Analysis of extracellular calcium and volume changes in the compound eye of the honeybee drone,Apis mellifera

Summary Superfused slices of drone retina were used for a quantitative analysis of light-induced changes in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and extracellular space (ECS) volume. 20-ms light flashes elicited biphasic changes in [Ca²⁺]_o. For a saturating flash a brief, initial decrease was followed by a transient increase of 120±34 M. Long, dim steps of light (5 min) produced either a decrease or an increase in [Ca²⁺]_o depending strongly on the previous illumination. Brighter continuous lights caused the [Ca²⁺]_o to increase transiently by 1.4 mM to a peak from which it decayed to a plateau, up to 0.6 mM above the dark concentration.Light flashes (20 ms) caused a shrinkage in ECS volume not exceeding 4%. Thus, changes in [Ca²⁺]_o were almost completely due to Ca²⁺ fluxes between the ECS and adjacent cells. Continuous lights caused a shrinkage in ECS volume rarely exceeding 16%–20%. Thus, less than 15% of the measured Ca²⁺ changes could be attributed to shrinkage of the ECS. These data confirm that the ECS functions as a source and a sink for Ca²⁺ mobilized by light. For comparison, we also made a few measurements of changes in [Ca²⁺]_o in the retina ofCalliphora.Abbreviations [Ca ²⁺]_i intracellular free Ca²⁺ concentration - [Ca ²⁺]_o extracellular free Ca²⁺ concentration - ECS extracellular space - ER endoplasmic reticulum - TMA ⁺ tetramethylammonium ion     

Light-induced changes in extracellular calcium concentration in the compound eye of Calliphora,Locusta and Apis

Summary Ion-selective microelectrodes inserted into the compound eyes of Calliphora, Locusta and Apis were used to monitor the changes in extracellular concentration of Ca²⁺ (Ca_o) brought about by a 1-min exposure to white light (maximal luminous intensity ca. 10³ cd/m²).In the blowfly retina such stimulation causes a decrease in Ca_o. At high light intensities the Ca_o signal is phasic, falling over about 6 s to a transient light-induced minimum (Ca_o= -6.2% ± 0.4%, n = 20, SE) and then rising to an approximately stable plateau (-3.3% ± 0.6%). In migratory locusts the light-induced minimum corresponds to a Ca_o of -13.8% ± 1.6% (n = 10), and at the plateau the Ca_o decrease is-13.2% ± 1.5%. In honey-bees Ca_o at first decreases only slightly, by -2.6% ± 1.0% (n = 10); by the end of the 1-min stimulus the extracellular concentration averages 33.6% ± 14.6% above the dark level.The results suggest a relationship between the position of the characteristic curve of the photoreceptor in the dark-adapted state, the occurrence of quantum bumps, and light-induced increases or decreases in Ca_o. Therefore the species differences might be interpreted as a consequence of differences in the intracellular dark concentration of Ca²⁺.Abbreviations Ca_i intracellular Ca²⁺ concentration - Ca_o extracellular Ca²⁺ concentration     

Light-induced extracellular calcium and sodium concentration changes in the retina of Calliphora: involvement in the mechanism of light adaptation

Summary Ion-selective microelectrodes inserted into the compound eyes of Calliphora were used to monitor the changes in extracellular concentration of Ca²⁺ and Na⁺ (Ca_o, Na^o) brought about by a 1-min exposure to white light (maximal luminous intensity 0.1 cd/cm²).Using Ringer solution as the reference (Ca²⁺ = 1 mM), the dark concentration of the calcium in the retina was found to be (1.4 ± 0.4) mM (n=12). Stimulation with light reduces Ca_o. At intensities near maximal the Ca_o signal is phasic, reaching a transient minimum about 6 s after light onset and then rising to a nearly stable plateau below the dark level (-3.3% ± 2.6%). Ca_o signals measured in the white-eyed mutant (chalky), which lacks pigment granules, are comparable to those in the wild type.Conclusions: (a) There are no extracellular Ca²⁺ binding sites that regulate light adaptation, such as were postulated by Hochstrate and Hamdorf (1985). (b) Ca²⁺ influx into the photoreceptors seems to be necessary for light adaptation, (c) The pigment granules have no major function in intracellular calcium regulation.The time course of the Na_o signals resembles that of the Ca_o signals. Because the percentage concentration change is small, light-induced extracellular Na⁺-depletion cannot contribute to a reduced response amplitude at light adaptation.Abbreviations Ca _i intracellular Ca²⁺ concentration - Ca _o extracellular Ca²⁺ concentration - Kino extracellular K⁺ concentration - Na _o extracellular Na⁺ concentration     

Characterization of calcium fluxes across the envelope of intact spinach chloroplasts

Calcium fluxes across the envelope of intact spinach chloroplasts (Spinacia oleracea L.) in the light and in the dark were investigated using the metallochromic indicator arsenazo III. Light induces Ca²⁺ influx into chloroplasts. The action spectrum of light-induced Ca²⁺ influx and the inhibitory effect of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) indicate an involement of photosynthetic electron transport in this process. The driving force for light-induced Ca²⁺ influx is most likely a change in the membrane potential component of the proton motive force. This was demonstrated by the use of agents modifying the membrane potential (lipophilic cations, ionophores, different KCl concentrations). The activation energy of the observed Ca²⁺ influx is about 92 kJ mol^-1. Verapamil and nifedipine, two Ca²⁺-channel blockers, have no inhibitory effect on light-induced Ca²⁺ influx, but enhance ferricyanide-dependent oxygen evolution. Inhibition of Ca²⁺ influx by ruthenium red reduces the light-dependent decrease in stromal NAD⁺ level.Abbreviations and symbols Chl chlorophyll - DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea - FCCP earbonyl cyanide p-trifluoromethoxyphenylhydrazone - PGA 3-phosphoglyceric acid - ABA⁺ tetrabutylammonium chloride - TPP⁺ tetraphenylphosphonium chloride - E membrane potential     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

External calcium requirements for light induction of chlorophyll accumulation and its enhancement by red light and cytokinin pretreatments in excised etiolated cucumber cotyledons

Excised etiolated cucumber (Cucumis sativus L.) cotyledons that were depleted of external Ca²⁺ by equilibration with a Ca²⁺ buffer, which maintained the free Ca²⁺ concentration at 10^–8 M, failed to accumulate chlorophyll upon a 2-h exposure to white light. Increasing amounts of chlorophyll accumulation occurred at increasing external Ca²⁺ concentrations within the range of 10^–7-10^–3 M. Preillumination with red light or pretreatment with benzyladenine, which enhanced the rate of light-induced chlorophyll accumulation in control cotyledons, did not overcome the block to light-induced chlorophyll accumulation caused by the depletion of external Ca²⁺. Etiolated cotyledons that were treated with the Ca²⁺ ionophore, A23187, and then equilibrated with 10^–5 M Ca²⁺, accumulated significantly more chlorophyll during exposure to light than did untreated cotyledons. The enhancing effect of A23187 was approximately equal to that caused by red-light pretreatment. Etiolated cotyledons that were exposed to the Ca²⁺ channel-blocking agent, Nd³⁺ (neodymium), in the presence of 10^–5 M Ca²⁺, did not exhibit an enhancement of chlorophyll accumulation by red-light pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by cytokinin pretreatment. Conversely, etiolated cotyledons that were equilibrated with 10^–5 M Ca²⁺ in the presence of nifedipine, a blocker of some Ca²⁺ channels, did not exhibit an enhancement of chlorophyll accumulation by cytokinin pretreatment, although they accumulated control levels of chlorophyll upon exposure to light and showed control levels of enhancement of chlorophyll accumulation by red-light pretreatment. These results indicate that external Ca²⁺ is required for chlorophyll accumulation by excised etiolated cucumber cotyledons during the first 2 h of light exposure, and that an influx of external Ca²⁺ is required for the enhancing effect of redlight and cytokinin. The differential abilities of Nd³⁺ and nifedipine to block the effects of red-light and cytokinin pretreatments suggests that enhancement of chlorophyll accumulation by red-light and cytokinin may involve different classes of Ca²⁺ channels.Abbreviations A23187 antibiotic 23187 calcium ionophore - Chl chlorophyll - nifedipine 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester We thank Randy Wayne for advice and encouragement.     

Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity

Single Ca²⁺ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl₂ gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca²⁺ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca²⁺ channel antagonist). Extracellular AlCl₃ (70 M, pH 4.9) almost completely blocked the channel.     

Calcium- and inositol polyphosphate-sensitivity of the calcium-sequestering endoplasmic reticulum in the photoreceptor cells of the honeybee drone

Summary The photoreceptor cells in the honeybee drone contain an elaborate Ca²⁺-sequestering endoplasmic reticulum (ER). We measured Ca-oxalate formation within the ER of permeabilized retinal slices with a microphotometer and studied the kinetics of Ca²⁺-uptake into the ER and the properties of Ins(1,4,5)P₃-induced Ca²⁺-release.The ATP-dependent Ca²⁺-uptake mechanism has a high affinity for Ca²⁺: Uptake rate was half maximal at Ca²⁺ _free 0.6 M.Addition of Ins(1,4,5)P₃ caused a persistent depression of Ca-oxalate formation due to Ca²⁺ -release from the ER. The Ins(1,4,5)P₃-dependent Ca²⁺-release mechanism has a high affinity (half maximal rate with 0.2 M Ins(1,4,5)P₃) and a high specificity for Ins(1,4,5)P₃: Ins(2,4,5)P₃ was 6 times, Ins(1,3,4,5)P₄ was 15 times less potent in inducing Ca²⁺-release. 3 M Ins(1,4)P₂ had no detectable effect. The sensitivity for Ins(1,4,5)P₃ was maximal between 280 nM and 1.6 M Ca²⁺ _free and decreased at higher and lower Ca²⁺-concentrations.Our data show that the ER in invertebrate photoreceptor cells is an effective Ca²⁺ -sink and an Ins(1,4,5)P₃-sensitive Ca²⁺-source. We support the idea (Payne et al. 1988) that the ER-network close to the photoreceptive membrane, the submicrovillar cisternae (SMC), are the light- and Ins(1,4,5)P₃-sensitive Ca²⁺-stores.Abbreviations ER endoplasmic reticulum - Ins(1,4,5)P ₃ D-inositol 1,4,5-trisphosphate - Ins(1,3,4)P ₃ D-inositol 1,3,4-trisphosphate - Ins(2,4,5)P ₃ D-inositol 2,4,5-trisphosphate - Ins(1,4)P ₂ D-inositol 1,4-bisphosphate - Ins(1,3,4,5)P ₄ D-inositol 1,3,4,5-tetrakisphosphate - SMC submicrovillar cisternae - [Ca ²⁺]_i intracellular free Ca²⁺-concentration     

Effects of intracellular Ca2+ chelation on the light response in Drosophila photoreceptors

In order to test the hypothesis that excitation in Drosophila photoreceptors is mediated by Ca²⁺ released from internal stores, the Ca²⁺ buffers EGTA, BAPTA and di-bromo-BAPTA (DBB) were introduced into dissociated photoreceptors via whole-cell recording pipettes. All buffers were preloaded with Ca²⁺ to provide the same free Ca²⁺ concentration (250 nM). EGTA (up to 18 mM free buffer) had only weak effects upon voltage-clamped flash responses in normal Ringer's solution (1.5 mM Ca ₀ ²⁺ ), and no effect in Ca²⁺-free solution. The maximum BAPTA concentration tested (14.4 mM free BAPTA) reduced the initial rate of rise by ca. 5000-fold in normal Ringer's solution; by ca. 500-fold in Ca²⁺free solution; and only ca. 60-fold in the absence of Mg²⁺, which preferentially blocks one component of the light-sensitive current. Although BAPTA delayed the time-to-peak in normal Ringer's solution, responses in Ca²⁺ free Ringer's solution were accelerated. These results support the role of Ca²⁺ influx in regulating sensitivity and response kinetics; however, in view of the high concentrations required to attenuate responses in Ca²⁺ free Ringer's solution, the role of Ca²⁺ release in excitation remains unclear. DBB was ca. 2–3 fold more potent than BAPTA, and at concentrations > 5 mM had a qualitatively different action, greatly delaying the time-to-peak. This suggests DBB may have distinct pharmacological actions or access to compartments inaccessible to BAPTA.The only current activated by introducing 5–500 M Ca²⁺ (buffered with nitrilo-triacetic acid) was electrogenic Na⁺/Ca²⁺ exchange. When this was blocked by removing Nao ₀ ⁺ , a novel cationic conductance was activated. However, its properties did not resemble those the light-activated conductance, and thus do not support the hypothesis that Ca²⁺ is sufficient for excitation.Abbreviations BAPTA bis-(o-aminophenoxy)-ethane-N,N,N-tetracetic acid - DBB Di-bromo-bapta - NTA nitrilo-triacetic acid - InsP ₃ inositol 1,4,5-trisphosphate     

Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone

We have measured Ca_i at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca²⁺ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Ca_i was 90 nM after 5 min of dark adaptation. A saturating light step caused Ca_i to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Ca_i which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Ca_i From our Fluo-3 measurements it appears that the latency of the Ca²⁺ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca²⁺ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER endoplasmic reticulum - IP₃ Inositol 1,4,5-trisphosphate - SMC submicrovillar cisternae     

Phytochrome-mediated uptake of calcium in Mougeotia cells

Ca²⁺ is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca²⁺ in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of ⁴⁵Ca²⁺ and low temperature autoradiography: 10–20 cells within 40–60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2–10 times more Ca²⁺ than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca²⁺. These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca²⁺ and secondly, that the accumulation of radioactive Ca²⁺ in red light irradiated cells is an expression of an increased intracellular concentration of Ca²⁺. This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca²⁺ persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca²⁺ in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca²⁺-extrusion.Abbreviations APW artificial pond water - EGTA ethylene glycol-bis-(-amino ethyle ether) N,N-tetraacetic acid - R red irradiation - D darkness - FR far-red irradiation - Pfr physiologicallyactive form of phytochrome - Pr physiologically inactive form of phytochrome This paper is part of a Ph. D. Thesis submitted to the University of Erlangen-Nürnberg by E.M. Dreyer     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Sensitivity changes of photoreceptor cells of Hirudo medicinalis caused by changes in extracellular calcium concentration

Extracellular recordings from the vacoule of photoreceptor cells of Hirudo medicinalis L. were performed using microelectrodes. The cells were adapted by white light flashes given at constant intervals (20 s). Response height versus relative intensity curves obtained from the same cell in physiological saline (PS) and in bathing solutions of either a) lowered calcium contents (2 ΜM/1 or less) or b) raised calcium contents (15 mM/1) were compared. The cells' adaptation state in PS was operationally defined by the ratio Q=h _A /h _S where h _A is the response height evoked by the adapting flashes, and h _S is the corresponding saturation response height. Sensitivity changes were measured by the half saturation intensity shift. Lowering extracellular calcium resulted in:

1. The response height increased and the shape of the response became more rounded and prolonged.
2. The total resistance between the vacuole and outside decreased from 8.2±1.4 MΩ (n=6) in PS to 4.6±0.4 MΩ (n=5). The resistance was independent of the cells' adaptation state.
3. A change of the cells' sensitivity occured either in direction to light adaptation or in direction to dark adaptation. It depended functionally on the ratio Q:

a) if Q was less or equal to about 0.6 the cells' sensitivity increased. b) if Q was greater than 0.6 the cells' sensitivity diminished. Raising extracellular calcium decreased the sensitivity of all cells tested independent of their adaptation states in PS. The results can be interpreted under the assumptions that 1. the sensitivity of leech photoreceptor cells is inversely proportional to the intracellular free calcium concentration and Z. intracellular calcium can interact with extracellular calcium in relatively dark adapted cells whereas in relatively light adapted cells the raise of intracellular free calcium is mainly effected by a release from intracellular stores. It is assumed that a Q value of about 0.6 separates relatively light adapted cells from relatively dark adapted cells.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Photolysis of caged Ca2+ induces trichocyst discharge inParamecium caudatum

Summary Trichocyst discharge, ciliary reversal, and cell body contraction inParamecium spp. have all been claimed to be regulated by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the cortical region of the cell. We injected caged Ca²⁺ intoP. caudatum cells and applied ultraviolet (UV) light to the cell for 125 ms. This did not induce trichocyst discharge but did induce both ciliary reversal and cell body contraction. A re-application of UV for 125 ms triggered trichocyst discharge. These results demonstrate that (1) trichocyst discharge and ciliary reversal and cell body contraction are controlled by [Ca²⁺]_i and (2) the threshold of [Ca²⁺]i for trichocyst discharge is higher than those for the other two functions.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ICL infraciliary lattice - [Ca²⁺]_i intracellular Ca²⁺ concentration - NP-EG o-nitrophenyl EGTA - PMT photomultiplier tube - UV ultraviolet     

Lead Reduces Depolarization-Induced Calcium Entry in Cultured DRG Neurons Without Crossing the Cell Membrane: Fura-2 Measurements

1. Cultured dorsal root ganglion neurons of rat pups were depolarized by exposure to 50 mM K⁺ and the rise of [Ca²⁺]_i was measured using fura-2 as an indicator.2. Lead in the extracellular solution reduced the rise of [Ca²⁺]_i in a concentration-dependent manner, with a threshold concentration of 0.25 M. More than 80% of the calcium entry was prevented by 5 M lead. The IC₅₀ and the Hill coefficient were 1.3 M and 1, respectively.3. This effect was considered to be due to a reduction of VACCCs, since applications of NMDA did not result in any rise of [Ca²⁺]_i.4. Since Pb²⁺ itself changes the fura-2 signal in a typical and characteristic manner, fura-2 is also an indicator for Pb²⁺. No changes in fura-2 signals were detected when lead (5 M) was applied for several minutes in the absence of calcium, indicating that Pb²⁺ did not enter the cells.5. Thus it is concluded that lead prevents calcium entry by reducing VACCCs but does not cross the cell membrane itself.     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

Binding sites for horseradish peroxidase on the cell surface

Summary The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH₃ cells, and isolated rat liver cells was suppressed by 30 M monosialoganglioside, by 30 M trisialoganglioside, or by 5 mM CMP-neurminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyltransferase. The addition of 2 mM Mn²⁺ to the incubation medium with HRP suppressed the reaction for surfacebound HRP, and the addition of 10–20 mM Ca²⁺ intensified the reaction. The addition of 2 mM Zn²⁺ caused less inhibition than that of 2 mM Mn²⁺, and the addition of 2 mM Co²⁺ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.