Effect of HgCl2 on acetylcholine,carbachol, and glutamate currents ofAplysia neurons

Summary 1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl₂) on acetylcholine-, carbachol-, and glutamate-activated currents onAplysia neurons. Hg²⁺ was applied with microperfusion.2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl^– dependent and reversed at about –60 mV, while the slower component was K⁺ dependent and reversed at greater than –80 mV.3. Hg²⁺ (0.1–10 µM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl^– current in medial neurons. With only a 1-min preapplication of Hg²⁺, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 µM Hg²⁺.4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1–10 µM Hg²⁺ caused a complete and irreversible blockade of K⁺-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl₂ that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 µM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl₂ as well.5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg²⁺ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg²⁺ toxicity.     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Pb2+ reduces voltage- andN-methyl-d-aspartate (NMDA)-activated calcium channel currents

Summary 1. While intracellular calcium concentrations are closely regulated, two types of ion channels in neurons allow calcium influx: both voltage-activated and NMDA-activated channels are significantly permeable to calcium. In this study we compare the effects of lead (Pb²⁺) on currents carried through voltage-activated calcium channels and NMDA-activated channels.2. Pb²⁺ reduces voltage-activated calcium channel currents elicited by a voltage jump from –80 to 0 mV at 0.1 to 1 µM, with an IC₅₀ of 0.64 µM and a Hill slope of 1.22. This effect was partially reversible and not voltage dependent. Sodium and potassium currents were relatively unaffected at Pb²⁺ concentrations sufficient to block calcium channel currents by more than 80%. Pb²⁺ is, thus, a potent, reversible and selective blocker of voltage-dependent calcium channel currents.3. A fast reversible and slow irreversible blocking action of Pb²⁺ was found on NMDA-activated currents. When Pb²⁺ was applied simultaneously with aspartate and glycine (Asp/Gly), the inward currents were rapidly and reversibly reduced in a dose-dependent manner with a minimum effective concentration below 2 µM and a total blockade (>80%) with 100 µM Pb²⁺. The IC₅₀ was 45 µM and the Hill coefficient 1.1. Preincubation with 50 µM Pb²⁺ resulted in a greater reduction in the response to Asp/Gly/Pb²⁺. This effect was reversed within 2 to 5 sec of wash. The lack of voltage dependence suggests that Pb²⁺ does not block the channel but rather alters the binding of agonists. Prolonged superfusion of a cell with the Asp/Gly/Pb²⁺-containing external solution resulted in a slow and irreversible decrease in the Asp/Gly activated current. No clear threshold concentration is found for this slow and irreversible effect of Pb²⁺. This slow action might be more important for neurotoxic effects of Pb²⁺.     

Cadmium and copper interactions on the accumulation and distribution of Cd and Cu in birch (Betula pendula Roth) seedlings

The effect of different external cadmium (Cd) and copper (Cu) regimes on the concentration of Cd and Cu in roots and shoots of birch (Betula pendula Roth.) seedlings was investigated. The seedlings were grown for 12 days in a weak nutrient solution (containing all essential nutrient elements including 0.025 µM Cu) at pH 4.2 with combinations of additional 0–2 µM CdCl₂ and 0–2 µM CuCl₂. Root and shoot concentrations of Cu were decreased by Cd in all treatments which included 0.1–2 µM of additional Cu in the treatment solution. When no extra Cu was added, only the shoot concentration of Cu was decreased by Cd whereas the root concentration was not affected. The shoot concentration of Cd was decreased by 0.5 and 2 µM of additional Cu in the treatment solution. The root concentration of Cd was decreased by Cu only when the concentration of additional Cu in the treatment solution was equal to or exceeded the concentration of Cd.     

A culture unit system for the study of responses of mycorrhizal and non-mycorrhizal seedling to treatments

The time-dependence of Mn accumulation was confirmed in potato foliage (Solanum tuberosum. L.cv. Norland) grown in solution culture. Older leaves grown at 0.61 mM Mn had substantially higher Mn concentrations than younger leaves and stem samples. Levels of Mn in older leaves increased steadily from 4000 µg g^–1 at one week to 8–10,000 µg g^–1 at 6 weeks, but were relatively constant in the emerging leaves. Even foliage grown at low Mn levels (0.01 mM Mn) had 4 fold gradients in Mn concentration from younger (40 µg g^–1) to older leaves (180 µg g^–1).At 0.61 mM Mn, concentrations of 3–4000 µg g^–1 in the youngest fully-developed leaves did not bring about any decline in yield, and levels of up to 5000 µg g^–1 occurred in individual potato leaves before Mn toxicity symptoms were observed. Potato foliage grown at the high Mn had similar leaf numbers, but showed an increased stem length and smaller leaves than foliage grown at 0.01 mM Mn. In particular, the leaf area of the middle and lower leaf fractions were affected by the high Mn level.The ability of rapidly growing plants to withstand high concentrations of Mn is discussed in relation to the pattern of dry matter and Mn accumulation shown by potato foliage.     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Intracellular chloride activity of leech neurones and glial cells in physiological,low chloride saline

Leech blood apparently contains considerably less chloride than generally used in physiological experi ments. Instead of 85–130 mM Cl^– used in experimental salines, leech blood contains around 40 mM Cl^– and up to 45 mM organic anions, in particular malate. We have reinvestigated the distribution of Cl^– across the cell membrane of identified glial cells and neurones in the central nervous system of the leech Hirudo medicinalis L., using double-barrelled Cl^–- and pH-selective micro electrodes, in a conventional leech saline, and in a saline with a low Cl^– concentration (40 mM), containing 40 mM malate. The interference of anions other than Cl^–to the response of the ion-selective microelectrodes was estimated in Cl^–-free salines (Cl^– replaced by malate and/or gluconate). The results show that the absolute intracellu lar Cl^– activities (aCl_i) in glial cells and neurones, but not the electrochemical gradients of Cl^– across the glial and the neuronal cell membranes, are altered in the low Cl^–, malate-based saline. In Retzius neurones, aCl_i is lower than expected from electrochemical equilibrium, while in pressure neurones and in neuropil glial cells, aCl_i is distributed close to its equilibrium in both salines, re spectively. The steady-state intracellular pH values in the glial cells and Retzius neurones are little affected (0.1 pH units) in the low Cl^–, malate-based saline.     

Distinct Responses of Osphradial Neurons to Chemical Stimuli and Neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10^–2 M) and L-aspartate (10^–5 M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10^–6 M), -aminobutyric acid (GABA; 10^–6 M) and the GABA_B agonist baclofen (10^–6 M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10^–6 M) and FMRFamide (10^–6 M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10^–6 M) but picrotoxin (10^–5 M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10^–6 M) and 5HT (10^–6 M) but were inhibited by GABA (10^–6 M). FMRFamide (10^–6 M) had a biphasic response. The Ach effect was blocked by hexamethonium (10^–6 M) and tetraethylammonium (10^–6 M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.     

Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “Electrogenic” transport revealed by current-voltage and difference-current-voltage analysis

Summary The steady-state, current-voltage (I–V) characteristics of stomatal guard cells fromVicia faba L. were explored by voltage clamp using conventional electrophysiological techniques, but with double-barrelled microelectrodes containing 50mm K⁺-acetate. Attention was focused, primarily, on guard cell response to metabolic blockade. Exposures to 0.3–1.0mm NaCN and 0.4mm salicylhydroxamic acid (SHAM) lead consistently to depolarizing (positive-going) shifts in guard cell potentials (V _m ), as large as +103 mV, which were generally complete within 60–90 sec (mean response half-time, 10.3±1.7 sec); values forV _m in NaCN plus SHAM were close or positive to –100 mV and well removed from the K⁺ equilibrium potential. Guard cell ATP content, which was followed in parallel experiments, showed a mean half-time for decay of 10.8±1.9 ([ATP] _t=0, 1.32±0.28mm; [ATP] _{t=60–180sec}, 0.29±0.40mm). In respiring cells, theI–V relations were commonly sigmoid aboutV _m or gently concave to the voltage axis positive toV _m . Inward- and outward-rectifying currents were also observed, especially near the voltage extremes (nominally –350 and +50 mV). Short-circuit currents (atV=0 mV) were typically about 200–500 mA m^–2. The principal effect of cyanide early on was to linearize theI–V characteristic while shifting it to the right along the voltage axis, to decrease the membrane conductance, and to reduce the short-circuit current by approx. 50–75%. The resulting difference-current-voltage (dI–V) curves (±cyanide) showed a marked sensitivity to voltages negative from –100 mV and, when clamp scans had been extended sufficiently, they revealed a distinct minimum near –300 mV before rising at still more negative potentials. The difference currents, along with changes in guard cell potential, conductance and ATP content are interpreted in context of a primary, ATP-consuming ion pump. FittingdI–V curves to reaction kinetic model for the pump [Hansen, U.-P., et al. (1981)J. Membrane Biol. 63:165; Blatt, M.R. (1986)J. Membrane Biol. 92:91] implicates a stoichiometry of one (+) charge transported outward for each ATP hydrolyzed, with pump currents as high as 200 mA m^–2 at the free-running potential. The analysis indicates that the pump can comprise more than half of the total membrane conductance and argues against modulations of pump activity alone, as an effective means to controlling K⁺ transport for stomatal movements.     

A laboratory study of feeding and assimilation in Euchlanis dilatata lucksiana

Feeding of Euchlanis dilatata lucksiana, a brachionid rotifer isolated from the Loosdrecht Lakes (The Netherlands), was examined in the laboratory using ¹⁴C-labelled food. The gut-filling time at a food concentration of 9.6 µg C ml^–1 was about 15 minutes. Animals which were fed on 3 size fractions of the lake seston (< 7 µm, 7–15 µm, and 15–33 µm) showed a clear preference (Jacobs' selectivity index) for the largest size fraction. This fraction was composed predominantly of filamentous cyanobacteria. For animals weighing 0.37–0.49 µm C ind.^–1 the daily ration (daily food consumption per unit body weight × 100) ranged from 50 to 100% at food levels of 2 mg C l^–1 and below, but increased to 150–250% at food concentrations of 5 mg C l^–1 and above. The assimilation efficiency was 100% up to 5 mg C l^–1 of food, but decreased to about 80% at higher food levels.     

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellateNoctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellateNoctiluca miliaris were examined under voltage-clamp conditions.Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of –80 mV to a potential level more positive than –50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na⁺ concentration was lowered. The transient inward current is assumed to be responsible for the Na⁺-dependent positive spike in non-clamped specimens ofNoctiluca.Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of –20 mV to a potential level more negative than –30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl^– concentration was lowered. The transient outward current is responsible for the Cl^–-dependent negative spike in non-clamped specimens ofNoctiluca.Abbreviations ASW artificial seawater - TRP tentacle regulating potentials - TTX tetrodotoxin     

Dynamics of chloride and potassium currents during the action potential in Chara studied with action potential clamp

TheCl^– and K⁺ currents underlying the action potential (AP) in the giant alga Chara were directly recorded with the action potential clamp method. An electrically triggered action potential was recorded and repetitively replayed as command voltage to the same cell under voltage clamp. The resulting clamp current was close to zero. Only the initial rectangular current used for stimulation was approximately reproduced by the clamp circuit. Inhibition of Cl^– channels with niflumic acid or ethacrynic acid and of K⁺ channels with Ba²⁺ evoked characteristic compensation currents because the amplifier had to add the selectively inhibited currents. Integration of the compensation currents revealed a mean flux through Cl^– and K⁺ channels of 3.3 10^–6 and 2.1 10^–6 mole M^–2 AP^–1 respectively. The dynamics of CI^– and K⁺ channel activation/inactivation were obtained by converting the relevant clamp currents to ionic permeabilities using the Goldman-Hodgkin-Katz current equation. During the AP the Cl^– permeability reaches a peak 370 ms, on average, after termination of the stimulating pulse. The following inactivation proceeds 3.6 times slower than the activation. The increase in K⁺ permeability lags behind the rise in Cl^– permeability, reaching a peak approximately 2 s after the latter.     

Responses of cortical neurons to local application of excitatory amino acids to their dendrites and soma

The efficacy of excitation induced by iontophoretic application of excitatory amino acids to the soma or different parts of the dendritic tree has been compared in experiments performed on parietal cortex slices. Spike activity was recorded extracellularly from single nerve cells of layer V. In total, the responses of 125 neurons were analyzed. Upon application of glutamate and aspartate to the neuronal soma and the majority of dendrites, latencies of excitatory responses did not exceed 500 msec. In 18% of cases, neuronal responses to transmitter application to basal and apical dendrites had longer (2–3 sec) latencies. The maximum intensity of responses was observed when excitatory amino acids had been applied to the soma or proximal parts of dendrites. If applied at a distance of over 100 µm to basal and 300 µm to apical dendrites, glutamate and aspartate elicited cellular responses whose intensity was 2–3 times lower than that of the responses induced by application to the soma. The maximum distances at which somatic spike responses could be recorded were 350 µm and 800 µm for basal and apical dendrites, respectively. Different latencies of the responses to somatic and dendritic applications of excitatory amino acids in some neurons, as well as high efficacy of responses to stimulation of remote parts of dendritic tree, may indicate nonidentity of electrical properties of dendritic and somatic membranes.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 437–446, November–December, 1993.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Effects of Nitrendipine and Nimodipine on Low-Threshold Ca<Superscript>2+</Superscript> Channels in Thalamic Neurons of the Rat

We compared the effects of two dihydropyridines extensively used in clinical medicine, nimodipine and nitrendipine, on Ca²⁺ channels of two subtypes providing the fast and slow components of low-threshold Ca²⁺ currents in isolated neurons of the nucl. lateralis dorsalis of the thalamus of the rat; a patch-clamp technique in the whole-cell configuration was used. The fast component of the Ca²⁺ current demonstrated a higher sensitivity to nimodipine and nitrendipine (IC₅₀ = 0.6 and 0.1 µM, respectively) than the slow component (IC₅₀ = =1.09 and 0.18 µM, respectively). Maximum decreases in the current amplitude, A _max, caused by nimodipine and nitrendipine were 74 and 94% for the fast component and 55 and 80% for the slow component, respectively. Both tested agents evoked a shift of the inactivation curves of the fast component toward more negative potentials, whereas they did not significantly modify the inactivation characteristics of the slow component. Both dihydropyridines weakly influenced the characteristics of stationary activation of low-threshold Ca²⁺ channels and the blocking intensity demonstrated a voltage dependence.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 3–10, January–February, 2005.     

Physiological background for using freshwater mussels in monitoring copper and lead pollution

In studying the effect of copper (10 ± 0.57 µg Cu l^–1 and 100 ± 3.01 µg Cu l^–1) and lead (50 ± 1.12 µg Pb l^–1 and 500 ± 12.5 µg Pb l^–1) on the filtration activity of Anodonta cygnea L. it was found that both heavy metals resulted in significant shortening of the active periods, but little change occurred in the length of the rest periods. The concentrations of copper and lead were measured in the gill, foot, mantle, adductor muscle and kidney for 840 hours of exposure to 10.9 ± 5 µg Cu l^–1 and 57.0 ± 19 µg Pb l^–1 as well as during subsequent depuration. Uptake was observed after 72 hours of exposure. The highest copper concentration (59.1 ± 16.2 µg Cu g^–1) was measured at 672 h in the mantle, and the highest lead value (143 ± 26.1 µg Pb^–1) was obtained in the kidney. Depuration of copper was fastest from the foot, and from the adductor muscle for lead. The gill had the longest half-depuration time (> 840 h for copper and > 672 h for lead).     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Rotifers as predators on small ciliates

Clearance rates of Synchaeta pectinata, Brachionus calyciflorus and Asplanchna girodi on Tetrahymena pyriformis (46 µm in length) at a density of 10 cells ml^–1, in the presence of algal food, were 2.5 to 6.1 ml rot.^–1 day^–1. Clearance rates of these rotifers were, respectively, about 2, 3, and 13 times lower on Strobilidium gyrans (58 µm in length) than on T. pyriformis, indicating that the saltations of S. gyrans are an effective escape response. Clearance rates of S. pectinata were considerably lower on Colpidium striatum (81 µm) than on S. gyrans, suggesting that S. pectinata may not be able to ingest ciliates of this size. S. pectinata had a clearance rate of 19 ml rot.^–1 day^–1 on S. gyrans at a density of 1.2 cells ml^–1, in the absence of edible algal food. Rotifers may prey extensively on ciliates in natural plankton communities, ingesting 25 to 50 individuals in the 45–60 µm size range day^–1.