Lanthanum-induced changes in gravicurvature, calcium balance, and statocyte ultrastructure of pea roots

A study to investigate the effects of La" ions on graviresponsiveness, statocyte ultrastructure, and calcium balance were carried out on 5-day seedlings of Pisum sativum. The results indicate that this blocker of some Ca2+ channels 1) inhibits the root graviresponsiveness, 2) disturbs statocyte polarity and changes qualitative and quantitative characteristics of some cellular organelles and compartments, 3) results in the disturbance of calcium homeostasis. Taken together, the data are consistent with the hypothesis, concerning the role of Ca2+ in gravitational signal transduction.     

Changes in carbohydrate composition in wheat and pea seedlings induced by calcium deficiency

Wheat (Triticum aestivum L. cv Jubilar) seedlings were grown for 10 days in hydroponics with or without calcium. In the leaves, Ca deficiency caused the level of ethanol soluble carbohydrate to increase between 2-and 10-fold, enhanced dark respiration and decreased CO₂ fixation capacity. Sucrose was the major carbohydrate to accumulate in wheat roots.     

Effect of calcium channel blockers on experimentally induced seizures in rats

Chemically different classes of calcium channel blockers were examined in rats for their effects on behavior, tolerability and protection against maximal electroshock seizures (MES) and pentylenetetrazol (PTZ) induced seizures. In MES test at doses (mg/kg, ip) that were devoid of side effects, felodipine, 50, afforded 100% protection, while nimodipine, 5; pimozide, 10; and thioridazine, 25, showed 50 to 66% protection. Nifedipine, 10, and diltiazem, 50, showed 30 and 66% protection respectively, but were associated with side effects. Verapamil and loperamide were ineffective against MES and PTZ induced seizures. Nimodipine, 1 mg/kg, ip, was the most potent agent and produced 100% protection against PTZ. Equieffective doses were pimozide, 25, felodipine, 50, and thioridazine, 50. The rest of the calcium channel blockers showed marginal to moderate activity against chemoshock. The data obtained suggest that some calcium channel blockers possess anticonvulsant activity and may be considered as adjuvant therapeutic agents in epileptics refractory to conventional antiepileptic medication.     

Augmented behavioral response and enhanced synaptosomal calcium transport induced by repeated cocaine administration are decreased by calcium channel blockers

Recent studies suggest that calcium influx via L-type calcium channels is necessary for psychostimulant-induced behavioral sensitization. In addition, chronic amphetamine upregulates subtype Cav1.2-containing L-type calcium channels. In the present studies, we assessed the effect of calcium channel blockers (CCBs) on cocaine-induced behavioral sensitization and determined whether the functional activity of L-type calcium channels is altered after repeated cocaine administration. Rats were administered daily intraperitoneal injections of either flunarizine (40 mg/kg), diltiazem (40 mg/kg) or cocaine (20 mg/kg) and the combination of the CCBs and cocaine for 30 days. Motor activities were monitored on Day 1, and every 6th day during the 30-day treatment period. Daily cocaine administration produced increased locomotor activity. Maximal augmentation of behavioral response to repeated cocaine administration was observed on Day 18. Flunarizine pretreatment abolished the augmented behavioral response to repeated cocaine administration while diltiazem was less effective. Measurement of tissue monoamine levels on Day 18 revealed cocaine-induced increases in DA and 5-HT in the nucleus accumbens. By contrast to behavioral response, diltiazem was more effective in attenuating increases in monoamine levels than flunarizine. Cocaine administration for 18 days produced increases in calcium uptake in synaptosomes prepared from the nucleus accumbens and frontal cortex. Increases in calcium uptake were abolished by flunarizine and diltiazem pretreatment. Taken together, the augmented cocaine-induced behavioral response on Day 18 may be due to increased calcium uptake in the nucleus accumbens leading to increased dopamine (DA) and serotonin (5-HT) release. Flunarizine and diltiazem attenuated the behavioral response by decreasing calcium uptake and decreasing neurochemical release.     

Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and methyl-salicylate and their relation with calcium signalling

Ca²⁺ plays a critical role as second messenger in the signal–response coupling of plant defence responses, and methyl-jasmonate and methyl-salicylate are important components of signal transduction cascades activating plant defences. When intact axenic non-induced seedling roots of sunflower were treated with different Ca²⁺ concentrations up to 1 mM, there was no significant increase in O ₂ ^.? generation or DMAB–MBTH peroxidase (extracellular, ECPOX) activities in the apoplast, probably because these roots had enough Ca²⁺ in their exo- and endocellular reservoirs. Both activities were strongly inhibited by the RBOH–NADPH oxidase inhibitor DPI and by the Ca²⁺ surrogate antagonist La³⁺, but the voltage-dependent Ca²⁺ channel blocker verapamil was only inhibitory at concentrations higher than those active on animal L-type Ca²⁺ channels. Concentrations >5 mM EGTA (chelating Ca²⁺ in the apoplast) and Li⁺ (inhibiting PI cycle dependent endogenous Ca²⁺ fluxes) also inhibited both activities. W7, inhibitor of binding of Ca–CaM to its target protein, enhanced both activities, but the inactive analogue W5 showed a similar effect. Our data suggest that Ca²⁺ from exocellular and, to a lesser extent, from endocellular stores is involved in oxidative activities, and that RBOH–NADPH oxidase is the main system supporting them. Ca²⁺ activation of the PM cytosolic side of RBOH–NADPH oxidase is probably the key to Ca²⁺ involvement in these processes. Roots induced by MeJA or MeSA showed significant enhancement of both oxidative activities, as corresponding to the oxidative burst evoked by the two phytohormones in the root apoplast. But while ECPOX activity showed a response to the effectors similar to that described above for non-induced roots, O ₂ ^.? generation activity in the apoplast of induced roots was insensitive to EGTA, verapamil and Li⁺, the inhibitors of exogenous and endogenous Ca²⁺ fluxes; only DPI and La³⁺ were inhibitory. As exogenously added 0.1 mM Ca²⁺ also increased O ₂ ^.? generation, we propose that, in these roots, activation of RBOH–NADPH oxidase by Ca²⁺ could be regulated by Ca²⁺ sensors in the apoplast.     

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.Abbreviations 9-HFCA 9-hydroxyfluorenecarboxylic acid - NPA naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - IAA indole-3-acetic acid     

T-type calcium channel blockers - new and notable

Since cloning of the T-type or Ca(V)3.n calcium channel family in 1998-1999 much progress was made in investigation of their regulation. Most effective metal Ca(V)3 channel blockers are trivalent cations from lanthanide group together with transition metals La(3+) and Y(3+). Divalent cations Zn(2+), Cu(2+) and Ni(2+) inhibit Ca(V)3.2 channels more efficiently than Ca(V)3.1 and Ca(V)3.3 channels via second high-affinity binding site including histidine H191 specific for the Ca(V)3.2 channel. Dihydropyridines and phenylalkylamines in addition to block of L-type calcium channel can inhibit Ca(V)3 channels in clinically relevant concentration.     

Voltage-gated calcium channel blockers deregulate macroautophagy in cardiomyocytes

Voltage-gated calcium channel blockers are widely used for the management of cardiovascular diseases, however little is known about their effects on cardiac cells in vitro.We challenged neonatal ventricular cardiomyocytes (CMs) with therapeutic L-type and T-type Ca²⁺ channel blockers (nifedipine and mibefradil, respectively), and measured their effects on cell stress and survival, using fluorescent microscopy, Q-PCR and Western blot. Both nifedipine and mibefradil induced a low-level and partially transient up-regulation of three key mediators of the Unfolded Protein Response (UPR), indicative of endoplasmic (ER) reticulum stress. Furthermore, nifedipine triggered the activation of macroautophagy, as evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), decreased levels of polyubiquitin-binding protein p62/SQSTM1 and ubiquitinated protein aggregates, that was followed by cell death. In contrast, mibefradil inhibited CMs constitutive macroautophagy and did not promote cell death. The siRNA-mediated gene silencing approach confirmed the pharmacological findings for T-type channels.We conclude that L-type and T-type Ca²⁺ channel blockers induce ER stress, which is divergently transduced into macroautophagy induction and inhibition, respectively, with relevance for cell viability. Our work identifies VGCCs as novel regulators of autophagy in the heart muscle and provides new insights into the effects of VGCC blockers on CMs homeostasis, that may underlie both noxious and cardioprotective effects.     

Dihydropyridines as potent calcium channel blockers in neuronal cells

Nicardipine, one of the dihydropyridine derivatives, in a nanomolar concentration range suppressed the high K+ -induced neurotransmitter release from cultured neuronal cells (chick embryonic neural retina cells and clonal rat pheochromocytoma cells). The high K+ -induced Ca2+ uptake into pheochromocytoma cell was also blocked by nicardipine in the same concentration range. [3H]Nitrendipine, another dihydropyridine derivative, bound specifically to pheochromocytoma cell homogenate in a saturable manner. We concluded that dihydropyridines block and bind to the high K+ -sensitive Ca2+ channels in neuronal cells.     

Lead discovery and optimization of T-type calcium channel blockers

A series of compounds were designed as T-type calcium channel blocker containing 6 or 5 pharmacophore features from structure-based virtual screening. To optimize the suggested structure, over 130 derivatives were synthesized and their inhibitory activities on T-type calcium channel were assayed using in vitro screening system with alpha1(G) and alpha1(H) clones. For the compounds with higher activities in FDSS assay system, the efficacy was measured by patch-clamp method. Among the library with 5 features, alkaneamide derivatives (7b, 9j, 11b, 11g, 11h) with 4-arylsubstituted piperazine showed better IC(50) values than Mibefradil.     

The effect of calcium channel blockers on blood platelet function, especially calcium uptake

The effects of organic and inorganic calcium antagonists on washed platelets from rat and human have been studied. Platelet aggregation was assessed by turbidimetry. Endogenous serotonin release was measured on the same sample by means of electrochemically treated carbon fiber electrodes. The organic calcium antagonist, nitrendipine, and the inorganic calcium channel blockers (Co2+, Mn2+, Cd2+, La3+) drastically inhibited rat and human platelet aggregation induced by thrombin, ADP or adrenaline in the presence of 0.32 mM Ca2+. In our conditions, the thrombin-induced release of endogenous serotonin was found to be external Ca2+-dependent and completely inhibited by 20 microM nitrendipine or 1 mM Cd2+. In addition, Ba2+ or Sr2+ ions can be substituted for Ca2+ to bring about platelet aggregation as well as endogenous serotonin secretion. In Ba2+ or Sr2+-containing media, rat platelet aggregation and/or serotonin secretion can be inhibited by either nitrendipine or Cd2+. Finally, we have also studied the thrombin- and external Ca2+-dependence of radiolabeled calcium uptake by rat platelets. We found that the thrombin-induced 45Ca uptake was inhibited by either 18 microM nitrendipine or 1 mM Cd2+. These results provide strong evidence for the existence of an influx of divalent cations (Ca2+, Sr2+, Ba2+) triggering platelet function. They also suggest, although they do not prove, that the translocation of these cations occurs through an agonist-operated channel as proposed by Hallam and Rink (FEBS Lett. 186 (1986) 175-179).     

Growth inhibition of human cancer cells in vitro by T-type calcium channel blockers

This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equipotent with Cisplatin against some human cancers in the micromolar range.     

Phototropism and gravitropism in lateral roots of Arabidopsis

Gravitropism and, to a lesser extent, phototropism have been characterized in primary roots, but little is known about structural/functional aspects of these tropisms in lateral roots. Therefore, in this study, we report on tropistic responses in lateral roots of Arabidopsis thaliana. Lateral roots initially are plagiogravitropic, but when they reach a length of approximately 10 mm, these roots grow downward and exhibit positive orthogravitropism. Light and electron microscopic studies demonstrate a correlation between positive gravitropism and development of columella cells with large, sedimented amyloplasts in wild-type plants. Lateral roots display negative phototropism in response to white and blue light and positive phototropism in response to red light. As is the case with primary roots, the photoresponse is weak relative to the graviresponse, but phototropism is readily apparent in starchless mutant plants, which are impaired in gravitropism. To our knowledge, this is the first report of phototropism of lateral roots in any plant species.     

Growth and gravitropism of corn roots in solution

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m⁻³ Ca²⁺. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.     

Potentiation of analgesia and reversal of tolerance to morphine by calcium channel blockers

Effect of four calcium channel blockers (CCBs) belonging to different chemical classes, alone and in combination with morphine was investigated on two models of pain sensitivity, i.e. formalin and tail flick tests in mice. All the studied CCBs, i.e. diltiazem, flunarizine, nimodipine and verapamil inhibited formalin-induced pain responses; however, with verapamil, though there was a trend towards a reduction of paw-licking response to formalin, it was not found to be statistically significant. In contrast, none of the CCBs affected the tail flick latency at any of the doses studied. Morphine, a mu-receptor agonist exerted a significant analgesic effect in formalin as well in tail flick tests. Pretreatment with all CCBs significantly enhanced the analgesic effect of morphine in both tests of nociception. Further, concomitant administration of one of the CCBs, diltiazem with morphine prevented the development of tolerance to the latter. However, combination of diltiazem with morphine, like morphine alone was found to be ineffective in morphine tolerant animals. Results, thus, show that CCBs produced an analgesic effect of their own in formalin-induced tonic pain and potentiated the analgesic activity of morphine. They also modulated opioid-induced tolerance.