The trajectories of the cells were determined from digitized pictures (phase contrast). The basic results are: (i) The track velocity is a constant as shown by means of the velocity autocorrelation function. (ii) The chemokinetic signal transduction/response mechanism is described in analogy to enzyme kinetics. The model predicts a single gaussian for the track velocity distribution density as measured. (iii) The mean drift velocity induced by an electric field, is the product of the mean track velocity and the polar order parameter. (iv) The galvanotactic dose-response curve was determined and described by using a generating function. This function is linear in E for E < E ₀ = 0.78 V/mm with a galvanotaxis coefficient K _G of (–0.22 V/mm)^–1 at 2.5 mM Ca⁺⁺. For E > E ₀ the galvanotactic response is diminished. This inhibition is described by a second term in the generating function (–K _G · K _I (E – E ₀)) with an inhibition coefficient K _I of 3.5 (v) The characteristic time involved in directed movement is a function of the applied electric field strength: about 30 s at low field strengths and below 10 s at high field strengths. The characteristic time is 32.4 s if the cells have to make a large change in direction of movement even at large field strength (E jump). (vi) The lag-time between signal recognition and cellular response was 8.3 s. (vii) The galvanotactic response is Ca⁺⁺ dependent. The granulocytes move towards the anode at 2.5 mM Ca⁺⁺ towards the cathode at 0.1 mM Ca⁺⁺. (viii) The directed movement of granulocytes can be described by a proportional-integral controler. Offprint requests to: H. Gruler     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Spontaneous and repetitive driver potentials in crab cardiac ganglion neurons

Summary Driver potentials (DP), TTX-resistant voltage-activated slow depolarizations probably involving Ca⁺⁺-influx, have previously been shown to play an essential role in the organization of spike bursts in crustacean cardiac ganglia. The work reported here suggests that the DP system may also constitute an important component of the ganglionic oscillator.DPs were recorded intracellularly from neurons in ganglia isolated from two brachyurans,Portunus sanguinolentus and Podophthalmus vigil. InPortunus, full-sized DPs can be evoked in TTX by brief stimulus pulses only at invervals of several seconds; a single DP is triggered during a long (3-s) current pulse, and spontaneous DPs never occur. In contrast, nearly one half ofPodophthalmus preparations show spontaneous high-frequency trains of DPs lasting up to 5 min and recurring at irregular intervals. In allPodophthalmus preparations repetitive DPs are evoked during a 3-s pulse, and increase in frequency as current strength is increased. Portunus ganglia can be made to exhibit repetitive DPs in response to current if perfused with TEA (which suppresses a rectifying K⁺-current) in a medium with reduced Na⁺ (which is likely to enhance the inward Ca⁺⁺-current); neither TEA alone nor low-Na⁺ alone permits repetitive DPs. IfPortunus large cells are first conditioned at more negative voltages than normal, subsequent depolarizing current tends to induce large oscillations, and a second full DP may result in normal medium containing TTX.InPodophthalmus ganglia, single evoked DPs rise more rapidly and are shorter in duration than inPortunus; they are less effectively suppressed by Mn⁺⁺, but show similar Ca⁺⁺-dependency at lowered Ca⁺⁺ levels. Conditioning hyperpolarization slows the rate of rise of single evoked DPs and delays the onset of repetitive DPs during a 3-s pulse. A fast-K⁺ current appears to be more strongly activated inPodophthalmus.The results suggest (1) that oscillatory capability of the DP system may itself play an important role in the generation of rhythmic output by cardiac ganglia; and (2) differences in the V-dependence of Ca^{+ +}, delayed rectifying K⁺ and fast K⁺ currents may be responsible in diverse species for different tendency of the DP system to oscillate in TTX.Abbreviations DP driver potential - Rm membrane resistance - TEA tetraethylammonium - TTX tetrodotoxin - Vm membrane potential     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C

The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca⁺⁺ ([Ca⁺⁺] _bl ), and closing them by returning [Ca⁺⁺] _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca⁺⁺] _bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca⁺⁺ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca⁺⁺ being necessary to allow TJ recovery. A step rise of apical Ca⁺⁺ concentration ([Ca⁺⁺] _ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca⁺⁺ entering the open TJs. The specific condition of having Ca⁺⁺ only in the apical solution and the TJs located midway between the Ca⁺⁺ source (apical solution) and the Ca⁺⁺-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca⁺⁺] _ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca⁺⁺. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca⁺⁺ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca⁺⁺ removal but induces a prompt blockade of TJ oscillations induced by apical Ca⁺⁺, oscillations which reappear again when H7 is removed. Received: 9 May 2000/Revised: 30 August 2000     

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.     

Controlling the urge for a Ca2+ surge: all-or-none Ca2+ release in neurons

Changes in the intracellular calcium concentration ([Ca²⁺]_i) convey signals that are essential to the life and death of neurons. Ca²⁺-induced Ca²⁺-release (CICR), a process in which a modest elevation in [Ca²⁺]_i is amplified by a secondary release of Ca²⁺ from stores within the cell, plays a prominent role in shaping neuronal [Ca²⁺]_i signals. When CICR becomes regenerative, an explosive increase in [Ca²⁺]_i generates a Ca²⁺ wave that spreads throughout the cell. A discrete threshold controls activation of this all-or-none behavior and cellular context adjusts the threshold. Thus, the store acts as a switch that determines whether a given pattern of electrical activity will produce a local or global Ca²⁺ signal. This gatekeeper function seems to control some forms of Ca²⁺-triggered plasticity in neurons. BioEssays 21:743–750, 1999. © 1999 John Wiley & Sons, Inc.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation

Cardiac transverse (t)‐tubules are altered during disease and may be regulated by stretch‐sensitive molecules. The relationship between variations in the degree and duration of load and t‐tubule structure remains unknown, as well as its implications for local Ca²⁺‐induced Ca²⁺ release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart–lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t‐tubule structure were assessed using confocal‐microscopy, patch‐clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart‐only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time‐dependent manner. Ca²⁺ release synchronicity was reduced at 8 weeks moderate unloading only. Ca²⁺ sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t‐tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t‐tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca²⁺ transient, increased Ca²⁺ spark frequency or impaired t‐tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t‐tubule structure in a time‐ and degree‐dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

Effects of different ions on resting polarization and on the mass receptor potential of carotid body chemosensors

The carotid body and its own nerve were removed from cats anesthetized with sodium pentobarbital and placed in an air gap system; the carotid body was bathed in modified Locke's solution equilibrated with 50% O₂ in N₂, pH 7.43 at 35°C. The sensory discharges, changes in “resting” receptor polarization and the mass receptor potential evoked by ACh or NaCN were recorded with nonpolarizable electrodes placed across the gap. Receptor potentials and sensory discharges evoked by ACh showed an appreciable increase in amplitude and frequency when the preparation was bathed in eserinized Locke. Eserine did not change appreciably the responses evoked by NaCN. Excessive depolarization elicited by either ACh or NaCN was accompanied by sensory discharge block. Removal of K⁺ ions from the bathing solution induced receptor hyperpolarization and an increase in the amplitude of the evoked receptor potentials. An increase of K⁺ concentration had the opposite effect. Reduction of Na⁺ or NaCl to one half, or total removal of this salt, induced an initial reduction and later disappearance of the sensory discharges, some receptor hyperpolarization and a reduction in the amplitude of the evoked receptor potentials. Reduction or removal of Ca⁺⁺ produced receptor depolarization, a marked depression of the evoked receptor potentials, an increase in the frequency of the sensory discharges and a reduction in the amplitude of the nerve action potentials. High Ca⁺⁺ or Mg⁺⁺ had little or no effect on action potential amplitude or resting polarization, but decreased sensory discharge frequency and the evoked receptor potentials. Total or partial replacement of Ca⁺⁺ with Mg⁺⁺ induced complex effects: (1) receptor depolarization which occurred in low Ca⁺⁺, was prevented by addition of Mg⁺⁺ ions; (2) the amplitude of the evoked receptor potentials was depressed; (3) the nerve discharge frequency was reduced as it was in high Mg⁺⁺ solutions; and (4) the amplitude of the nerve action potentials was reduced as it was in low Ca⁺⁺ solutions. Temperature had a marked effect on the chemoreceptors since a t high temperatures the receptors were depolarized and the discharge frequency increased. The baseline discharge and responses evoked by ACh or NaCN were depressed at low temperatures. The results are discussed in terms of possible receptor mechanisms influenced by the different ions.     

Protein Kinase Inhibitors and the Dynamics of Tight Junction Opening and Closing in A6 Cell Monolayers

This study focuses, in A6 cell monolayers, on the role of protein kinases in the dynamics of tight junction (TJ) opening and closing. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺-switch assay (FCSA), which consisted of opening the TJs by removing basolateral Ca⁺⁺ (Ca⁺⁺ _bl), and closing them by returning Ca⁺⁺ _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the evaluation of the effects of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in response to the FCSA followed single-exponential time courses. A rise of apical Ca⁺⁺ (Ca⁺⁺ _ap) causes a reduction of TJ opening rate in an FCSA or even a partial recuperation of G, an effect that is interpreted as mediated by Ca⁺⁺ _ap entering the open TJs. Protein kinase C (PKC) inhibition by H7 at low concentrations caused a reduction of the rate of junction opening in response to Ca⁺⁺ _bl removal, without affecting junction closing, indicating that PKC in this preparation is a key element in the control of TJ opening dynamics. H7 at 100 μm completely inhibits TJ opening in response to Ca⁺⁺ _bl withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase, a process that can be halted again by H7 reintroduction into the bathing solution. Differently from the condition in which Ca⁺⁺ is absent from the apical solution, in which H7 halts the process of G increase in response to a FCSA, when Ca⁺⁺ is present in the apical solution, addition of H7 during G increase in an FCSA not only induces a halt of the G increase but causes a marked recuperation of the TJ seal, indicated by a drop of G, suggesting a cooperative effect of Ca⁺⁺ and H7 on the TJ sealing process. Staurosporine, another PKC inhibitor, differently from H7, slowed both G increase and G decrease in an FCSA. Even at high concentrations (400 nm) staurosporine did not completely block the effect of Ca⁺⁺ withdrawal. These discrepancies between H7 and staurosporine might result from distinct PKC isoforms participating in different steps of TJ dynamics, which might be differently affected by these inhibitors. Immunolocalizations of TJ proteins, carried out in conditions similar to the electrophysiological experiments, show a very nice correlation between ZO-1 and claudin-1 localizations and G alterations induced by Ca⁺⁺ removal from the basolateral solution, both in the absence and presence of H7. Received: 18 April 2001/Revised: 16 July 2001     

Mitogen-activated Ca++ channels in human B lymphocytes

Two complementary experimental methods have been used to examine mitogen-induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen-induced changes in [Ca⁺⁺]_i and transmembrane potential. Activation of human B cells with anti-μ antibodies resulted in a biphasic rise in [Ca⁺⁺]_i, the second phase being mediated by the influx of extracellular Ca⁺⁺. Ca⁺⁺ influx was inhibited by high [K⁺]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti-μ antibodies to the bath solution, inward currents were observed at negative voltages. Whole-cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than ?40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP₃ into the intracellular solution, suggesting that IP₃ generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti-μ and IP³ induced currents were blocked by 1 mM La⁺⁺⁺, which is known to block Ca⁺⁺ channels. These results strongly support the presence of membrane Ca⁺⁺ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca⁺⁺ influx. These mechanisms work in concert to regulate the level of [Ca⁺⁺]_i during the early phases of human B cell activation. © 1993 Wiley-Liss, Inc.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Sub-MHz bursts of nanosecond pulses excite neurons at paradoxically low electric field thresholds without membrane damage

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20–200 kHz, with up to 20–30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-μs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3–5 %. nsEP bursts were compared with single “long” pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2–3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-μs pulse decreased to 57 ± 8 V/cm for a 100-μs burst of 100-ns, 0.25-MHz pulses (p < 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.