How modeling can reconcile apparently discrepant experimental results: The case of pacemaking in dopaminergic neurons

Midbrain dopaminergic neurons are endowed with endogenous slow pacemaking properties. In recent years, many different groups have studied the basis for this phenomenon, often with conflicting conclusions. In particular, the role of a slowly-inactivating L-type calcium channel in the depolarizing phase between spikes is controversial, and the analysis of slow oscillatory potential (SOP) recordings during the blockade of sodium channels has led to conflicting conclusions. Based on a minimal model of a dopaminergic neuron, our analysis suggests that the same experimental protocol may lead to drastically different observations in almost identical neurons. For example, complete L-type calcium channel blockade eliminates spontaneous firing or has almost no effect in two neurons differing by less than 1% in their maximal sodium conductance. The same prediction can be reproduced in a state of the art detailed model of a dopaminergic neuron. Some of these predictions are confirmed experimentally using single-cell recordings in brain slices. Our minimal model exhibits SOPs when sodium channels are blocked, these SOPs being uncorrelated with the spiking activity, as has been shown experimentally. We also show that block of a specific conductance (in this case, the SK conductance) can have a different effect on these two oscillatory behaviors (pacemaking and SOPs), despite the fact that they have the same initiating mechanism. These results highlight the fact that computational approaches, besides their well known confirmatory and predictive interests in neurophysiology, may also be useful to resolve apparent discrepancies between experimental results. © 2011 Drion et al.

吲哚生物碱Desbromoarborescidine A 的全合成及烯胺C-亚磺酰化反应研究

从新几内亚核桃木的树皮中分离得到的吲哚类喹诺里西定生物碱10-Desbromoarborescidine A，因发现其具有阻滞钙离子通道的活性而倍受关注。10-Desbromoarborescidine A由A、B、C、D四个环组成，只有一个手性中心，是吲哚生物碱中结构较简单的一种，常作为此类生物碱全合成方法的模型化合物。但迄今为止，能高效而简便的实现手性10-Desbromoarborescidine A不对称全合成方法线路不多，大多数以不对称诱导的方式建立其手性中心，手性催化的方式仅有一例金属催化。从逆合成分析可知，Desbromoarborescidine A的全合成可以通过亚胺不对称催化还原进行关键的手性中心构建，而本课题组在之前的研究中通过手性有机小分子催化剂的发展，已将三氯硅烷氢转移还原亚胺发展成了一类简便实用、高效、高对映选择性并具有优良底物适应范围的不对称催化反应，我们希望以这一反应作为关键手段，发展一条Desbromoarborescidine A及其类似物不对称合成新路线。 根据我们设计的新路线，首先成功合成了其关键中间体，然后我们进行了关键的不对称催化尝试。用本实验室已有的高性能有机小分子催化剂虽得到了较好的对应选择性，但是产率很低。同时，为了验证整条线路的可行性，我们也用消旋的中间体进行拉通线路的尝试。但不幸的是，在脱除保护基时遇到了很大困难。尝试换不同的保护基，或改变脱保护基的顺序，都未能成功合成目标产物。究其原因可能是由于吲哚的特殊性造成的，吲哚类亚胺与常规的芳香亚胺有较大的差异，其NH基团无论保护还是不保护，对与其2位相联接的C=N双键均有很大的影响，导致其不对称催化还原难以进行。另外，由于所设计的还原产物含有处在吲哚苄位的胺基，稳定性较差，造成保护基脱除困难。 烯胺C-亚磺酰化反应是本课题组最近发现的一个新反应，之前未见文献报道。本研究对该反应进行了反应条件优化和底物扩展，发现带Cbz，Ac，COt-Bu，CO2Et，Bz等保护基的一系列环状和非环状烯胺在亚磺酸钠、DMAc和MeSiCl3的共同作用下能高效高产率生成β-胺基烯基亚砜类新化合物，为合成多官能团化的烯基亚砜新化合物提供了一条简便实用的途径。 The main constituent of Dracontomelum mangiferum B1, indoloquinolizidine alkaloid 10-Desbromoarborescidine A, has drawn great attention due to its calcium channel blocking activity. Its molecular structure is relatively simple compared with the other alkaloids of the same type, which has only one chiral center, albeit with four cycles A, B, C, and D. This compound is often used as a model target for exploring different strategies for the total synthesis of indole alkaloids. Nevertheless, so far there still lack practical and highly efficient methods for the asymmetric total synthesis of 10-Desbromoarborescidine A. Most of the current available methods rely on stoichiometric asymmetric synthesis for the construction of the chiral center. There is only one example reporting utilization of asymmetric catalysis, but with transition metal complex as the catalyst. Our retrosynthetic analysis shows that catalytic asymmetric reduction of imine could be used as the key step for the construction of the chiral center of Desbromoarborescidine A. Since in the previous studies our group has developed the asymmetric reduction of imines by trichlorosilane into a practical and highly efficient and enantioselective method using newly designed chiral organocatalysts, we hope to apply this method to develop a novel synthetic route for the total synthesis of Desbromoarborescidine A and its analogues in this study. According to the newly designed synthetic route, we first accomplished the synthesis of the key intermediates which was then examined for the critical asymmetric catalysis. The asymmetric reduction using the highly efficient organocatalysts, developed in our lab afforded high ee but poor yield. We tried different reaction conditions to improve the yield, but failed to get any good results. Simultaneously, to vertify the feasibility of the synthetic route we designed, we also tired to go through the route toward the racemic synthesis of Desbromoarborescidine A. But unfortunately, protection and deprotection proved to be big hurdles. All the different protection groups and different sequences of protection and deprotection we tried failed to get us through the designed synthetic sequence and furnish the final product. Most likely, the indole part is the culprit behind the failures.The NH group of the indole, no matter protected or not, may impact the catalytic asymmetric reduction of C-N double bond connected with 2-C. Additionally, the reduction product we designed contains an amino group in the β-position of the indole, which may cause problems due to its instability. C-sulfenylation of enamines is a novel reaction discovered recently by our group, which has not been seen before in the literature. In this study, optimization of the reaction conditions and exploration of the substrate scope were further undertaken for this reaction, which reveal that a series of enamines with N-Cbz, Ac, COt-Bu, CO2Et protection groups could all undergo smooth C-sulfinylations with the comined use of sodium benzene sulphinate, DAMc and MeSiCl3, efficiently furnishing the β-amino vinylsulfoxide products in high yield, affording a practical and highly efficient methods for synthesis of functional vinylsulfoxides.

Study on the GABA gated channels in the neurosecretory cells of MTXO in the eyestalks of Eriocheir sinensis

The responses to rapid application of gamma-aminobutyric acid (GABA) and the GABA receptor characteristics of MTXO neurosecretory cells in the eyestalks of Chinese mitten-handed crab (Eriocheir sinensis) were examined by whole-cell patch clamp. Under current clamp mode, the depolarization and hyperpolarization were evoked from the three types of neurosecretory cells in response to the GABA (0.1 mmol/L) depending on the Nernst Cl- potential. Under voltage clamp mode, the inward Cl- channel currents (I-GABA) were resolved from all three types of neurosecretory cells in response to GABA (0.01similar to5 mmol/L). The GABA currents were activated within 1 200 ms and peaked within 800 ms. No obviously desensitization was observed during GABA application. The dose-response curve showed usual S-shape, with a just-discernible effect at 0.01 mmol/L and near-saturation at 0.5 mmol/L. The GABA currents had reversal potentials that followed Nernst Cl- potentials when [Cl-] was varied. The pharmacological results revealed that the GABA receptor of the crab neurosecretory cells was sensitive to the Cl- channel blockers picrotoxin and niflumic acid (0.5 mmol/L), insensitive to GABA(A) receptor antagonist bicuculline and GABA(C) receptor agonist cis-4-aminocrotonic acid (CACA 1 mmol/L) and trans-4-aminocrotonic (TACA 1 mmol/L).

INVOLVEMENT OF CA(2+) SIGNALING PATHWAY DURING OOCYTE MATURATION OF THE NORTHERN QUAHOG MERCENARIA MERCENARIA

In many molluses, it has been found that Ca2+ signaling pathway is involved in the resumption of meiotic maturation in oocytes. To better understand the possible role of Ca2+ signaling pathway in regulating meiotic maturation in oocytes of the northern quahog Mercenaria mercenaria, free extracellular Ca2+, A23187 (calcium ionophore), verapamil (calcium channel blocker), and trifluoperazin (calmodulin antagonist) were used to incubate oocytes or serotonin-induced oocytes by pharmacological methods. Results show that extracellular Ca2+ (50 similar to 200 mM) and A23187 (1 similar to 10 mu M) can stimulate the meiotic maturation. In addition, verapamil (1 similar to 100 mu M) and trifluoperazin (10 similar to 1,000 mu M) could inhibit serotonin-induced oocyte maturation. Therefore, Ca2+ is essential for the reinitiation of meiotic maturation in oocytes of the northern quahog. Moreover, an increase i [Ca2+]i can promote meiotic maturation.

Reversible inactivation of macaque frontal eye field.

The macaque frontal eye field (FEF) is involved in the generation of saccadic eye movements and fixations. To better understand the role of the FEF, we reversibly inactivated a portion of it while a monkey made saccades and fixations in response to visual stimuli. Lidocaine was infused into a FEF and neural inactivation was monitored with a nearby microelectrode. We used two saccadic tasks. In the delay task, a target was presented and then extinguished, but the monkey was not allowed to make a saccade to its location until a cue to move was given. In the step task, the monkey was allowed to look at a target as soon as it appeared. During FEF inactivation, monkeys were severely impaired at making saccades to locations of extinguished contralateral targets in the delay task. They were similarly impaired at making saccades to locations of contralateral targets in the step task if the target was flashed for < or =100 ms, such that it was gone before the saccade was initiated. Deficits included increases in saccadic latency, increases in saccadic error, and increases in the frequency of trials in which a saccade was not made. We varied the initial fixation location and found that the impairment specifically affected contraversive saccades rather than affecting all saccades made into head-centered contralateral space. Monkeys were impaired only slightly at making saccades to contralateral targets in the step task if the target duration was 1000 ms, such that the target was present during the saccade: latency increased, but increases in saccadic error were mild and increases in the frequency of trials in which a saccade was not made were insignificant. During FEF inactivation there usually was a direct correlation between the latency and the error of saccades made in response to contralateral targets. In the delay task, FEF inactivation increased the frequency of making premature saccades to ipsilateral targets. FEF inactivation had inconsistent and mild effects on saccadic peak velocity. FEF inactivation caused impairments in the ability to fixate lights steadily in contralateral space. FEF inactivation always caused an ipsiversive deviation of the eyes in darkness. In summary, our results suggest that the FEF plays major roles in (1) generating contraversive saccades to locations of extinguished or flashed targets, (2) maintaining contralateral fixations, and (3) suppressing inappropriate ipsiversive saccades.

The effects of blood pressure lowering on development of cognitive impairment and dementia in patients without apparent prior cerebrovascular disease

BACKGROUND: Hypertension and cognitive impairment are prevalent in older people. It is known that hypertension is a direct risk factor for vascular dementia and recent studies have suggested hypertension also impacts upon prevalence of Alzheimer's disease. The question is therefore whether treatment of hypertension lowers the rate of cognitive decline. OBJECTIVES: To assess the effects of blood pressure lowering treatments for the prevention of dementia and cognitive decline in patients with hypertension but no history of cerebrovascular disease. SEARCH STRATEGY: The trials were identified through a search of CDCIG's Specialised Register, CENTRAL, MEDLINE, EMBASE, PsycINFO and CINAHL on 27 April 2005. SELECTION CRITERIA: Randomized, double-blind, placebo controlled trials in which pharmacological or non-pharmacological interventions to lower blood pressure were given for at least six months. DATA COLLECTION AND ANALYSIS: Two independent reviewers assessed trial quality and extracted data. The following outcomes were assessed: incidence of dementia, cognitive change from baseline, blood pressure level, incidence and severity of side effects and quality of life. MAIN RESULTS: Three trials including 12,091 hypertensive subjects were identified. Average age was 72.8 years. Participants were recruited from industrialised countries. Mean blood pressure at entry across the studies was 170/84 mmHg. All trials instituted a stepped care approach to hypertension treatment, starting with a calcium-channel blocker, a diuretic or an angiotensin receptor blocker. The combined result of the three trials reporting incidence of dementia indicated no significant difference between treatment and placebo (Odds Ratio (OR) = 0.89, 95% CI 0.69, 1.16). Blood pressure reduction resulted in a 11% relative risk reduction of dementia in patients with no prior cerebrovascular disease but this effect was not statistically significant (p = 0.38) and there was considerable heterogeneity between the trials. The combined results from the two trials reporting change in Mini Mental State Examination (MMSE) did not indicate a benefit from treatment (Weighted Mean Difference (WMD) = 0.10, 95% CI -0.03, 0.23). Both systolic and diastolic blood pressure levels were reduced significantly in the two trials assessing this outcome (WMD = -7.53, 95% CI -8.28, -6.77 for systolic blood pressure, WMD = -3.87, 95% CI -4.25, -3.50 for diastolic blood pressure).Two trials reported adverse effects requiring discontinuation of treatment and the combined results indicated a significant benefit from placebo (OR = 1.18, 95% CI 1.06, 1.30). When analysed separately, however, more patients on placebo in SCOPE were likely to discontinue treatment due to side effects; the converse was true in SHEP 1991. Quality of life data could not be analysed in the three studies. There was difficulty with the control group in this review as many of the control subjects received antihypertensive treatment because their blood pressures exceeded pre-set values. In most cases the study became a comparison between the study drug against a usual antihypertensive regimen. AUTHORS' CONCLUSIONS: There was no convincing evidence from the trials identified that blood pressure lowering prevents the development of dementia or cognitive impairment in hypertensive patients with no apparent prior cerebrovascular disease. There were significant problems identified with analysing the data, however, due to the number of patients lost to follow-up and the number of placebo patients given active treatment. This introduced bias. More robust results may be obtained by analysing one year data to reduce differential drop-out or by conducting a meta-analysis using individual patient data.

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Pharmacological characterisation of neuropeptide F (NPF)-induced effects on the motility of Mesocestoides corti (syn. Mesocestoides vogae) larvae

Neuropeptide F is the most abundant neuropeptide in parasitic flatworms and is analogous to vertebrate neuropeptide Y. This paper examines the effects of neuropeptide F on tetrathyridia of the cestode Mesocestoides vogae and provides preliminary data on the signalling mechanisms employed. Neuropeptide F ( greater than or equal to 10 muM) had profound excitatory effects on larval motility in vitro. The effects were insensitive to high concentrations (I mM) of the anaesthetic procame hydrochloride suggesting extraneuronal sites of action. Neuropeptide F activity was not significantly blocked by a FMRFamide-related peptide analog (GNFFRdFamide) that was found to inhibit GNFFRFamide-induced excitation indicating the occurrence of distinct neuropeptide F and FMRFamide-related peptide receptors. Larval treatment with guanosine 5'-O-(2-thiodiphosphate) trilithium salt prior to the addition of neuropeptide F completely abolished the excitatory effects indicating the involvement of G-proteins and a G-protein coupled receptor in neuropeptide F activity. Addition of guanosine 5'-O-(2-thiodiphosphate) following neuropeptide F had limited inhibitory effects consistent with the activation of a signalling cascade by the neuropeptide. With respect to Ca2+ involvement in neuropeptide F-induced excitation of M. vogae larvae, the L-type Ca2+-channel blockers verapamil and nifedipine both abolished neuropeptide F activity as did high Mg+ concentrations and drugs which blocked sarcoplasmic reticulum Ca2+-activated Ca2+-channels (ryanodine) and sarcoplasmic reticulum Ca2+ pumps (cyclopiazonic acid). Therefore, both extracellular and intracellular Ca2+ is important for neuropeptide F excitation in M. vogae. With resepct to second messengers, the protein kinase C inhibitor chelerythrine chloride and the adenylate cyclase inhibitor MDL-2330A both abolished neuropeptide F-induced excitation. The involvement of a signalling pathway that involves protein kinase C was further supported by the fact that phorbol-12-myristate-13-acetate,known to directly activate protein kinase C, had direct excitatory effects on larval motility. Although neuropeptide F is structurally analogous to neuropeptide Y, its mode-of-action in flatworms appears quite distinct from the common signalling mechanism seen in vertebrates. (C) 2003 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Feedback via Ca²⁺-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle

PURPOSE: To investigate the role of feedback by Ca²?-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²?](i) and the amplitude and frequency of Ca²?-waves in retinal arterioles. The Ca²?-activated Cl?-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²?-activated K?-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²?](i). The voltage-operated Ca²?-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²?](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²?-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²?, also require positive feedback via Ca²?-activated Cl?-channels and L-type Ca²?-channels.

PLATYHELMINTH FMRFAMIDE-RELATED PEPTIDES (FARPS) CONTRACT SCHISTOSOMA-MANSONI (TREMATODA, DIGENEA) MUSCLE-FIBERS IN-VITRO

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10(-9) and 10(-7) M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10(-8)-10(-6) M and 10(-7)-10(-5) M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 mu M FMR-D-Famide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0.5 mu M EGTA. was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high Kf-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

The Role of K+ and Cl- Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow.

Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1).

Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10µl) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1µM) and iberiotoxin (4µM), and the IKv-inhibitor correolide (40µM) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide.

Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

An efficacy and mechanism evaluation study of Levosimendan for the Prevention of Acute oRgan Dysfunction in Sepsis (LeoPARDS): Protocol for a randomized controlled trial

Background

Organ dysfunction consequent to infection (‘severe sepsis’) is the leading cause of admission to an intensive care unit (ICU). In both animal models and early clinical studies the calcium channel sensitizer levosimendan has been demonstrated to have potentially beneficial effects on organ function. The aims of the Levosimendan for the Prevention of Acute oRgan Dysfunction in Sepsis (LeoPARDS) trial are to identify whether a 24-hour infusion of levosimendan will improve organ dysfunction in adults who have septic shock and to establish the safety profile of levosimendan in this group of patients.

This is a multicenter, randomized, double-blind, parallel group, placebo-controlled trial. Adults fulfilling the criteria for systemic inflammatory response syndrome due to infection, and requiring vasopressor therapy, will be eligible for inclusion in the trial. Within 24 hours of meeting these inclusion criteria, patients will be randomized in a 1:1 ratio stratified by the ICU to receive either levosimendan (0.05 to 0.2 μg.kg^-1.min^-1 or placebo for 24 hours in addition to standard care. The primary outcome measure is the mean Sequential Organ Failure Assessment (SOFA) score while in the ICU. Secondary outcomes include: central venous oxygen saturations and cardiac output; incidence and severity of renal failure using the Acute Kidney Injury Network criteria; duration of renal replacement therapy; serum bilirubin; time to liberation from mechanical ventilation; 28-day, hospital, 3 and 6 month survival; ICU and hospital length-of-stay; and days free from catecholamine therapy. Blood and urine samples will be collected on the day of inclusion, at 24 hours, and on days 4 and 6 post-inclusion for investigation of the mechanisms by which levosimendan might improve organ function. Eighty patients will have additional blood samples taken to measure levels of levosimendan and its active metabolites OR-1896 and OR-1855. A total of 516 patients will be recruited from approximately 25 ICUs in the United Kingdom.

This trial will test the efficacy of levosimendan to reduce acute organ dysfunction in adult patients who have septic shock and evaluate its biological mechanisms of action.

Combination aerosol therapy to treat Burkholderia cepacia complex

Burkholderia cepacia infection in cystic fibrosis (CF) patients is associated with significant morbidity and mortality, yet no definitive treatment is currently available. This report describes a new approach to treat B. cepacia infection in CF patients, using a combination of amiloride and tobramycin aerosols. Four adults with the typical clinical syndrome of CF were recruited after repeated positive sputum cultures for B. cepacia. Aerosols of amiloride and tobramycin were given three times daily for 1-6 months, and repeated sputum cultures were collected to assess efficacy. Three of the four patients treated with the combined therapy eradicated B. cepacia from their sputum cultures for at least 2 yrs, and there were no adverse events. This novel combination may provide a new therapeutic option for Burkholderia cepacia infections. Furthermore, the strategy of combining antibiotics with ion transport agents may have ramifications for the treatment of other multi-resistant organisms.