Prominent contribution of L-type Ca2+ channels to cutaneous neurovascular transmission that is revealed after spinal cord injury augments vasoconstriction

In patients with spinal cord injury (SCI), somatosympathetic reflexes produce exaggerated decreases in skin blood flow below the lesion. This hypoperfusion appears to result from an increased responsiveness of cutaneous arterial vessels to neural activation. Here we investigated the mechanisms that underlie SCI-induced enhancement of neurovascular transmission in a cutaneous vessel, the rat tail artery. Isometric contractions of arterial segments from T11 spinal cord transected and sham-operated rats were compared 6 wk postoperatively. SCI more than doubled the amplitudes of contractions of arteries in response to moderate frequencies of nerve stimulation (0.1 to 1 Hz). In arteries from SCI rats, but not those from sham-operated rats, the L-type Ca(2+) channel blocker nifedipine (1 μM) reduced the amplitudes of nerve-evoked contractions. Furthermore, while the sensitivity to the agonists phenylephrine (α(1)-adrenoceptor selective) and clonidine (α(2)-adrenoceptor selective) did not differ significantly between arteries from SCI and sham-operated rats, nifedipine had a greater inhibitory effect on contractions to both agents in arteries from SCI rats. Although sensitivity to clonidine was unchanged, SCI selectively reduced the contribution of postjunctional α(2)-adenceptors to nerve-evoked contractions. In arteries from unoperated rats, the L-type channel agonist BAY K 8644 (0.1 μM) produced a similar enhancement of nerve-evoked contraction to that produced by SCI and also selectively reduced the contribution of α(2)-adrenceptors to these responses. Together the findings demonstrate that the SCI-induced enhancement of neurovascular transmission in the rat tail artery can largely be accounted for by an increased contribution of L-type Ca(2+) channels to activation of the vascular smooth muscle.     

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca<Superscript>2+</Superscript>-channels and ryanodine receptors

L-type Ca²⁺-channel blockers, verapamil (5 μM) and nifedipine (10 μM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca²⁺-channel activator BAY K 8644 (1 μM) has a decreased mediator secretion level. Nifedipine (10 μM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 μM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 μM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 μM).     

Bay K8644及nifedipine对大鼠下丘脑神经元L—型Ca^2＋通道特性的影响

采用神经元急性分离和膜片箍技术以及细胞贴附式方式记录通道活动,探讨DHP类Ca^2 通道激动剂Bay K8644及拮抗剂nifedipine对下丘脑神经元L－型Ca^2 通道的影响,结果显示,在Bay K8644作用下,通道开放形式发生变化,明显可见多级开放;通道平均开放时间,平均开放概况显著增加,但单通道电导无明显变化。nifedipine的作用与Bay K8644相反。结果提示,Bay K8644对下丘脑神经元L－型Ca^2 通道有明显激动作用 nifedipine有显著抑制作用。     

A role for Ca(v)1.3 in rat intestinal calcium absorption

Active Ca²⁺ absorption through epithelial Ca²⁺ channels TRPV5/6 in duodenum is activated by hyperpolarisation. However, when diet and Ca²⁺ are plentiful, digestion products cause depolarisation. We therefore used homology-based PCR from a rat jejunal mucosal cDNA preparation to reveal the presence of the neuroendocrine L-type isoform Ca_v1.3α₁. Immunocytochemical labelling and immunoblotting localised Ca_v1.3α₁ protein in apical membrane from proximal jejunum to mid ileum. Perfusion studies in vivo with 1.25 mM luminal Ca²⁺ revealed L-type channel activity. Inhibition of glucose absorption with phloridzin strongly inhibited ⁴⁵Ca²⁺ absorption; absorption was inhibited by nifedipine and Mg²⁺ and activated by Bay K 8644, none of which affect TRPV5/6. At 10 mM Ca²⁺, nifedipine inhibited ⁴⁵Ca²⁺ absorption with a time course similar to that at 1.25 mM Ca²⁺: absorption was therefore channel-mediated rather than paracellular. We suggest that in times of dietary sufficiency, Ca_v1.3 may mediate a significant route of Ca²⁺ absorption into the body.     

T－2毒素对心肌细胞三型钙通道的阻滞作用

用膜片钳连细胞电压钳法, 在培养的Wistar大鼠单个心肌细胞上记录了T-2毒素对B、L和T三型Ca²⁺通道单通道电活动的影响. 结果表明, T-2毒素浓度为10mg/L时, 心肌细胞B、L和T三型Ca²⁺通道均受到明显的阻滞, 其阻滞作用表现为使Ca²⁺通道的开放概率减小, 开放时间缩短, 关闭时间延长, 而对流过Ca²⁺通道的Ba²⁺流幅值无影响.     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998     

Identification of a sphingosine-sensitive Ca2+ channel in the plasma membrane of Leishmania mexicana

The disruption of the intracellular Ca²⁺ homeostasis of Leishmania mexicana represents a major target for the action of drugs, such as amiodarone and miltefosine. However, little is known about the mechanism of Ca²⁺ entry to these cells. Here we show the presence of a Ca²⁺ channel in the plasma membrane of these parasites. This channel has many characteristics similar to the human L-type voltage-gated Ca²⁺ channel. Thus, Ca²⁺ entry is blocked by verapamil, nifedipine and diltiazem while Bay K 8644 opened this channel. However, different to its human counterpart, sphingosine was able to open this channel, while other well known sphingolipids had no effect. This fact could have important pharmacological implications.     

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca²⁺ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100 μM). In the presence of Bay K8644 (100 nM), magnolol (10–100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and N_ώ-nitro-l-arginine methyl ester (l-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3–100 μM) inhibited the L-type Ca²⁺ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca²⁺ channel activity.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

The calcium agonist Bay K 8644 stimulates secretion from a pituitary cell line

The dihydropyridine calcium agonist Bay K 8644 acts in a dose-dependent manner to increase prolactin secretion from the GH₄C₁ pituitary cell line. Enhanced secretion was observed at agonist concentrations as low as 10 nM. In the continued presence of Bay K 8644 secretion remained elevated for at least 30 min. The effect of the agonist was Ca²⁺-dependent and competitively antagonized by dihydropyridine antagonists. Apparently Bay K 8644 acts at the dihydropyridine binding site associated with GH₄C₁ Ca²⁺ channels to enhance Ca²⁺ influx and stimulate secretion from these cells. This is the first report demonstrating that the newly discovered Ca²⁺ agonist can, by itself, stimulate secretion from a cell.     

Morphine Activates ω-Conotoxin-Sensitive Ca2+ Channels to Release Adenosine from Spinal Cord Synaptosomes

Abstract: Morphine-induced release of adenosine from the spinal cord is believed to contribute to spinal antinociception. Although this release is Ca²⁺ dependent, little is known of the nature of this dependence. In this study, the effects of the dihydropyridine L-type Ca²⁺ channel agonist Bay K 8644 and the antagonist nifedipine, the N-type Ca²⁺ channel antagonist ω-conotoxin, and ruthenium red, a blocker of Ca²⁺ influx induced by capsaicin, on release of adenosine evoked by morphine were determined. The effect of partial depolarization with a minimally effective concentration of K⁺ on morphine-evoked release of adenosine also was examined. Morphine 10^?5-10^?4M produced a dose-dependent enhancement of adenosine release from dorsal spinal cord synaptosomes. Following the addition of 6 mM K⁺ (total K⁺ concentration of 10.7 mM), 10^?6M morphine also enhanced release, and an additional component of action at 10^?8M was revealed. Release was Ca²⁺-dependent as it was not observed in the absence of Ca²⁺ and presence of EGTA. Bay K 8644 (10 nM) and nifedipine (100 nM) had no effect on the release of adenosine evoked by morphine, but ω-conotoxin (100 nM) markedly reduced such release in both the absence and the presence of the additional 6 mM K⁺. Morphine-evoked adenosine release was not altered in the presence of a partially effective dose of capsaicin, nor by ruthenium red. These results indicate that morphine can stimulate two distinct phases of adenosine release from the spinal cord (nanomolar and micromolar), and that both phases of release are due to Ca²⁺ entry via ω-conotoxin-sensitive N-type Ca²⁺ channels.     

Calciseptine, a Ca2+ Channel Blocker, Has Agonist Actions on L-type Ca2+ Currents of Frog and Mammalian Skeletal Muscle

Calciseptine is a natural peptide consisting of 60 amino acids with four disulfide bonds. The peptide is a natural L-type Ca²⁺-channel blocker in heart and other systems, but its actions in skeletal muscle have not been previously described. The aim of this study is to characterize the effects of calciseptine on L-type Ca²⁺ channels of skeletal muscle and on contraction. Whole-cell, patch-clamp experiments were performed to record Ca²⁺ currents (I _Ca) from mouse myotubes, whereas Vaseline-gap voltage-clamp experiments were carried out to record I _Ca from frog skeletal muscle fibers. We found that calciseptine acts as a channel agonist in skeletal muscle, increasing peak I _Ca by 37% and 49% in these two preparations. Likewise, the peptide increased intramembrane charge movement, though it had little effect on contraction. The molecular analysis of the peptide indicated the presence of a local, electrostatic potential that resembles that of the 1,4-dihydropyridine agonist Bay K 8644. These observations suggest that calciseptine shares the properties of 1,4-dihydropyridine derivatives in modulating the permeation of divalent cations through L-type channels. Received: 18 December 2000/Revised: 16 July 2001     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Increase of atrial ANP release by 2,3-butanedione monoxime in beating rabbit atria

2,3-Butanedione monoxime (BDM) is a chemical phosphatase and has been known to dissociate mechanical contraction in the excitation–contraction coupling via inhibition of myofibrillar ATPase. BDM has also been found to decrease sarcolemmal L-type Ca²⁺ channel activity and intracellular Ca²⁺ in cardiac myocytes. It has been shown that Ca²⁺ entry via L-type Ca²⁺ channels decreased atrial myocyte atrial natriuretic peptide (ANP) release. The purpose of the present study was to address the effects of BDM in the regulation of ANP release. Experiments were performed in perfused beating rabbit atria. BDM accentuated atrial myocyte ANP release concomitantly with a decrease in atrial stroke volume and pulse pressure in a concentration-dependent manner. The BDM-induced activation of ANP release was attenuated by the treatment with nifedipine, an inhibitor of L-type Ca²⁺ channels. BDM further decreased atrial stroke volume and pulse pressure in the presence of nifedipine. Blockade of function of the sarcoplasmic reticulum with thapsigargin plus ryanodine slightly but not significantly attenuated the BDM-induced activation of ANP release. These data show that BDM is a potent stimulator for the ANP release and also suggest that the mechanism by which BDM activates atrial myocyte ANP release is related to inhibition of the L-type Ca²⁺ channel activity. The present finding also suggests that the effects of ANP released may be considered in an occasion of uncoupling by BDM of the excitation–contraction coupling of cardiomyocytes.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

The ion Ca²⁺ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca²⁺ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca²⁺ channel blocker was used. In contrast, none of the Ca²⁺ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca²⁺ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [³H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca²⁺ channels in neurotransmitter release. Total high voltage Ca²⁺ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca²⁺ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.     

Bay K 8644 induce enhancement of K+ current in both single heart cell and smooth muscle cell

Summary The effects of the Ca²⁺ agonist Bay K 8644 on outward potassium currents have been studied in single ventricular cells of chick embryo and aortic single cells of rabbit using the whole-cell patch clamp technique. Bay K 8644 was found to increase l_K in both heart and aortic single cells. This effect of Bay K 8644 on both muscle was reversed by Mn²⁺ and blocked by 20 mM TEA. The Bay K 8644 potassium I/V curve of single heart cell had a N shape, which is Ca²⁺ dependent. These data strongly suggest that Bay K 8644 increases a g_K(ca) in both aortic and heart muscle.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

Calcium Entry in Toxoplasma gondii and Its Enhancing Effect of Invasion-linked Traits

During invasion and egress from their host cells, Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca²⁺) concentration. Our work with Toxoplasma gondii provides evidence for Ca²⁺ influx from the extracellular milieu leading to cytosolic Ca²⁺ increase and enhancement of virulence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn²⁺ and Ba²⁺ uptake do not support a canonical store-regulated Ca²⁺ entry mechanism. Ca²⁺ entry was blocked by the L-type Ca²⁺ channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca²⁺ and by the specific L-type Ca²⁺ channel agonist Bay K-8644. Our results demonstrate that Ca²⁺ entry is critical for parasite virulence. We propose a regulated Ca²⁺ entry mechanism activated by cytosolic Ca²⁺ that has an enhancing effect on invasion-linked traits.     

Suppressive effect of an activator of ATP-dependent potassium channels, flocalin, on electrical and contractile activities of smooth muscles of the guinea-pig ureter

In experiments on isolated segments or strips obtained from the guinea-pig ureter, we showed, using a sucrose-gap technique, that application of an activator of ATP-dependent potassium channels (K_ATP), (flocalin (PF-5), suppresses generation of action potentials (APs) by ureter smooth muscle cells (SMCs). Pre-incubation of the ureter preparations under study in Krebs solution containing 1 to 10 μM PF-5 results initially in a decrease in the frequency of oscillations preceding an AP plateau, shortening of this plateau, and, later on, complete inhibition of AP generation. In the presence of PF-5, spikes induced by hyperpotassium depolarization were also inhibited, while a tonic component of such depolarization underwent a mild decrease. The data obtained indicate that PF-5 modulates the entry of Ca²⁺ ions through L-type voltage-dependent channels in the SMC membrane. Shortening of the plateau and suppression of the spikes initiated by application of an activator of voltage-dependent L-type potassium channels, Bay K 8644, can be considered a confirmation of the modulatory influence of PF-5 on voltage-dependent L-type potassium channels. It seems possible that Bay K 8644 can be used under experimental conditions for initiation and long-lasting modulation of APs generated by the ureter SMC instead of corresponding neurotransmitters. We hypothesize that voltage-dependent entry of Ca²⁺ ions into SMCs depends significantly on the PF-5-induced activation of K_ATP channels of the ureter SMCs. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 403–409, September–December, 2005.