Distinct effects of ω-toxins and various groups of Ca-entry inhibitors on nicotinic acetylcholine receptor and Ca channels of chromaffin cells

The effects of ω-toxins and various Ca²⁺ antagonist subtypes on the ⁴⁵Ca²⁺ entry into bovine adrenal medullary chromaffin cells stimulated via nicotinic acetylcholine receptors or via direct depolarization with K⁺, have been compared. The conditions selected to stimulate the ⁴⁵Ca²⁺ entry consisted of a 60-s period of exposure of cells to 100 μM of the nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium or to 70 mM K⁺. The N-type voltage-dependent Ca²⁺ channel blockers ω-conotoxin GVIA and MVIIA (1 μM) inhibited ⁴⁵Ca²⁺ entry stimulated by dimethylphenylpiperazinium or K⁺ by around 25–30%. The P-type Ca²⁺ channel blocker ω-agatoxin IVA (10 nM) did not affect the dimethylphenylpiperazinium nor the K⁺ responses; 1 μM (Q-channel blockade) inhibited both responses by around 50%. The N/P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC (1 μM) inhibited the K⁺ evoked ⁴⁵Ca²⁺ entry by 70%, while dimethylphenylpiperazinium was blocked by 50% (P<0.001). The L-type Ca²⁺ channel blockers nifedipine, furnidipine, diltiazem or verapamil (3 μM each) inhibited much more the dimethylphenylpiperazinium than the K⁺ response. The dimethylphenylpiperazinium signal was blocked 71, 88, 89, and 53%, respectively, by nifedipine, furnidipine, diltiazem and verapamil, and the K⁺ response by 38, 29, 22, and 10%. Combined ω-conotoxin MVIIC (1 μM) and furnidipine (3 μM) blocked 100% of the K⁺ evoked ⁴⁵Ca²⁺ entry. However, combined ω-conotoxin GVIA (1 μM), and furnidipine left unblocked 50% of the K⁺ response. The ‘wide spectrum' Ca²⁺ channel antagonists flunarizine or dotarizine (3 μM each) blocked the dimethylphenylpiperazinium and the K⁺ responses to a similar extent (50%); cinnarizine (3 μM) inhibited more the dimethylphenylpiperazinium (82%) than the K⁺ response (21%). At 3 μM, the highly lipophilic β-adrenoceptor antagonist (±)-propranolol, reduced by 68% the dimethylphenylpiperazinium signal and by 23% the K⁺ signal. Other high lipophilic β-adrenoceptor antagonists such as metoprolol and labetalol, reduced little the dimethylphenylpiperazinium and the K⁺ responses. The highly lipophilic agent penfluridol blocked the dimethylphenylpiperazinium response by 30% and the K⁺ response by 50%. One of the least lipophilic compounds tested, (+)-lubeluzole, blocked by 40% the dimethylphenylpiperazinium and the K⁺ responses. These data are compatible with the idea that the various ω-toxin peptides used to separate pharmacologically the different voltage-dependent Ca²⁺ channels expressed by neurones, do not block the neuronal nicotinic acetylcholine receptor ion channel. In contrast the L-type Ca²⁺ channel blockers do block the nicotinic acetylcholine receptor ionophore. Lipophilicity of the compounds is not a requirement for Ca²⁺ channel or nicotinic acetylcholine receptor blockade.     

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

The Ca channel agonist Bay K 8644 induces central respiratory depression in cats, an effect blocked by naloxone

We analyzed the respiratory effects induced by the Ca²⁺ channel agonist Bay K 8644 and its enantiomers, Bay R 5417 and Bay R 4407, applied to the ventral medullary surface of cats. Bay K 8644 (10 to 100 μg) and Bay R 5417 (50 to 200 μg) elicited a dose-dependent respiratory depression. Naloxone (0.1 mg/kg), but not D-naloxone, reversed these effects, indicating that an endogenous opioid mechanism was involved. Bay R 4407 (100 μg) was ineffective. The respiratory depressant effects induced by Bay K 8644 and its (−) enantiomer were a consequence of their agonist properties on the L-type Ca²⁺ channel, since (1) the activity of Bay K 8644 was stereospecific, and (2) nimodipine prevented the effect. We suggest that potent activation of Ca²⁺ channels or other mechanisms by high doses of Ca²⁺ agonists elicits the release of endogenous opioid peptides in medullary respiration-related structures.     

Cromakalim inhibits contractions of the rat isolated mesenteric bed induced by noradrenaline but not caffeine in Ca-free medium: Evidence for interference with receptor-mediated Ca mobilization

The effects of the K⁺ channel opener cromakalim on phasic contractions induced by noradrenaline and caffeine were studied in the rat isolated mesenteric bed. In the presence of 1.4 mM Ca²⁺, 1-s pulses of noradrenaline increased the perfusion pressure of the preparation concentration dependently (midpoint at 92 ± 10 μM noradrenaline). Cromakalim (0.3 and 1 μM) inhibited these contractions in a non-competitive manner. Contractions elicited by 1-s pulses of noradrenaline (100 μM) were inhibited by the dihydropyridine Ca²⁺ antagonist isradipine by maximally 24 ± 1%, indicating that only a minor component of this contraction depended on Ca²⁺ entry via dihydropyridine-sensitive Ca²⁺ channels. Cromakalim was a much more effective inhibitor of these contractions (maximum inhibition by 80%, midpoint of the inhibition curve at 171 ± 15 nM). The effect of cromakalim was stereoselective, inhibited by the sulphonylurea glibenclamide, and abolished in partially depolarizing media (KC1 = 35 and 50 mM). In Ca²⁺-free medium, cromakalim inhibited the contraction induced by noradrenaline (100 μM) by maximally 69 ± 4%, with a midpoint at 58 ± 14 nM. The effect of cromakalim was again stereoselective, inhibited by glibenclamide, and abolished in the presence of 50 mM KC1. Contractions induced by caffeine (10 and 100 μM) were not affected by cromakalim (1 μM). The results indicate that, in rat mesenteric resistance vessels, cromakalim interferes with the ability of noradrenaline, but not caffeine, to mobilize Ca²⁺ from intracellular stores. The antivasoconstrictor effect of cromakalim against noradrenaline is inhibited by glibenclamide and appears to be linked to the ability of cromakalim to hyperpolarize the cell membrane.     

Inhibitory effects of cilnidipine on peripheral and brain N-type Ca2+ channels expressed in BHK cells

We investigated differences in electrophysiological characteristics between peripheral and central N-type Ca²⁺ channels, containing _1B-a and a_1B-c, respectively. In addition, we examined the inhibitory effects of cilnidipine, a dihydropyridine (DHP) derivative, on both channels. Both _1B subunits were transiently expressed in BHK cells, and then analyzed using the whole-cell patch-clamp technique. The current–voltage relationship showed that _1B-c currents were activated at more negative potentials than _1B-a currents. The voltage-dependent steady-state inactivation and activation showed that the V_1/2 values for inactivation and activation of _1B-c (−88.5±1.3 and −33.2±1.3 mV) were both significantly more negative than those for _1B-a (−83.3±1.3 and −27.9±2.3 mV). Despite the different electrophysiological characteristics of these two N-type channels, cilnidipine blocked both with similar potency within the range 0.1 to 10 μM. Furthermore, cilnidipine had no effect on the I–V relationships or the steady-state inactivation curves. Our data indicate that the spliced positions of _1B-a and a_1B-c may affect not only their voltage-sensing abilities but also the kinetics of channel activation and inactivation. The data also suggest that cilnidipine binds to sites independent of those controlling voltage-sensing and channel kinetics in these _1B subunits.     

Delta(9)-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Δ⁹-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca²⁺]_i spiking by Win55,212-2. In control experiments, 0.1 μM Win55,212-2 inhibited [Ca²⁺]_i spiking by 93 ± 5%. Win55,212-2 produced significantly less inhibition of [Ca²⁺]_i spiking following 18–24 h treatment with 1 μM THC (48 ± 5%) or treatment with 1 μM Win55,212-2 (29 ± 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 μM, remained stable between 1 and 7 days of preincubation and shifted the EC₅₀ of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.     

Adenosine A2 receptor activation facilitates Ca uptake by rat brain synaptosomes

Adenosine has been shown to increase the release of neurotransmitters by stimulation of adenosine A₂ receptors. This effect probably depends on Ca²⁺ entry into presynaptic nerve terminals. In the present work the ability of the mixed adenosine A₁/A₂ agonist, 2-chloroadenosine, to stimulate Ca²⁺ uptake into rat brain synaptosomes was investigated. ⁴⁵Ca²⁺ uptake was induced by 20 μM veratridine. In the absence of other drugs, 2-chloroadenosine (1 μM) decreased ⁴⁵Ca²⁺ uptake into synaptosomes. Blocking the adenosine A₁ receptor with 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 2-chloroadenosine (1 μM) increased rather than decreased the uptake of ⁴⁵Ca²⁺ into synaptosomes. The excitatory effect of 2-chloroadenosine observed in the presence of DPCPX was reversed by 200 nM of ω-agatoxin-IVA, a specific P-type Ca²⁺ channel antagonist, but not by L-type (nifedipine, 100 nM to 1 μM; methoxyverapamil 1-10 μM) or N-type (ω-conotoxin GVIA, 500 nM) Ca²⁺ channel antagonists. The adenosine A_2A selective agonist, 2-p-(2-carboxyethyl)-phenethylamino-5′-N-ethyl-carboxamido-adenosine (CGS 21680), did not significantly modify Ca²⁺ uptake induced by veratridine. In contrast, the selective adenosine A₂ receptor agonist, N⁶-(2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl)-adenosine (DPMA), in concentrations ranging from 10 nM to 1 μM increased Ca²⁺ uptake induced by veratridine. The selective adenosine A₂ receptor antagonist 3,7-dimethyl-1-propargylxanthine (DPMX) at a concentration of 10 μM antagonized the stimulatory effect of DPMA (0.1 μM) on ⁴⁵Ca²⁺ uptake. In conclusion, activation of adenosine A₂ receptors increases Ca²⁺ uptake by synaptosomes depolarized by veratridine, which could explain the increase of neurotransmitter release observed when A₂ receptors are activated.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca²⁺ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced, Ca²⁺-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 μM glutamate for 15 min, at 22°C, in the absence of Mg²⁺, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC₅₀ 300 μM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 μM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA- (20 μM) stimulated current was inhibited by 700 μM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at −50 mV, 100 μM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg²⁺-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca transport proteins and causing dysregulation of Ca homeostasis

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca²⁺ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca²⁺ within these cells in the 5–60 μM concentration range (EC₅₀, 21 μM). TBBPA also causes cell death (LC₅₀, 18 μM) partly via apoptosis, involving Ca²⁺-dependent mitochondrial depolarisation. Studies on intracellular Ca²⁺ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA) at low concentrations (IC₅₀, 0.4 to 1.2 μM) and also activate the Ryanodine receptor Ca²⁺ channel within the 0.4–4 μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca²⁺ signalling, by directly affecting Ca²⁺ transport proteins.     

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl₂DHA) raises cytoplasmic Ca²⁺ in synaptosomes and blocks neural GABA_A receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl₂DHA (50 μM) almost completely blocked native GABA_A currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (1, β2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl₂DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10 μM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl₂DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca²⁺ release). 12,14-Cl₂DHA was unable to stimulate phosphate labelling of [³H]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca²⁺ release via an IP₃ mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl₂DHA clearly blocks GABA_A currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca²⁺ via an unidentified recognition site (or receptor) found in neuronal cells.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

Effects of verapamil, diltiazem, nisoldipine and felodipine on sarcoplasmic reticulum

Verapamil, diltiazem, nisoldipine and felodipine, calcium antagonist drugs with different chemical structures, were studied for their effects on activities of sarcoplasmic reticulum (SR) isolated from dog cardiac and rabbit skeletal muscles. Nisoldipine and felodipine exerted biphasic actions on both cardiac and skeletal SR Ca²⁺-ATPase with maximum activation of 40–60% occurring at 20–40 μM for nisoldipine and 30–40% occurring at 15–30 μM for felodipine. At higher drug concentrations, Ca²⁺-ATPase was inhibited. In the presence of oxalate the maximum activation of the Ca²⁺ uptake rates at 5–20 μM nisoldipine were 30–50% for cardiac SR and 80–100 μM of the drug were 300–500% for skeletal SR. Felodipine inhibited the rate of Ca²⁺ uptake by dog cardiac SR, but activated Ca²⁺ uptake by rabbit skeletal SR with a maximum of 30–50% at 12–25 μM. At higher concentrations of the two drugs the rate of Ca²⁺ uptake was inhibited. In the absence of oxalate, i.e., limited tranport, nisoldipine shortened the duration of time that Ca²⁺ was bound to the cardiac and skeletal SR, while the rate of release of Ca²⁺ from skeletal SR was stimulated. Felodipine at low concentrations similarly caused a premature release of Ca²⁺ from skeletal SR at a rapid rate; at high concentrations both drugs did not alter Ca²⁺ binding but delayed Ca²⁺ release. Unlike nisoldipine and felodipine, verapamil and diltiazem inhibited the rates of Ca²⁺ transport both in cardiac and skeletal SR. The two drugs inhibited Ca²⁺-ATPase in cardiac SR but activated the enzyme in skeletal SR. Thus, these drugs caused complex and different effects on cardiac and skeletal SR, possibly resulting from perturbations of the lipid environment of the SR Ca²⁺-ATPase.     

Participation of intracellular sites in the action of Ca channel blockers

The action of phenylalkylamine Ca²⁺ channel blockers D890 and D888 on Ca²⁺ uptake and neurotransmitter amino acid release were studied. D890, the quaternary derivative of D600, did not inhibit veratrine-induced ⁴⁵Ca²⁺ uptake or the release of neurotransmitter amino acids from rat cerebrocortical synaptosomes, except at high concentrations (200 μM) when it was probably acting extracellularly in a non-specific manner. This contrasted with the more potent (10–50 μM) inhibitory actions of D600 and D888, and may be due to the inability of D890 to cross the synaptic plasma membrane. (−)D888 was shown to cross cell membranes and accumulate in the intracellular compartment of cerebrocortical slices and synaptosomes, where it was associated predominantly with the soluble cytoplasmic fraction.     

5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.     

Inhibition of voltage-gated ca channel activity in small cell lung carcinoma by the ca/calmodulin-dependent protein kinase inhibitor KN-62 (1-[n,o-bis(5-isoquinolinesulfonyl)-n-methyl-L-tyrosyl]-4-phenylpiperazine)

Although small cell lung carcinoma (SCLC) cells express both voltage-gated Ca²⁺ channels (VGCC) and second messenger-operated Ca²⁺ channels (SMOCC), little is known about the factors that regulate the activity of these channels in SCLC cells. Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) type II has been implicated recently in regulating Ca²⁺ channel activity in other cell types. Because of this, we investigated the effects of the specific CaM kinase antagonist 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tryosyl]-4-phenylpiperazine (KN-62) on Ca²⁺ channel activity in SCLC cells. Incubation with 10 μM KN-62 for 20 min inhibited depolarization-dependent ⁴⁵Ca²⁺ influx by 96.1 ± 2.1% in four independent SCLC cell lines, and by 42.2 ± 6.8% in the NCI-H146 SCLC cell line. Similar inhibitory effects of KN-62 were observed when Fura-2 was used to measure depolarization-dependent Ca²⁺ influx. These results indicate that KN-62 potently inhibits VGCC activity in SCLC cells. In contrast, KN-62 (10 μM, 20 min) did not inhibit significantly Ca²⁺ mobilization induced by muscarinic acetylcholine receptor (mAChR) activation in SCLC cells. This indicates that SMOCC are less susceptible than VGCC to inhibition by KN-62 in SCLC cells. Because mAChR activation also inhibits VGCC activity in SCLC cells, we examined the effects of KN-62 on the mAChR-mediated inhibition of VGCC activity. To do this, we measured depolarization-dependent ⁴⁵Ca²⁺ influx in SCLC cells incubated with submaximal concentrations of KN-62 and the mAChR agonist carbachol. Treatment of cells with both drugs resulted in almost twice as much inhibition of VGCC activity as in cells treated with only one of the drugs. This indicates that inactivation of CaM kinase with KN-62 does not suppress the ability of mAChR agonists to inhibit VGCC activity.     

A novel aspect of lindane testicular toxicity: in vitro effects on peritubular myoid cells

The in vitro effects of the insecticide lindane have been investigated in rat testis peritubular myoid cells (PMCs). Upon PMC exposure to lindane, polarity increase and decrease of dipole dynamics were seen at the membrane level (EC50 20 μM), leading to a partial dissipation of the membrane intrinsic dipole potential. The initial membrane depolarization was increased by Cl⁻ efflux and limited by Ca²⁺-activated repolarizing currents. Concomitantly, lindane produced an increase in [Ca²⁺]_i (EC50 125 μM) resulting from both Ca²⁺ release from an inositol 1,4,5-trisphosphate-sensitive intracellular store and a voltage-dependent Ca²⁺ influx from the extracellular medium. Of particular interest from a toxicologic point of view, insecticide concentrations well below those effective in altering ion homeostasis potently inhibited both [Ca²⁺]_i increase and contraction induced by the natural agonists vasopressin and endothelin-1 (IC50s < 10 μM). These data demonstrate that PMCs are highly susceptible to lindane and suggest that the insecticide may exert testicular toxicity by interfering with hormone-regulated PMC function.     

Stimulatory and inhibitory effects of inorganic lead on calcineurin

Calcineurin is a phosphatase with activity dependent on both Ca²⁺/calmodulin binding to the catalytic A subunit and Ca²⁺ binding to the regulatory B subunit. We have previously shown that Pb²⁺ activates calmodulin with a threshold of about 100 pM free Pb²⁺, and that Pb²⁺ and Ca²⁺ are roughly additive in calmodulin activation (Kern et al., NeuroToxicology 21, 353–364 (2000)). In the present study, we evaluated the effects of Pb²⁺, with and without Ca²⁺ and calmodulin, on calcineurin activity. In calmodulin-containing, Ca²⁺-free solutions, Pb²⁺ activated calcineurin with a threshold of about 100 pM free Pb²⁺. Maximum calcineurin activity (comparable to that induced by 10 μM Ca²⁺) was reached at about 200 pM free Pb²⁺. Higher Pb²⁺ concentrations reduced activity, although some activity remained even at 2000 pM free Pb²⁺. Combined with subsaturating Ca²⁺ concentrations, as little as 20 pM free Pb²⁺ enhanced calcineurin activity, but free Pb²⁺ concentrations greater than 200 pM still reduced activity below maximum. Extremely high Ca²⁺ concentrations (10 μM) completely reversed the inhibition of activity by 2000 pM free Pb²⁺. In the absence of calmodulin, Ca²⁺ slightly stimulated calcineurin activity. Pb²⁺ did not substitute for Ca²⁺ in calmodulin-free activation; in fact, high concentrations of Pb²⁺ inhibited Ca²⁺-mediated activation. We tentatively conclude that low concentrations of free Pb²⁺ activate calcineurin by activating calmodulin. Higher concentrations reduce calcineurin activity, perhaps by binding to the B subunit.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.