Disposition of TF-PEG-Liposome-BSH in tumor-bearing mice.

BNCT requires high concentration and selective delivery of (10)B to the tumor cell. To improve the drug delivery in BNCT, we conducted a study by devising TPLB. We administrated three types of boron delivery systems: BSH, PLB and TPLB, to Oral SCC bearing mice. Results confirmed that (10)B concentration is higher in the TPLB group than in the BSH group and that TPLB is significantly effective as boron delivery system.

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity.

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca(2+)-activated K(+) current [I (K(Ca))] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K(+) current [I (K(ATP))] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for I (K(ATP)) and I (K(Ca)) was 50 and 29.5%, respectively, whereas at F19, I (K(ATP)) is reduced to 14%, and I (K(Ca)) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of K(ATP) and K(Ca) channels.

A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.

Low threshold T-type calcium current in rat embryonic chromaffin cells.

1. The gating kinetics and functions of low threshold T-type current in cultured chromaffin cells from rats of 19-20 days gestation (E19-E20) were studied using the patch clamp technique. Exocytosis induced by calcium currents was monitored by the measurement of membrane capacitance and amperometry with a carbon fibre sensor. 2. In cells cultured for 1-4 days, the embryonic chromaffin cells were immunohistochemically identified by using polyclonal antibodies against dopamine beta-hydroxylase (DBH) and syntaxin. The immuno-positive cells could be separated into three types, based on the recorded calcium current properties. Type I cells showed exclusively large low threshold T-type current, Type II cells showed only high voltage activated (HVA) calcium channel current and Type III cells showed both T-type and HVA currents. These cells represented 44 %, 46 % and 10 % of the total, respectively. 3. T-type current recorded in Type I cells became detectable at -50 mV, reached its maximum amplitude of 6.8 +/- 1.2 pA pF(-1) (n = 5) at -10 mV and reversed around +50 mV. The current was characterized by criss-crossing kinetics within the -50 to -30 mV voltage range and a slow deactivation (deactivation time constant, tau(d) = 2 ms at -80 mV). The channel closing and inactivation process included both voltage-dependent and voltage-independent steps. The antihypertensive drug mibefradil (200 nM) reduced the current amplitude to about 65 % of control values. Ni(2+) also blocked the current in a dose-dependent manner with an IC(50) of 25 microM. 4. T-type current in Type I cells did not induce exocytosis, while catecholamine secretion by exocytosis could be induced by HVA calcium current in both Type II and Type III cells. The failure to induce exocytosis by T-type current in Type I cells was not due to insufficient Ca(2+) influx through the T-type calcium channel. 5. We suggest that T-type current is expressed in developing immature chromaffin cells. The T-type current is replaced progressively by HVA calcium current during pre- and post-natal development accompanying the functional maturation of the exocytosis mechanism.

Calcium signals in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.

Development of multiple calcium channel types in cultured mouse hippocampal neurons.

The development of multiple calcium channel activities was studied in mouse hippocampal neurons in culture, using the patch-clamp technique. A depolarizing pulse (40-50 ms duration) from the holding potential of -80 mV to levels more depolarized than -40 mV produced a low threshold T-type current. The T-type current was observed in 52% of four days in vitro neurons. The number of neurons which expressed T-type current decreased with age of culture, so that the current was detected in only 18% of neurons after 16 days in vitro. The T-type current densities varied between 1.9 pA/pF and 3.29 pA/pF in the mean values during the period studied (4-16 days in vitro). A depolarizing pulse from -80 mV to levels more depolarized than -35 mV evoked a high threshold calcium channel current. The high threshold current density increased in the mean values from 3.9 pA/pF in four days in vitro neurons to 28 pA/pF in 16 days in vitro neurons. We have then examined the effect of nifedipine, omega-Agatoxin IVA and omega-conotoxin GVIA on the high threshold current. Nifedipine (1-5 microM) sensitive current density stayed in the range of 1.9-2.1 pA/pF during 4-16 days in vitro, while omega-Agatoxin IVA (200 nM) sensitive current density increased in the mean values from 1.54 pA/pF in four days in vitro neurons to 21.5 pA/pF in 16 days in vitro neurons. The omega-conotoxin GVIA sensitive N-type channel current was maximum at eight days in vitro (5.44 pA/pF) and it reduced progressively to reach almost half (2.46 pA/pF) in 16 days in vitro neurons. These results showed that diverse subtypes of calcium channels change in density during the early period of culture. We suggest that the temporal expression of each type of channel may be linked to the development of neural activities.

The action of diltiazem and gallopamil (D600) on calcium channel current and charge movement in mouse Purkinje neurons.

The dihydropyridines (DHP) receptor forms a high threshold L-type calcium channel in various excitable cells. In skeletal and cardiac muscle cells, a DHP receptor antagonist blocks not only the voltage-gated calcium current but also immobilizes the charge movement linked to the receptor. The DHP receptor is also present in cerebellar Purkinje neurons. Previously, we showed that nifedipine immobilizes a part of the charge movement but has no effect on the calcium channel current recorded in freshly dissociated mice Purkinje neurons. We report here the effect of other families of DHP receptor antagonists, benzothiazepines and phenylalkylamines, on the physiological properties of this receptor in mouse Purkinje neurons.

Effect of lipophilic ions on the intramembrane charge movement and intracellular Ca2+ release in fetal mouse skeletal muscle cells.

The effects of lipophilic ions on the intramembrane charge movement and intracellular calcium transient were studied using freshly dissociated skeletal muscle cells from mice fetuses. The lipophilic cations Rhodamine 6G and tetraphenylphosphonium (TPP) immobilized part of the intramembrane charge movement in a dose-dependent manner, and inhibited both calcium transient and contraction evoked by membrane depolarization. In contrast, the lipophilic anion 1-anilinonaphthalene-8-sulfonic acid (ANS) had no effect on intramembrane charge movement. We suggest that the lipophilic cations block the voltage-sensing mechanism for the excitation-contraction (E-C) coupling mechanism.

Effect of SR33805 on barium current and asymmetric intramembrane charge movement in freshly dissociated mouse cerebellar Purkinje neurons.

SR33805 is a novel calcium channel blocker that binds selectively and with high affinity to the alpha 1 subunit of the L-type calcium channel. The binding site for SR33805 is distinct from other classical calcium channel blockers although they interact allosterically. The block by SR33805 of the neuronal L-type calcium current has been reported [Romey, G. and Lazdunski, M., J. Pharmacol. Exp. Ther., 271 (1994) 1348-1352.]. In Purkinje neurons, the L-type calcium current is nearly absent. Nevertheless, we have shown the presence of intramembrane charge movement related to the dihydropyridines (DHP) receptor in these neurons. We show here that SR33805 has no effect on barium currents recorded in Purkinje cells but is a very potent blocker of intramembrane charge movement. It reduces charge movement to 48% of control with an IC50 of 0.5 nM.

cAMP-dependent modulation of L-type calcium currents in mouse diaphragmatic cells.

The regulation of calcium channels by cAMP-dependent phosphorylation was investigated in the diaphragm muscle. Experiments were performed on dissociated costal diaphragmatic cells from 16- to 17-day-old fetal mice. The ionic current through calcium channels was measured using the whole cell clamp technique with barium as the charge carrier. A depolarizing pulse delivered from a holding potential of -80 mV elicited a low-threshold dihydropyridine (DHP)-insensitive T-type current and a high-threshold DHP-sensitive L-type current. Agents that either increase intracellular cAMP levels (forskolin, 10(-4) M, and dibutyryladenosine 3'-5' cyclic monophosphate, 10(-4) M) or inhibit cAMP degradation (theophylline, 10(-4) M) produced relative increases in L-type current amplitude of 24.4 +/- 13.8%, 13.4 +/- 4.6%, and 15.9 +/- 2.8% (p < 0.05), respectively. Current intensity increased after application of the beta-adrenergic agonist isoproterenol (10(-5) M, 16.5 +/- 3.6%, P < 0.005). None of these agents affected the T-type current. These results suggest that L-type calcium channel activities of the diaphragm muscle are regulated by cAMP-dependent phosphorylation.

Nifedipine-sensitive intramembrane charge movement in Purkinje cells from mouse cerebellum.

1. The intramembrane charge movement was recorded in freshly dissociated Purkinje cells from 14- to 18-day-old mouse cerebellum using the whole-cell voltage clamp technique. 2. After pharmacological elimination of all ionic currents, a depolarizing pulse from a holding potential of -80 mV revealed a transient capacitive outward current at the onset and a transient inward current at the end of the pulse. The amount of charge transferred at the onset (Qon) was equivalent to that moved at the end of the pulse (Qoff). The decay time course of Qon can be fitted by a single exponential curve with a maximum time constant of 1.89 +/- 0.35 ms at 20 mV (n = 11). 3. The charge movement had an S-shaped dependence on test membrane potential, according to a two-state Boltzmann function. The maximum amount (Qmax) of Qon that could be moved was 17.46 +/- 0.83 nC muF-1; the membrane potential at which half the charge movement occurred (V) was 13.48 +/- 2.20 mV and the slope factor (k) was 16.83 +/- 0.84 mV (n = 27). 4. Phenylglyoxal (2 mM), an arginine-specific modifying reagent, reduced Qmax to 60% of control after 20 min treatment. 5. The charge movement was partially immobilized by nifedipine in a dose-dependent manner with an IC50 of 70 nM. The fraction of the nifedipine-sensitive component was 39% of the total charge movement. The potential dependence of the nifedipine-sensitive charge movement could be expressed by a Boltzmann function with values of 7.00 +/- 0.53 nC muF-1 for Qmax, 31.44 +/- 4.23 mV for V and 21.53 +/- 3.18 mV for k (n = 8). 6. The P-type calcium channel specific inhibitor, omega-Aga IVA (250 nM), had no effect on intramembrane charge movement. 7. The above results show that part of the intramembrane charge movement in Purkinje cells may be related to a conformational change of DHP receptors upon membrane depolarization.

Ca2+ entry through acetylcholine receptor channel in dysgenic myotubes.

Skeletal muscles of mutant mice with "muscular dysgenesis" are characterized by excitation-contraction uncoupling resulting from the absence of dihydropyridine receptors. However contraction of the dysgenic myotubes can be evoked by afferent nerve stimulation or by ionophoretic application of acetylcholine (ACh) on the muscle. These contractions are elicited by Ca2+ entry through the ionic channel of the ACh receptor at multiple synaptic contacts. In the present paper, the calcium entry through ACh receptors was compared in cultured normal and dysgenic myotubes. At elevated external calcium concentration (110 mM), the elementary slope conductance of the ACh-activated ionic channel of dysgenic myotubes did not differ from that found in normal myotubes. We conclude that dysgenic muscle contraction induced by nerve stimulation does not result from an abnormal Ca2+ entry across ACh receptors. We discuss the possible involvement of sustained high threshold calcium current (Idys) and of the calcium induced calcium release mechanism in the contractile response related to synaptic activity of dysgenic myotubes.

Asymmetric intramembrane charge movement in mouse hippocampal pyramidal cells.

Intramembrane charge movement was recorded from freshly dissociated hippocampal pyramidal cells from mice using the whole cell clamp technique. Once the ionic currents were suppressed, a depolarizing pulse from a holding potential of -80 mV elicited a capacitive transient outward current at onset and a capacitive inward current at offset of the pulse. The amount of charge displaced at the onset of the pulse (Qon) was equivalent to the charge moved at repolarization (Qoff). The relationship between the amount of charge moved and pulse potential could be expressed by a simple two states Boltzmann equation: Q = Qmax/(1 + exp[-(V-V1/2)/k]), where Qmax is the maximum charge, V1/2 the membrane potential at which Q is half of Qmax and k is a slope factor. On average, Qmax was 10.90 +/- 0.62 nC/microF, V1/2 was 1.70 +/- 2.90 mV, and k was 18.80 +/- 1.20 mV (n = 16). Phenylglyoxal (10 mM), an arginine modifying reagent, reduced the maximum amount of charge movement to 14% of control. The inhibitory effect of phenylglyoxal was time dependent and the decline time course of maximum amount of charge movement could be fitted by a single exponential curve with a time constant of 5.79 min. The dihydropyridine (DHP) receptor antagonist, nifedipine, immobilized 54% of the charge movement. These results suggest that a part of the charge movement reflects the conformational change of the DHP receptors upon membrane depolarization.

Effect of SR33557 on intramembrane charge movement in normal and 'muscular dysgenesis' mouse skeletal muscle cells.

It has been reported that the indolizinsulphone SR33557, which binds to a site on the alpha 1 subunit of the dihydropyridine receptor, blocks both L-type calcium channel activity and contraction in skeletal muscle. Moreover, we know that charge movement plays a key role in the mechanism of excitation-contraction coupling and in controlling the opening of L-type calcium channels. We demonstrate here that SR33557 reduces intramembrane charge movement in skeletal muscle from normal mice with an IC50 of approximately 10 nM. The drug does not completely inhibit charge movement since approximately 20% of total charge movement persists even in the presence of 30 microM SR33557. However, the SR33557-sensitive charge component is more important than the dihydropyridine-sensitive one. Surprisingly, SR33557 also reduces intramembrane charge movement in dysgenic myotubes which are characterized by a very strong reduction of the number of dihydropyridine binding sites. In these muscles, 10 microM SR33557 reduces approximately 40% of total charge movement. These observations suggest the presence of a new component of charge movement which is sensitive to SR33557 but insensitive to nifedipine. This component is also present in dysgenic myotubes, and it could be produced by the lower molecular weight alpha 1 subunit described by Malouf, N. N., McMahon, D. K., Hainsworth, C. N. and Kay, B. K. (1992) (Neuron, 8, 899-906).

Extracellular Ca(2+)-dependent and independent calcium transient in fetal myotubes.

Spatio-temporal changes in the intracellular calcium concentration [Ca2+]i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca2+ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca2+]i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 microM lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca2+]i by stimulating Ca2+ release from the sarcoplasmic reticulum independently of extracellular Ca2+ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca2+ entry through calcium channels was suppressed.

Anti-IgE induces the opening of non selective cation channels on human basophils.

Basophils play a major role in allergic reactions-particularly in late phase reactions-by releasing histamine and other mediators of inflammation. Although transmembrane ion fluxes are thought to play an important role in the modulation of histamine release, little is known about ion pathways through the basophil membrane. We thus studied human basophils from normal subjects (n = 25 cells) with the patch-clamp method. We observed that IgE-dependent activation of human basophils led to the opening of non selective cation channels with a 20pS conductance. This was obtained when the patch pipette was applied onto the cell surface and sealed onto it in order to measure transmembrane currents on a small surface of intact basophils (cell-attached configuration). Non selective channels with the same 20pS conductance were also observed when a membrane patch was detached from basophil and its inner side placed in a Ca(2+)-containing medium (inside-out configuration). These data are a first contribution of the patch-clamp method in the understanding of ion movements in human basophils.

Ciguatoxin, extracted from poisonous morays eels, causes sodium-dependent calcium mobilization in NG108-15 neuroblastoma x glioma hybrid cells.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) in cultured mouse NG108-15 neuroblastoma x glioma hybrid cells, using the fluorescent probe fura-2, revealed that 5-25 nM ciguatoxin (CTX) increased [Ca2+]i either in cells bathed in standard medium or after removal of external Ca2+ by a Ca(2+)-free medium supplemented with EGTA. Tetrodotoxin prevented the CTX increased [Ca2+]i suggesting that CTX-induced mobilization of intracellular Ca2+ depends on Na+ influx through voltage-gated Na channels. CTX-induced Ca2+ mobilization prevented subsequent action of bradykinin (1 microM) suggesting that CTX stimulates the inositol 1,4,5-trisphosphate-releasable Ca2+ store.

Intramembrane charge movement in developing skeletal muscle cells from fetal mice.

The development of intramembrane charge movement was studied in freshly isolated skeletal muscle cells from 13- to 19-day-old mouse fetuses. Charge movement was present in myotubes from 13-day-old fetuses. The relationship between charge movement and membrane potential could be described by a two-state Boltzmann equation. The amount of maximum charge movement (Qmax) increased substantially with the age of the fetuses from 2.84 +/- 0.39 nC/microF (n = 10) at day 13 to 10.01 +/- 0.97 nC/microF (n = 15) at day 19. Nifedipine (1 microM) consistently reduced Qmax by 33 +/- 2% (n = 37) of the control value at each age studied. Increasing the concentration of nifedipine to 20 microM had no further effect, suggesting that the charge movement in developing myotubes consists of at least two components: a nifedipine-sensitive charge movement (Qns) and a nifedipine-resistant one (Qnr). Both Qns and Qnr increased exponentially with a distinct enhancement of rate at day 16.

Regulation of human basophil activation. II. Histamine release is potentiated by K+ efflux and inhibited by Na+ influx.

Na+ and K+ are the major extra- and intracellular cations, respectively. We have thus studied the role of these ions on human basophil histamine release by modifying their transmembrane gradients or by increasing membrane ion fluxes using ionophores. 1) When external Na+ (reduced to 4 mM) was replaced by the nonpermeating Na+ substitute N-methyl-D-glucamine, the release of histamine was enhanced in 2 mM Ca2+ (from 37.5 +/- 8.0% in 140 mM Na+ to 68.5 +/- 9.1% in low Na+) and became possible in the presence of low Ca2+ (at 1 microM Ca2+: from 0.6 +/- 0.7% in 140 mM Na+ to 36.2 +/- 8.0% in low Na+); moreover, in low Na+, the release of histamine became partly independent on Ca2+ influx. 2) Increasing the Na+ influx with the cation channel-forming gramicidin D inhibited the release of histamine by 33.2 +/- 13.6% (n = 6) in an external Na(+)-dependent manner. 3) Decreasing K+ efflux using K+ channel blockers (4-aminopyridine, quinine, sparteine) inhibited histamine release in a dose-response manner. 4) The K+ ionophore valinomycin, which increases K+ efflux, slightly enhanced IgE-mediated histamine release when used alone, whereas it potentiated the release of histamine from leukocytes previously treated with 4-aminopyridine by 57.0 +/- 18.6% (n = 7). 5) Decreasing K+ efflux by increasing external K+ inhibited IgE-mediated release in a similar manner as Na+ did. The inhibitory effects of Na+ and high K+ were not additive, thus suggesting that both cations inhibited the release by a common mechanism. In conclusion 1) our data evidence that histamine release from human basophils is inhibited by Na+ influx and potentiated by K+ efflux; 2) they suggest that K+ channels are present on the basophil membrane and that Na+ and K+ fluxes act on histamine release most probably via modulation of membrane potential.