Biological Characteristics and Molecular Mechanisms of Fludioxonil Resistance in Fusarium graminearum in China.

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 µg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

[Effect of aerobic exercise and resistance exercise in improving non-alcoholic fatty liver disease: a randomized controlled trial].

Objective: To investigate the effect of dietary control combined with different exercise modes on plasma vaspin, irisin, and metabolic parameters in patients with non-alcoholic fatty liver disease (NAFLD) through a randomized open parallel-controlled study. Methods: The patients aged 30-65 years who visited Tianjin Third Central Hospital from January 2013 to December 2014 and were diagnosed with NAFLD by liver ultrasound and fat content determination were screening, and 474 patients were enrolled in this randomized controlled trial and divided into aerobic exercise group, resistance exercise group, and control group. All patients received dietary intervention. The three groups were compared in terms of biochemical parameters, fat content, NFS score, energy metabolic parameters, body composition index, and levels of vaspin and irisin at baseline and after 6 months of intervention. SPSS 19.0 was used for statistical analysis. The t-test, the Mann-Whitney U test, the chi-square test, and an analysis of variance were used for comparison between groups. The multiple imputation method was used for missing data, and the results were included in the intention-to-treat analysis. Results: There were no significant differences in age, sex, anthropometrical parameters, and biochemical parameters between the three groups at baseline. Compared with dietary control alone, aerobic exercise and resistance exercise helped to achieve significant reductions in waist circumference, diastolic pressure, percentage of body fat, volatile fatty acid, fasting blood glucose, homeostasis model assessment of insulin resistance, triglyceride, low-density lipoprotein cholesterol, free fatty acid, uric acid, alanine aminotransferase, and liver fat content after 6 months of intervention (P < 0.05). The aerobic exercise group had a significant increase in non-protein respiratory quotient and significant reductions in body mass index and aspartate aminotransferase after intervention, as well as a significant increase in resting energy expenditure and significant reductions in abdominal fat ratio and total cholesterol after 6 months of resistance exercise (P < 0.05). The aerobic exercise group and the resistance exercise group had a significant reduction in vaspin and a significant increase in irisin after intervention (P < 0.05), and the resistance exercise group had significantly greater changes in these two adipokines than the aerobic exercise group (P < 0.05). Conclusion: Exercise therapy is an effective method for the treatment of metabolism-associated diseases, and a combination of resistance and aerobic exercises is more reasonable and effective in clinical practice. As a relatively safe exercise mode, resistance exercise can also effectively improve the metabolic state of NAFLD patients.

A village-based multidisciplinary study on factors affecting the intensity of cystic echinococcosis in an endemic region of the Tibetan plateau, China.

SUMMARY: We investigated and quantified the factors which may affect the prevalence of cystic echinococcosis caused by Echinococcus granulosus in Rangtang County using a multidisciplinary approach. From a previously performed field survey, epidemiological data were linked with environmental data. Altitude and land surface temperature were extracted from remote-sensing images. Cumulative logistic regression models were used to identify and quantify the potential risk factors. The multiple regression models confirmed that yaks (χ 2 = 4·0447, P = 0·0443), dogs (χ 2 = 8·3455, P = 0·0039) and altitude (χ2 = 7·6223, P = 0·0058) were positively correlated with the prevalence of cystic echinococcosis, while land surface temperature may have a negative association. The findings showed that dogs and yaks play the most important role in the transmission of cystic echinococcosis, while altitude and land surface temperature may also be involved in the transmission.

[Progress of researches on infection with two species of Echinococcus causing human diseases in animal hosts and influencing factors].

Echinococcosis is a serious zoonotic parasitic disease caused by infections with larval Echinococcus. The life cycle of Echinococcus involves a variety of animal hosts, including hoofed animals and rodents as intermediate hosts and carnivores as definitive hosts. The transmission of human echinococcosis is closely associated with the life cycle of E. granulosus and E. multilocularis among animal hosts in nature. This review summarizes the recent advances in the prevalence and influencing factors of E. granulosus and E. multilocularis infections in animal hosts, so as to provide insights into precision control of echinococcosis.

[SWOT analysis of schistosomiasis elimination in Laos].

OBJECTIVE: To analyze the advantages, disadvantages, opportunities and challenges for schistosomiasis elimination in Laos, so as to propose the corresponding healthy policies and suggestions. METHODS: A SWOT analysis was performed to analyze the strength, weakness, opportunity and threat for the schistosomiasis elimination program in Laos, and the corresponding policy suggestions were proposed. RESULTS: The national schistosomiasis elimination program of Laos receives governmental emphases and great supports. A strategy based on mass drug administration was proposed and a sentinel site-bases surveillance system has been built for schistosomiasis elimination in Laos; however, there are several challenges for the national schistosomiasis elimination program in Laos, including insufficient financial supports, inadequate professional capability, weak schistosomiasis control awareness in community populations and difficulty in vector control. CONCLUSIONS: Persistent governmental leadership, increasing financial supports, strengthening professional team building and improving schistosomiasis control awareness in community populations are required to facilitate the progress towards schistosomiasis elimination in Laos.

Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in China.

OBJECTIVE: In China, Chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) were abandoned for the treatment of falciparum malaria 20 years ago due to resistance. Subsequent field studies showed a trend of declining CQ and SP resistance in the country. The main purpose of this study was to analyse the molecular markers of antimalarial resistance and thereby to assess the possibility of reintroduction of CQ or SP for falciparum malaria treatment. METHODS: Plasmodium falciparum field isolates were collected in 2006-2007 from Hainan and Yunnan provinces, China. Nested PCR-sequencing assays were applied to analyse the SNPs in four genes: P. falciparum chloroquine resistance transporter (pfcrt) gene, multi-drug resistance 1 (pfmdr1) gene, dihydrofolate reductase (dhfr) gene and dihydropteroate synthetase (dhps) gene. RESULTS: We found the widespread presence of point mutations in the dhfr and dhps genes which are associated with SP treatment failure. The molecular analyses also showed the fairly high prevalence of point mutation in the pfcrt gene which is linked to CQ resistance. CONCLUSION: The results of the present study indicate that CQ and SP should not be reintroduced for falciparum malaria treatment in the near future in China.

The relation between transmitter release and Ca2+ entry at the mouse motor nerve terminal: role of stochastic factors causing heterogeneity.

The relation between quantal transmitter release and presynaptic Ca2+/Ba2+ entry at the mouse neuromuscular junction was studied, making use of the finding that in the presence of Ba2+ trains of nerve stimuli or brief nerve terminal depolarizations elicit "tails" of raised miniature end-plate potential frequency (fm) that reflect entry of Ba2+ per pulse, and hence effectiveness of pulses in opening Ca2+/Ba2+ channels; at the same time these pulses elicit end-plate potentials. With nerve stimulation in the presence of Ba2+ and Ca2+ and modulation of release by raised Mg2+ or bekanamycin, slopes of log quantal content (m) vs log apparent Ba2+ entry per pulse were close to 4, which is the same as the Hill coefficient for Ba2+ cooperativity derived from other data. With depolarizing pulses of varied intensity, however, similar plots gave slopes close to 2, with Ba2+ alone or in a mixture of Ca2+ and Ba2+. Thus, the relation between transmitter release and Ca2+ (or Ba2+) entry apparently depends upon how entry is varied; varying the numbers of channels opened is not the same as varying ion entry per channel. A mathematical model was developed to examine the consequences of heterogeneity of local Ca2+ (or Ba2+) between release sites, arising because of stochastic variation of number and time course of Ca2+ channels opened per site; the experimental results were consistent with this model. It was therefore concluded that release is normally governed by intracellular Ca2+ close to points of Ca2+ entry through channels; stochastic factors give rise to more release than if Ca2+ were homogeneously distributed. If Ca2+ channels are uniformly close to release sites the average number of channels opened per site per action potential may be as low as 4.

Contractile response of aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium is reduced in spontaneous hypertension.

Vascular responses of aortic rings to alpha 1-adrenergic stimulation by phenylephrine (Phe) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied in Ca(2+)-containing medium and Ca(2+)-free medium plus 50 mumol/L EGTA. Although there was no difference in the sustained force development between SHR and WKY vessels in response to 100 mmol/L KCl or 10 mumol/L Phe in Ca(2+)-containing medium, the transient contractile response to 10 mumol/L Phe in Ca(2+)-free medium was substantially smaller in SHR compared to that in WKY. Subsequent addition of 2.5 mmol/L Ca2+ restored the sustained contractile response to a similar level in both SHR and WKY vessels. The transient contractile response to Phe in Ca(2+)-free medium containing EGTA, presumably due to the release of intracellular Ca2+, decreased progressively with preincubation time in Ca(2+)-free medium, indicating intracellular Ca2+ depletion. Such a temporal change of aortic response was more pronounced in SHR than in WKY. The subsequent response to Ca2+ repletion in the presence of Phe, on the other hand, increased progressively with Ca(2+)-depletion period and was higher in SHR than in WKY. The rate of relaxation after washout of Phe was slower in SHR aorta compared to WKY aorta. These results, together with our earlier findings, collectively suggest that the previous known deficiency in Ca2+ pumping mechanisms of vascular muscle microsomes leading to a reduced functional size of intracellular Ca2+ pool may account for the smaller contractile response of SHR aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium and the slower rate of relaxation.

Abnormal contractile response of aortas from spontaneously hypertensive rats to Ca2+ after depletion of Ca2+ in Ca2+-free medium.

Vascular responses of aortic rings from spontaneously hypertensive rats (SHR) were compared to those of the normotensive Wistar-Kyoto rats (WKY) in three sets of experimental protocols. The responses to cumulative doses of KCl indicated that SHR aortic rings were hyperresponsive to low but not high doses of KCl compared to WKY aortic rings. After Ca depletion by prolonged incubation of the rat aortic rings with Ca2+-free, EGTA containing solution, Ca repletion resulted in contraction. The magnitude of such a contraction was dependent on the period of Ca depletion and was highly sensitive to dihydropyridine Ca channel blocker, nifedipine. Although the Ca-depleted aortic rings eventually developed to the same level of maximum tension development upon Ca repletion, it took a considerably shorter period of Ca depletion for SHR than for WKY aortic rings to reach the maximum contraction upon Ca repletion. Our findings support the view that cell membranes of vascular smooth muscle in hypertension are more excitable and more susceptible to membrane destabilization by Ca removal.

Alpha 1-adrenoceptors mediate the responses to BHT-920 and rauwolscine in dog mesenteric artery after partial depolarization by KCl.

In normal (5 mM KCl) HEPES buffer solution, BHT-920 and rauwloscine did not produce any contractile responses in dog mesenteric artery strips. However, when the preparation was bathed in 20 mM KCl HEPES buffer solution, BHT-920 and rauwloscine evoked significant contractile responses. These effects were markedly inhibited by parazosin which caused a parallel shift to the right of the concentration-response curve to BHT-920. In 45Ca uptake experiments carried out in the 20 mM KCl HEPES buffer solution BHT-920 and rauwolscine significantly increased 45Ca influxes which were reduced by prazosin. These results suggest that postsynaptic alpha 1-adrenoceptors in dog mesenteric artery mediate the contractile responses and the 45Ca influxes induced by BHT-920 and rauwloscine after partial depolarization by 20 mM KCl.

Effects of erythrocyte lysate of different incubation times on intracellular free calcium in rat basilar artery smooth-muscle cells.

OBJECT: The purpose of this study was to characterize substance(s) in the erythrocytes that increase intracellular free Ca++ concentration ([Ca++]i) in smooth-muscle cells and that therefore may be involved in the pathogenesis of vasospasm. METHODS: Because vasospasm occurs days after subarachnoid hemorrhage (SAH), the authors studied the effects of aged human erythrocyte hemolysate and its low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions on [Ca++]i in freshly isolated rat basilar artery smooth-muscle cells. Fresh hemolysate (Day 0) produced a biphasic response consisting of a transient peak and a sustained plateau increase in [Ca++]i, whereas hemolysate prepared from cells incubated for 3, 7, or 14 days induced only a transient response without a sustained phase. The effect of hemolysate declined with increasing incubation time. The HMW fraction and purified human oxyhemoglobin (OxyHb) did not evoke a response. The LMW fraction from Days 3, 7, or 14 produced no response at low concentrations (< 10%) and a transient response at high concentrations (> 20%), and the effect diminished with increasing incubation time. Unfractionated hemolysate or the LMW fraction of hemolysate incubated for 21 days produced no response. The combination of the 10% LMW fraction from Day 3 plus the 10% HMW fraction (Days 3. 7, 14, or 21) transiently increased [Ca++]i,. However, [Ca++]i was not changed by the 10% LMW fraction from Day 14 plus the 10% HMW fraction from Day 3 or 14. In the presence of OxyHb, [Ca++]i was increased by the 10% LMW fraction on Days 3 and 7, but not by the LMW fraction from Days 14 or 21. CONCLUSIONS: The decline over time in the effect of hemolysate on [Ca++]i indicates either that the time that substances are released from erythrocytes is important in the generation of vasospasm or that this experimental system as used is not representative of conditions present after SAH. The data indicate that the ability to elevate [Ca++]i in smooth-muscle cells with hemolysate is provided by multiple substances, including OxyHb. These substances may interact during specific times after incubation of erythrocytes in vitro.

Erigeron breviscapus prevents defective endothelium-dependent relaxation in diabetic rat aorta.

We examined the endothelium-dependent relaxation response to acetylcholine (Ach) in streptozotocin-induced diabetic rat aorta at the stages of 2- and 6-wks' duration in vitro, and compared with another two groups which were treated with dietary supplement of 0.1% Aminoquanidine (AG) and 0.5% Erigeron breviscapus (EB) from 1-week of diabetes induction. At the stage of 2-wks' duration of diabetes, relaxation responses to lower concentrations of Ach in 0.3 uM phenylepherine-precontracted aortas were diminished significantly (P<0.05) compared with age-matched control, but the maximal relaxation of Ach remained unchanged. At the stage of 6-wks' duration, diabetes caused an approximately 60% (P<0.001) deficit in maximum relaxation, and this was significantly (P<0.001) prevented in AG and EB treated groups. There was an approximately 40% enhancement in the maximum contractile response to phenylepherine with diabetes (P<0.05), which was unaffected significantly by AG and EB treatments. The data suggest that the defective endothelium-dependent relaxation in diabetic rat aorta occurred as early as 2-wks' duration of diabetes, and the treatments of AG and EB could protect vascular endothelium although the deficits in vascular smooth muscle contractile responses were not protected.

Comparison of the Ca2+ movement by activation of alpha1-adrenoceptor subtypes in HEK-293 cells.

We studied the Ca2+ movement induced by activation of alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes in transfected HEK-293 cells with the fura-2 probe. All these alpha1-AR subtypes induced both Ca2+ release and Ca2+ entry. The effect on Ca2+ release in alpha1b transfected HEK-293 cells was bigger than that in alpha1a and alpha1d transfected HEK-293 cells, and the effects on Ca2+ entry were the same in alpha1a, alpha1b and alpha1d transfected HEK-293 cells. The Ca2+ entry was inhibited by 1 mM NiSO4, but not by nifedipine. Cyclopiazonic acid (CPA) produced a biphasic Ca2+ signal response in Ca2+ medium, and only induced a transient response in Ca2+-free medium. After depletion of CPA-sensitive Ca2+ pool by 10 microM CPA in Ca2+-free medium, 10 microM adrenaline (Adr) still transiently increased [Ca2+]i in three different alpha1-adrenoceptor subtype transfected HEK-293 cells. However, after depletion of adrenaline-sensitive Ca2+ pool by 10 microM Adr, CPA transiently elevated [Ca2+]i only in alpha1a and alpha1d transfected HEK-293 cells, not in alpha1b transfected HEK-293 cells. U73122, a phospholipase C (PLC) inhibitor, inhibited both Ca2+ release and Ca2+ entry induced by activation of alpha1A alpha1B and alpha1D subtypes in transfected HEK-293 cells. These results suggest that HEK-293 cell line contains two functionally separate intracellular Ca2+ pools, CPA-sensitive and Adr-sensitive pools. Activation of alpha1B-AR stimulates Ca2+ release from both CPA-sensitive and Adr-sensitive Ca2+ pools. Alpha1A and alpha1D subtypes induce Ca2+ release only from Adr-sensitive Ca2+ pool.

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells.

OBJECTIVES: Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. MATERIALS AND METHODS: Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. RESULTS: ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) induced by ET-1. CONCLUSION: Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells.

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl- channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl- channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl- channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively.

Cyclopiazonic acid causes endothelium-dependent relaxation in rat aorta.

The effects of cyclopiazonic acid (CPA), a selective inhibitor of Ca(2+)-pump ATPase for endoplasmic reticulum (ER), on the contractility of rat aorta with and without intact endothelium were studied to investigate the possible involvement of endothelial ER Ca(2+)-pump in the release of endothelium-derived relaxing factor (EDRF), which is known to cause vascular relaxation or inhibition of phenylephrine (PE)-precontracted aorta. When added to the organ bath cumulatively, CPA concentration-dependently caused gradual development of contraction, which was much less in aortic rings with intact endothelium than in endothelium-denuded aortic rings. But CPA at low concentrations (1-3 mumol.L-1) induced vascular relaxation when added to PE (3 mumol.L-1)-precontracted aortic rings with intact endothelium, but not in denuded aortic rings. This relaxant effect of CPA is very similar to the effect of acetylcholine (ACh), which is well recognized to be mediated by the release of EDRF from the endothelium. NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, completely prevented the vascular relaxation induced by CPA or ACh and the inhibitory effect of L-NAME was partially reversed by L-arginine (L-Arg). Treatment of the aortic rings with nifedipine (Nif) 0.3 mumol.L-1 did not affect the relaxant effect of ACh or CPA on PE-induced contraction indicating that the Ca(2+)-entry to the endothelial cells as a result of receptor activation by ACh or ER Ca(2+)-pump inhibition by CPA was via channels other than L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetrandrine is not a selective calcium channel blocker in vascular smooth muscle.

The effects of tetrandrine (Tet) on the contractile properties of rat aortic ring preparations were studied to test the hypothesis that Tet is a Ca2+ antagonist acting on voltage-operated Ca2+ channels (VOC). The tests were performed on contractions induced by depolarizing concentrations of KCl and by alpha 1-adrenoceptor agonist, phenylephrine (Phe). These vascular effects of Tet were compared to those of nifedipine (Nif). We found that Tet behaved qualitatively similar to, but less potent than, Nif in that it inhibited KCl-induced contraction in a concentration-dependent fashion and its inhibitory effect was long-lasting. However, the effects on Phe-induced contraction of Tet was different from those of Nif in that the extracellular Ca(2+)-dependent contraction was inhibited by Tet, but not by Nif. Tet (60 mumol.L-1) completely inhibited the 45Ca2+ uptake induced by KCl and Phe in rat aortic muscle strips. When the aortic muscle contractile response was induced by addition of Ca2+ following depletion of intracellular stores by Phe in the presence of sarcoplasmic reticulum Ca(2+)-pump inhibitor, cyclopiazonic acid, Tet (60 mumol.L-1) was more effective than Nif 1 mumol.L-1 in inhibiting such a response to extracellularly added Ca2+. Furthermore, Tet, but not Nif, also significantly inhibited the contraction to Phe in Ca(2+)-free medium. Collectively, these results led us to conclude that Tet does not behave as a selective VOC blocker like Nif.

Multiple actions of cadmium on transmitter release at the mouse neuromuscular junction.

The action of cadmium ions on transmitter release was studied at the neuromuscular junction in mouse diaphragm. In the presence of raised K+, Cd2+ caused a parallel shift to the right of the graph of transmitter release rate (frequency of miniature end-plate potentials, fmepp) versus log [Ca2+], with no change in maximum or slope, indicating a competitive mode of action of Cd2+. The apparent dissociation constant for Cd2+ was 3 microM. In calcium-free solutions containing 15 mM K+, Cd2+ caused a rise in the fmepp, which subsequently slowly declined despite the continued presence of Cd2+. The rise in fmepp caused by Cd2+ could be interrupted, but not reversed, by washing out the Cd2+ with EDTA. Exposure of the preparation to 100 microM Cd2+ for 15 min or more resulted in a raised fmepp that persisted despite the removal of Cd2+ and exposure to 200 microM EDTA. Following such treatment, the graph of fmepp versus log [Ca2+] continued to be shifted to the right. The interaction of Ca2+ with the residual effect of Cd2+ indicates that Cd2+, in addition to its action to block Ca2+ entry into the terminal, may act as a competitor and perhaps as a partial agonist at intracellular sites that normally bind Ca2+ and govern transmitter release. If this is the case, then it must be supposed that, in raised K+, quantal release of transmitter represents intermittent intense activation of release sites with local high levels of Ca2+ rather than continuous low level activation.

Multiple potassium conductances at the mammalian motor nerve terminal.

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not affected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similarly, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the 'accommodation curve' indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activated gK, which may be distinguished pharmacologically.