Pharmacological characterization of ionic currents that regulate the pacemaker rhythm in a weakly electric fish

Electric organ discharge (EOD) frequency in the brown ghost knifefish (Apteronotus leptorhynchus) is sexually dimorphic, steroid-regulated, and determined by the discharge rates of neurons in the medullary pacemaker nucleus (Pn). We pharmacologically characterized ionic currents that regulate the firing frequency of Pn neurons to determine which currents contribute to spontaneous oscillations of these neurons and to identify putative targets of steroid action in regulating sexually dimorphic EOD frequency. Tetrodotoxin (TTX) initially reduced spike frequency, and then reduced spike amplitude and stopped pacemaker activity. The sodium channel blocker muO-conotoxin MrVIA also reduced spike frequency, but did not affect spike amplitude or production. Two potassium channel blockers, 4-aminopyridine (4AP) and kappaA-conotoxin SIVA, increased pacemaker firing rates by approximately 20% and then stopped pacemaker firing. Other potassium channel blockers (tetraethylammonium, cesium, alpha-dendrotoxin, and agitoxin-2) did not affect the pacemaker rhythm. The nonspecific calcium channel blockers nickel and cadmium reduced pacemaker firing rates by approximately 15-20%. Specific blockers of L-, N-, P-, and Q-type calcium currents, however, were ineffective. These results indicate that at least three ionic currents-a TTX- and muO-conotoxin MrVIA-sensitive sodium current; a 4AP- and kappaA-conotoxin SIVA-sensitive potassium current; and a T- or R-type calcium current-contribute to the pacemaker rhythm. The pharmacological profiles of these currents are similar to those of currents that are known to regulate firing rates in other spontaneously oscillating neural circuits.     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Electrophysiological and pharmacological evidence that peripheral type benzodiazepine receptors are coupled to calcium channels in the heart

PK 11195, an antagonist of the peripheral type benzodiazepine receptor, does not affect either the duration of the action potential or the tension of the guinea pig papillary muscle. However, it antagonized the effects of the calcium channel blockers, nitrendipine, verapamil, diltiazem, and of BAY K8644, a calcium channel agonist in this heart preparation. On the other hand, PK 11195 does not change the increase in the action potential duration provoked by the potassium channel blocker tetraethylammonium. RO5-4864, an agonist of the peripheral type benzodiazepine receptor, decreased the tension of the guinea pig papillary muscle. The effect was reversed by increasing extracellular Ca2+ concentrations up to 4 mM. These results suggest that in the heart the peripheral type benzodiazepine receptors are coupled to calcium channels.     

Effect of ion channel blockers and H<Superscript>+</Superscript>-ATPase inhibitors on generation of local electrical response in a cucumber leaf

Electric potential difference was measured with extracellular electrodes between the leaf surface of 2-week-old cucumber (Cucumis sativus L.) plants and soil solution. When the leaf region with a diameter of 5 mm was gradually cooled during a 105-s period to 8–9°C, the temperature drop induced a local (confined to the cooled area) nonpropagating pulse-wise electric activity. The cessation of cooling was followed by gradual (within 12–15 min) restoration of the initial potential difference. Two peaks of electric potential with amplitudes of 100–120 mV usually appeared upon cooling. The first depolarizing stage of the pulse activity was sensitive to inhibition of voltage-gated and mechanosensitive calcium channels of plasmalemma by lanthanum and gadolinium chlorides and to verapamil treatment. Furthermore, the inhibition of this stage by ruthenium red implies the release of calcium ions from intracellular stores. The initial slow depolarization was followed by a fast depolarizing shift, which was sensitive to La³⁺ and the anion channel inhibitor 4-acetamido-4′-isothiocyano-stilbene-2,2′-dilsulfonic acid. At the next stage repolarization developed, which was sensitive to potassium channel blockers, tetraethylammonium and quinine sulfate. The influence of ion channels blockers indicates that generation of local bioelectric response is based on fluxes of the same ion species that are involved in the action potential. The depolarization stage was due to the transient Ca²⁺ influx into the cytosol from the apoplast and intracellular stores, together with the anion efflux from the cell; the repolarization stage involved potassium ions. Both stages of electric pulse generation were retarded by the H⁺-ATPase inhibitors, sodium orthovanadate and dicyclohexylcarbodiimide, which implies the involvement of the proton pump in the origin of electric pulses examined.     

T-type calcium channels and tumor proliferation

The role of T-type Ca2+ channels in proliferation of tumor cells is reviewed. Intracellular Ca2+ is important in controlling proliferation as evidenced by pulses, or oscillations, of intracellular Ca2+ which occur in a cell cycle-dependent manner in many tumor cells. Voltage-gated calcium channels, such as the T-type Ca2+ channel, are well suited to participate in such oscillations due to their unique activation/inactivation properties. Expression of the T-type Ca2+ channels has been reported in numerous types of tumors, and has been shown to be cell cycle-dependent. Overexpression of the alpha1 subunit of T-type Ca2+ channels in human astrocytoma, neuroblastoma and renal tumor cell lines enhanced proliferation of these cells. In contrast, targeting of the alpha1 subunit of the T-type calcium channel via siRNA decreased proliferation of these cells. A Ca2+ oscillatory model is proposed involving potassium channels, Ca2+ stores and Ca2+ exchangers/transporters. A review of T-type channel blockers is presented, with a focus on mibefradil-induced inhibition of proliferation. The development of newer blockers with higher selectivity and less potential side effects are discussed. The conclusion reached is that calcium channel blockers serve as a potential therapeutic approach for tumors whose proliferation depends on T-type calcium channel expression.     

Electrical activity at motor nerve terminals of the mouse

1. Extracellular recording of the electrical activity of mammalian end-plate made it possible to distinguish three different parts on the presynaptic terminal. Bath or ionophoretic application of ionic channel blockers induced specific alterations of electric signals which revealed the localization of classical ionic channels. 2. Sodium channels are restricted to a small area, sharply located after the end of the last myelinated segment and potassium channels are distributed over the rest of the terminal branches. 3. The first direct evidence of the presence of calcium channels at the terminal part of the motor endings was obtained, when potassium channel activity was suppressed. They occupy the same region as potassium channels. 4. Finally, the differential distribution of ionic channels over the terminal and the time-course of calcium current are discussed in relation to transmitter release.     

Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50-100 microM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [35S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [125I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca2+ levels modulate LDL receptor metabolism in human skin fibroblasts.     

The effects of freezing on membrane electric potential in winter oilseed rape leaves

Extracellular ice formation in winter oilseed rape leaf discs (Brassica napus L. var. oleifera L. cv. Jantar) at different temperatures resulted in a transient membrane depolarization, which was followed by a decrease in membrane electric potential. In discs which underwent supercooling (no extracellular ice was formed), no membrane depolarization was observed. The inhibitors of calcium ion channels, gadolinium and lanthanum, decreased to some extend the amplitude of the frost-induced (−6 °C) depolarization and completely eliminated the decrease in membrane potential. Changes in membrane potential were associated with the increased electrolyte efflux, measured after thawing of the discs. No efflux from supercooled discs was observed. Application of calcium channel blockers decreased the level of the efflux induced by freezing at −6°C. It is suggested that membrane depolarization is one of the primary events induced by ice formation at a leaf surface. The possible reasons for changes in the membrane electric potential and their physiological consequences are discussed.     

Lipid factor (bVLF) from bovine vitreous body evokes in EGFR-T17 cells a Ca2+-dependent K+ current associated with inositol 1,4,5-trisphosphate-independent Ca2+ mobilization

Bovine vitreous lipid factor (bVLF) is a complex phospholipid isolated from bovine vitreous body with strong Ca(2+)-mobilizing activity. In this study, the effects of bVLF on membrane potential were investigated in EGFR-T17 fibroblasts with the whole-cell patch clamp technique on monolayer cells, as well as with the fluorescent dye bis-oxonol as membrane potential-sensitive probe on monolayer and suspension cells. bVLF induced a transient hyperpolarization characterized by an initial peak and subsequent return to resting membrane potential levels within 1-2 min. The increase of [Ca(2+)](i) was concomitant with an outward current responsible for the hyperpolarizing response. Results with: (a) high [K(+)](o) media; (b) the monovalent cation ionophore gramicidin; and (c) substitution of K(+) with Cs(+) in the intracellular solution were consistent with the involvement of K(+) channels. The bVLF-induced hyperpolarization was blocked by the K(+) channel blockers, quinine and tetraethylamonium chloride, and partially affected by 4-aminopyridine. The calcium ionophore ionomycin caused a similar hyperpolarization as bVLF. When intracellular calcium was buffered by adding BAPTA to the pipette solution, bVLF-activated outward current was prevented. Moreover, the hyperpolarization response was strongly reduced at low doses (3 nM) of specific Ca(2+)-activated K(+) channel blockers, charybdotoxin and iberiotoxin. Based on these observations we conclude that bVLF hyperpolarizes the cells via the activation of a Ca(2+)-dependent K(+) current. In addition, it was observed that bVLF did not have a significant effect on intercellular communication measured by a single patch-electrode technique. Thus, membrane potential changes appeared to belong to the earliest cellular responses triggered by bVLF, and are closely associated with phosphatidic acid-dependent [Ca(2+)](i) mobilization.     

Ionic currents that contribute to a sexually dimorphic communication signal in weakly electric fish

Weakly electric fish produce a communication signal, the electric organ discharge, that is species specific, and in many species, sexually dimorphic. Because the neural circuit that controls the electric organ discharge is relatively simple, it is an excellent model in which to study both the biophysical mechanisms underlying a rhythmic behavior and the neuroendocrine control of a sexually dimorphic behavior. By studying the effects of ion channel blockers on neurons in the medullary pacemaker nucleus, I pharmacologically characterized three ionic currents that influence the pacemaker rhythm, and thus electric organ discharge frequency, in the gymnotiform fish, Apteronotus leptorhynchus. These currents included a tetrodotoxin-sensitive sodium current; a potassium current that was sensitive to 4-aminopyridine; and a calcium current that was sensitive to nickel and cadmium, but resistant to specific blockers of L-, N-, P-, and Q-type calcium currents. The pharmacological profiles of the ionic currents in the pacemaker nucleus are similar to those of ionic currents involved in pacemaking in other neuronal oscillators. Because these ionic currents dramatically influence pacemaker firing frequency, which is directly related to electric organ discharge frequency, these ionic currents are likely targets of steroid hormone action in producing sexual dimorphisms in electric organ discharge frequency. Additional studies are needed to determine how these ionic currents interact to generate the electric organ discharge rhythm and to investigate the possibility that sexual dimorphism in the electric organ discharge results from the actions of gonadal steroids on these ionic currents. Accepted: 3 June 1999     

The influence of amlodipine and verapamil on ion and water transport in the nephron, skin and urinary bladder of amphibians.

1. The addition of amlodipine or verapamil into the lumen of the newt distal tubule led to the decrease of reabsorption of Na, Cl, Ca and of fluid. 2. The application of amlodipine to the outside of the frog skin caused large increases in potential difference (PD) and short circuit (SCC) similar to what is seen with Co2+. If both amlodipine and Co2+ were applied simultaneously to the outer surface the increases in PD and SCC were additive. 3. Verapamil added to the outer surface of the skin caused a reduction in PD which could be overcome by subsequent addition of amlodipine. 4. After addition of amlodipine to serosal or mucosal surfaces of the frog urinary bladder, the ability of vasopressin to increase osmotic permeability was markedly attenuated. 5. It is likely that the calcium channel blockers used here not only affect intracellular calcium levels by inhibiting entry through calcium channels, but they may also alter calcium dependent processes within the plasma membranes which modulate sodium transfer across epithelia.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

Diabetes mellitus and cardiac function

Cardiovascular complications are the most common causes of morbidity and mortality in diabetic patients. Coronary atherosclerosis is enhanced in diabetics, whereas myocardial infarction represents 20% of deaths of diabetic subjects. Furthermore, re-infarction and heart failure are more common in the diabetics. Diabetic cardiomyopathy is characterized by an early diastolic dysfunction and a later systolic one, with intracellular retention of calcium and sodium and loss of potassium. In addition, diabetes mellitus accelerates the development of left ventricular hypertrophy in hypertensive patients and increases cardiovascular mortality and morbidity. Treating the cardiovascular problems in diabetics must be undertaken with caution. Special consideration must be given with respect to the ionic and metabolic changes associated with diabetes. For example, although ACE inhibitors and calcium channel blockers are suitable agents, potassium channel openers cause myocardial preconditioning and decrease the infarct size in animal models, but they inhibit the insulin release after glucose administration in healthy subjects. Furthermore, potassium channel blockers abolish myocardial preconditioning and increase infarct size in animal models, but they protect the heart from the fatal arrhytmias induced by ischemia and reperfusion which may be important in diabetes. For example, diabetic peripheral neuropathy usually presents with silent ischemia and infarction. Mechanistically, parasympathetic cardiac nerve dysfunction, expressed as increased resting heart rate and decreased respiratory variation in heart rate, is more frequent than the sympathetic cardiac nerve dysfunction expressed as a decrease in the heart rate rise during standing.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Calcium activation of mougeotia potassium channels

Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation.     

Electrophysiology of phagocytic membranes: III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques.The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is ?90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero.Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore.The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10^?5 M.It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Electrophysiology of phagocytic membranes. III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques. The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is -90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero. Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore. The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10(-5) M. It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.     

The functional consequences of paraoxon exposure in central neurones of land snail, Caucasotachea atrolabiata, are partly mediated through modulation of Ca2+ and Ca2+-activated K+-channels

Toxicity of paraoxon has been attributed to inhibition of cholinesterase, but little is known about its direct action on ionic channels. The effects of paraoxon (0.3 microM-0.6 microM) were studied on the firing behaviour of snail neurones. Paraoxon significantly increased the frequency of spontaneously generated action potentials, shortened the afterhyperpolarization (AHP) and decreased the precision of firing. Short periods of high frequency-evoked trains of action potentials led to an accumulation in the depth and duration of post-train AHPs that was evidenced as an increase in time to resumption of autonomous activity. The delay time in autonomous activity initiation was linearly related to the frequency of spikes in the preceding train and the slope of the curve significantly decreased by paraoxon. The paraoxon induced hyperexcitability and its depressant effect on the AHP and the post-train AHP were not blocked by atropine and hexamethonium. Calcium spikes were elicited in a Na+ free Ringer containing voltage dependent potassium channel blockers. Paraoxon significantly decreased the duration of calcium spikes and following AHP and increased the frequency of spikes. These findings suggest that a reduction in calcium influx during action potential may decrease the activation of calcium dependent potassium channels that participate in AHP generation and act as a mechanism of paraoxon induced hyperexcitability.     

Organic calcium channel blockers generate the coexistence of two different types of action potentials in the same muscle membrane following chemical induction of excitability.

1. Following exposure to the sulfhydryl reagents known as alpha,beta-unsaturated carbonyl compounds, the ventroabdominal flexor muscles of the crustacean Atya lanipes, which are normally completely inexcitable, generate trains of overshooting calcium action potentials; the effects of organic calcium channel antagonists and potassium channel blockers on the chemically-induced trains of action potentials have been studied. 2. Verapamil and D600, at micromolar concentrations, elicit the appearance of slow, cardiac-like action potentials which coexist with the much faster chemically-induced calcium spikes, transforming the regular repetitive firing into a cyclic bursting pattern. 3. Bepridil (1 microM) decreases the frequency of firing of the action potentials, probably by increasing the threshold for the activation of a population of the chemically-induced calcium channels. 4. The potassium channel blockers, TEA (30-40 mM) and quinidine (100-200 microM), delayed the rate of repolarization of the chemically-induced action potentials; none of the potassium channel blockers, however, induced the appearance of repetitive spike activity.