[Surveillance of potential transmission factors of schistosomiasis in Xiuzhou District of Jiaxing City from 2013 to 2015].

OBJECTIVE: To understand the potential risk of schistosomiasis transmission in Xiuzhou District of Jiaxing City, so as to provide the scientific evidence for consolidating schistosomiasis control achievements. METHODS: Fixed and mobile surveillance sites were set up in Xiuzhou District of Jiaxing City from 2013 to 2015. Oncomelania hupensis snails was surveyed historical snail habitats, current snail habitats, and suspected snail habitats. The schistosome infections were identified using serological and parasitological testing among local residents and mobile populations. In addition, the survival and reproduction of snails imported into Xiuzhou District was observed, and the schistosome infection in wild reservoir hosts was detected. RESULTS: A total of 540.14 hm2 of settings were surveyed in Xiuzhou District, Jiaxing City from 2013 to 2015, and 1.65 hm2 of snail habitats were identified. The snail habitats were mainly located in dry lands, and no infected snails or importation of snails were found. During the period from 2013 to 2015, a total of 7 668 local residents and mobile populations were examined in Xiuzhou District, and no new local infections were detected; however, three imported schistosomiasis cases were identified. Field simulation experiment showed that the imported snails laid eggs and reproduced in Xiuzhou District, and no schistosome infections were found in wild animals. CONCLUSIONS: There are still residual Oncomelania snails and imported schistosomiasis patients in Xiuzhou District of Jiaxing City; therefore, the surveillance and management of local Oncomelania snails and imported schistosomiasis should be intensified to reduce the risk of schistosomiasis transmission.

Inhibition of ryanodine binding to sarcoplasmic reticulum vesicles of cardiac muscle by Zn(2+) ions.

Using the assay of [(3)H]ryanodine binding to the sarcoplasmic reticulum, the effect of Zn(2+) on ryanodine receptors (RyRs) of cardiac muscle was investigated. There was no obvious change in the binding at [Zn(2+)](f) of less than 0.2 microM. However, a decrease of the binding became significant with raising [Zn(2+)](f) to 0.5 microM. The inhibitory effect of Zn(2+) was [Zn(2+)](f)-dependent, with IC(50/ZnI) of 2.1+/-0.4 microM (mean+/-S.D.). Scatchard analysis indicates that both an increase of K(d) and a decrease of B(max) were responsible for Zn(2+)-induced decrease of the binding. The Hill coefficient for this inhibitory effect of Zn(2+) was between 0.8 and 1.2. The interactions of the effects of Zn(2+) and various modulators of RyR indicate that the inhibitory effect of Zn(2+) was mostly mediated through inhibiting Ca(2+) activation sites (CaA) on RyR. Since the [Zn(2+)](f) dependence was not clearly changed by [Ca(2+)](f), the inhibitory effect of Zn(2+) may not be due to competition of Zn(2+) with Ca(2+) for CaA and probably is indirect. The inhibitory effect of Zn(2+) could not be antagonized by 2 mM dithiothreitol, a thiol-reducing agent, suggesting that the binding of Zn(2+) ions to RyRs of cardiac muscle is not accompanied by obvious change of redox state of the RyRs. In comparison with that seen in skeletal muscle [3], the effects of Zn(2+) on ryanodine binding to the sarcoplasmic reticulum of cardiac muscle show several distinct differences. It is indicated that the effect of Zn(2+) on RyRs may be isoform-dependent. The physiological significance of the effects of Zn(2+) is discussed.

Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions.

With the use of a [(3)H]ryanodine binding assay, the modulation of skeletal muscle ryanodine receptor (RyR1) by Zn(2+) was investigated. In the presence of 100 microM free Ca(2+) concentration ([Ca(2+)](f)) as activator, the equilibrium [(3)H]ryanodine binding to heavy sarcoplasmic reticulum vesicles was biphasically modulated by Zn(2+). The binding was increased by a free Zn(2+) concentration ([Zn(2+)](f)) of less than 1 microM; a peak binding, approx. 140% of the control (without added Zn(2+)) was obtained at 0.3 microM [Zn(2+)](f). An inhibitory effect of Zn(2+) became obvious with a [Zn(2+)](f) of more than 1 microM; the [Zn(2+)](f) for producing half inhibition was 2.7+/-0.5 microM (mean+/-S.D.). Scatchard analysis indicated that the increase in the binding induced by low [Zn(2+)](f) was due to a decrease in K(d), whereas both an increase in K(d) and a possible decrease in B(max) were responsible for the decrease in binding induced by high [Zn(2+)](f). The binding in the presence of micromolar [Zn(2+)](f) showed a biphasic time course. In the presence of 3 microM [Zn(2+)](f), after reaching a peak with an increased rate of initial binding, the binding gradually declined. The decline phase could be prevented by decreasing [Zn(2+)](f) to 0.5 microM or by adding 2 mM dithiothreitol, a thiol-reducing agent. The [Ca(2+)](f) dependence of binding was changed significantly by Zn(2+), whereas Ca(2+) had no clear effect on the [Zn(2+)](f) dependence of binding. Moreover, some interactions were found in the effects between Zn(2+) and other RyR1 modulators. It is indicated that Zn(2+) can modulate the activation sites and inactivation sites for Ca(2+) on RyR1. The physiological significance of the effects of Zn(2+) on ryanodine binding is discussed.

Inhibition of calcium signaling in terminal and soma of carp retinal bipolar cells by GABA.

AIM: To investigate the effect of activation of gamma-aminobutyric acid (GABA) receptors on high K(+)-evoked Ca2+ signaling in the terminal and soma of carp retinal ON-type bipolar cells. METHODS: Freshly dissociated carp retinal cells were loaded with fluo-3AM and then the fluorescence measurements were performed on a confocal laser-scanning microscope. RESULTS: Ca2+ signaling evoked by high K+ 35 mmol/L was completely suppressed in both the terminal and soma of bipolar cells by GABA 100 mumol/L. However, different results were found in the terminal and soma when only one subtype of GABA receptors was activated. While activation of either GABAA or GABAC receptors totally suppressed Ca2+ signaling in the soma, a gradual elevation of [Ca2+]i appeared in the terminal. GABA 10 mumol/L could also completely suppress Ca2+ signaling in the soma, but could only partially reduce Ca2+ signaling in the terminal. CONCLUSION: Activation of both GABAA and GABAC receptors could completely inhibit high K(+)-evoked Ca2+ signaling in the terminal and soma of carp retinal ON-type bipolar cells. While activation of either GABAA or GABAC receptors alone still totally suppressed Ca2+ signaling in the soma, a gradual elevation of [Ca2+]i appeared in the terminal, which may be due to desensitization of GABA receptors.

Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken.

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.

Effects of phorbol esters on excitation-contraction coupling and protein kinase C activity of frog twitch muscle fibers.

By recording the calcium transients evoked by voltage-clamp depolarizing pulse with arsenazo III as a calcium indicator, it has been shown that 1 micromol/l phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, causes a transient potentiation and then a depression of the calcium transients of twitch muscle fibers in frogs. PDBu also produces an initial translocation and activation of PKC, which is followed by a down-regulation. To find out whether the effect of PDBu on the calcium transients depends on PKC, a correlated study of the effect of phorbol esters on calcium transients and PKC activity was performed. The calcium transients and PKC activity were similarly affected by PDBu in ordinary and cold-accommodated frogs, but the effects occurred more quickly in the latter. However, they still changed in parallel as in ordinary frogs. 1 or 10 micromol/l, 4-alpha-phorbol, a PKC-inactive analogue of phorbol ester, caused a partial depression of the calcium transients in cold-accommodated frogs, while PKC activity was not affected. Moreover, the transient potentiation of the calcium transients induced by 1 micromol/l PDBu could be antagonized by the PKC inhibitors 10 micromol/l chelerythrine chloride or 10 micromol/l polymyxin B (PMB). All these results suggest that: (1) the transient potentiation of calcium transients induced by PDBu is caused by activation of PKC; (2) phorbol ester can depress the calcium transients by a mechanism that is independent of PKC.

Effect of removal of external calcium on phosphoinositide hydrolysis in cultured myotubes of embryonic chicken.

The effect of removal of external Ca2+ on phosphoinositide hydrolysis was investigated in cultured myotubes from 9-day-old Leghorn embryonic chicken. In the myotubes exposed to Ca(2+)-free Ringer's solution, the turnover of phosphoinositide was exponentially decreased with a time constant of about 26 min. In the presence of external Ca2+, the hydrolysis of phosphoinositide was significantly increased by exposure to 80 mmol/L K+ solution. After removal of external Ca2+, 80 mmol/L K+ exposure caused a slight decrease of phosphoinositides hydrolysis in comparison with the control (normal Ringer). It is indicated that hydrolysis of phosphoinositide in cultured myotubes can be enhanced by high K+ exposure. External Ca2+ is essential for this effect, which is different from mature muscle fibres.

[Potentiation of caffeine-induced contracture by raising extracellular potassium in frog skeletal muscle].

The effect of raising extracellular potassium ([K+]o) on caffeine contracture was investigated, using small bundles dissected from frog anterior tibialis muscle. Elevating [K+]o from the control of 2 mmol/L to 10 or 25 mmol/L significantly potentiated the contracture induced by 3 mmol/L caffeine. The potentiation represented by PKC/PC, where PKC and PC are the peak tension of the caffeine contracture evoked in high and normal [K+]o respectively, was dependent on [K+]o and the duration of conditioning high K+ exposure. With 10 mmol/L [K+]o, the potentiation was gradually increased by prolonging conditioning exposure up to 10 min. On the contrary, with 25 mmol/L [K+]o the potentiation reached a maximum within only 1 min, and then subsided to the control. These different time courses of PKC/PC could not be accounted for by high K+ induced depolarization, but were in general consistence with the time courses of the change in myoplasmic free calcium induced by corresponding high [K+]o. It is suggested that, at least in frog skeletal muscle, the high [K+]o induced potentiation of caffeine contracture is mainly due to an increase of myoplasmic free calcium.

Caffeine-sensitive Ca2+ stores in carp retinal bipolar cells.

High K+- or caffeine-induced Ca2+ signal was studied in freshly dissociated carp retinal ON-type bipolar cells using a confocal laser-scanning microscope. In response to 35 mM K+ exposure, a rise in [Ca2+]i appeared in both the terminal and soma, but was absent after removal of external Ca2+ or in the presence of 100 microM nifedipine. It is indicated that, for high K+-induced increase of [Ca2+]i, Ca2+ influx through voltage-gated L-type Ca2+ channels is essential and Ca2+ entry through reversed Na+/Ca2+ exchange may be negligible. Interestingly, caffeine-induced elevation of [Ca2+]i was restricted to the soma, and could be abolished by 50 microM ryanodine, suggesting that caffeine-sensitive Ca2+ stores gated by ryanodine receptors were present in the soma but not in the terminal of bipolar cells. After treatment with 50 microM ryanodine for 20 min, the peak of the Ca2+ transients evoked by 35 mM K+ in the soma decreased to 48.2+/-5.7% of the control. The results suggest that depolarization-evoked Ca2+ influx can cause Ca2+ release from caffeine-sensitive Ca2+ stores, and in turn amplify Ca2+ signal in the soma of retinal bipolar cells.

Effects of high potassium and caffeine exposure on activities of Ca2+-dependent and Ca2+-independent protein kinase C in frog skeletal muscle.

With the use of histone III-S as a protein kinase C (PKC) substrate, the activities of total and Ca2+-independent PKC in frog skeletal muscle were measured, and their difference is designated Ca2+-dependent PKC. In resting muscle, the total PKC was almost equally associated with the cytosol and membrane, and the ratio of membrane to cytosol PKC was about 1. However, the distribution of PKC was subtype dependent. About 60% of Ca2+-dependent PKC was located in the cytosol, whereas Ca2+-independent PKC was mainly associated with the membrane. High potassium exposure not only caused a significant translocation of Ca2+-dependent PKC from the cytosol to the membrane, but also changed the distribution of Ca2+-independent PKC, although to a lesser extent. However, in the preparations exposed to caffeine, the translocation of PKC occurred only in a Ca2+-dependent subtype. In addition, the biphasic change in membrane-associated PKC seen in high K+ exposed muscles was absent with caffeine treatment. The possible mechanisms of these differences are discussed.

[Absence of detubulation in frog twitch muscle fibres exposed to protein kinase C activator].

It has been shown that excitation-contraction(e-c) uncoupling occurred in frog twitch muscle fibres exposed to phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator. To investigate if this uncoupling results from detubulation of the transverse tubules, intracellularly evoked action potentials were examined. The after-depolarization of the action potential, representing intact transverse tubules was gradually abolished by osmotic detubulation, while those obtained from the fibres incubated in 1 mumol/L PDBu for 12 or 24 h still possessed the after-depolarization. Thus, it showed that the transverse tubules were still intact in frog twitch muscle fibres after exposure to PDBu. The mechanism of e-c uncoupling induced by PDBu remains to be clarified.

[Structure and pharmacology of ryanodine receptors].

Ryanodine receptors (RyR) exist at the membrane of the intracellular calcium stores and function as the calcium-release channels in the vertebrate animal cells. In the mammalian animals three isoforms of RyR, skeletal type (RyR1), cardiac type (RyR2) and brain type (RyR3), have been identified, and they are encoded by three distinct genes, ryr1, ryr2 and ryr3, respectively. Meanwhile, in the non-mammalian vertebrate animals other three isoforms of RyR have been found: alpha RyR and beta RyR present in the skeletal muscle simultaneously, whose amino acid sequences exhibit the high identity to that of the mammalian RyR1 and RyR3 respectively, and another distinct isoform of RyR in the avian heart. In addition, a large variety of cells of both mammalian and non-mammalian vertebrate animal expresses all or two of the three isoforms of RyR simultaneously. Molecular structure and pharmacology of these isoforms of RyR are reviewed in this article.

3,4-Diaminopyridine induced hydrolysis of phosphoinositide in cultured neurons from embryo chick forebrain.

The effect of 3,4-diaminopyridine (DAP) on phosphoinositide hydrolysis in cultured neurons from embryo chick forebrain has been studied. DAP produced a dose- and time-dependent accumulation of inositol phosphates. At 1 mM DAP a maximal effect was obtained. In Ca2+ free medium, DAP-activated turnover of phosphoinositide was reduced, but was still significant. Blocking Ca2+ entry with 200 microM Cd2+ also did not abolish the DAP-induced accumulation of inositol phosphates. As a comparison the effect of high K+ exposure was investigated. High K+ enhanced phosphoinositide hydrolysis, and this effect was also reduced by excluding Ca2+ influx. Moreover, DAP had no additional effect on the high K(+)-induced hydrolysis of phosphoinositide. Using oxonol-V, a depolarization of the membrane potential was seen in the neurons bathed in DAP containing medium. It is suggested that the depolarization may play a role in DAP-activated phosphoinositide turnover in cultured neurons of the embryo chick forebrain, but that Ca2+ entry is not necessary for this effect.

Effect of activation of protein kinase C on excitation-contraction coupling in frog twitch muscle fibres.

Intracellular Ca2+ transients were recorded from frog twitch muscle fibres in response to voltage-clamp depolarizing pulses, using arsenazo III as an intracellular Ca2+ indicator. The effect of the activation of protein kinase C (PKC) on the Ca2+ transients was studied. With 1 microM phorbol 12,13-dibutyrate (PDBu), a PKC activator, the peak of the Ca2+ transients increased to about 120% of control during the first 0.5 h, and then decreased gradually to a plateau of 44% of control within the following 2 h. This effect of PDBu could be alleviated significantly by PKC inhibitors, 10 microM polymyxin B (PMB) or 30 microM 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). Moreover, PDBu caused an upward shift of the strength/duration curve. In Li(+)-loaded muscle fibres the Ca2+ transients could not fully recover after 80 mM K+ exposure for 15 min, while the post-K+ Ca2- transients could be completely restored in the fibres not loaded with Li+. In the presence of 10 microM PMB or 30 microM H-7, a full restoration of the post-K+ Ca2+ transients was seen in Li(+)-loaded fibres. PMB supplemented after high-K+ exposure also could result in a complete recovery of the post-K+ Ca2+ transients in Li(+)-loaded fibres. The role of PKC in modulating excitation-contraction coupling in frog twitch muscle fibres is clearly indicated, but the mechanism(s) and physiological significance remain to be established.

[Construction of microfluorometer and its application for measuring intracellular free calcium].

A microfluorometer used for measuring intracellular free calcium has been constructed. This system was based on an XSJ-2 epifluorescence microscope with the following modifications. First, the AC mercury lamp was replaced by a DC xenon lamp to provide a stable light source. Second, an excitation filter wheel incorporating two interference filters was inserted in the light path allowing the biological sample to be excited alternately by lights of two different wavelength. The running of the whole system was under the control of a microcomputer. Such a system has successfully been used to monitor intracellular calcium changes with fluorescent calcium-sensitive dye Fura-2. Some examples of application are presented.

Effects of 3,4-diaminopyridine on myoplasmic calcium and phosphoinositide hydrolysis in frog sartorius muscle fibers.

Using Ca(2+)-selective microelectrodes, the effects of 3,4-diaminopyridine (DAP) on myoplasmic calcium ([Ca2+]i) were studied in frog satorius muscle fibers. DAP induced an increase of [Ca2+]i in a concentration-dependent manner. DAP 1 mmol.L-1 increased the [Ca2+]i from control 166 +/- 41 nmol.L-1 to 416 +/- 69 nmol.L-1 (n = 10). In the absence of external calcium, DAP still enhanced [Ca2+]i. [Ca2+]i of the fibers bathed in Ca(2+)-free Ringer's solution containing DAP 1 mmol.L-1 was 152 +/- 43 nmol.L-1, which was significantly higher than 77 +/- 35 nmol.L-1 of [Ca2+]i of the fibers in Ca(2+)-free Ringer's solution. In addition, DAP promoted the hydrolysis of phosphoinositides, and DAP-induced hydrolysis was more in the presence of external calcium. It is suggested that, through enhancing the hydrolysis of phosphoinositides, DAP released Ca2+ from intracellular Ca2+ store in frog sartorius muscle fibers.

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K+) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the "Independence" hypothesis is correct, tension during the decay of K+ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K+ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K+ contracture tension are not independent.

Depression of calcium transients after exposure to high K+ solution in Li(+)-loaded frog twitch muscle fibres and its reversal by exogenous myo-inositol.

Using Arsenazo III as a myoplasmic calcium indicator, we have studied the calcium transients evoked by voltage-clamp depolarizing pulses in frog twitch muscle fibres which had been temporarily depolarized by 80 mmol/L K+ in the absence or presence of myoplasmic Li+. After the high K+ exposure, for either a short (15 min) or long (1 h) time, the post-K+ calcium transients could gradually be restored to the level of the pre-K+ ones, if the fibres were not loaded with Li+. In contrast, the post-K+ calcium transients of Li(+)-loaded fibres could not fully recover, and were depressed in a Li+ concentration-dependent manner. The mean amplitude of the post-K+ responses recorded more than 3.5 h after 15 min high K+ exposure was reduced to 56% of pre-K+ control in the fibres which had been loaded with Li+ in 20 mmol/L Li+ Ringer's solution. This depression could be prevented or partially reversed by exogenous myo-inositol. More depression could be induced by 1 h high K+ exposure, but the presence of exogenous myo-inositol could not clearly prevent the post-K+ calcium transients from reduction. Assuming that high K+ exposure caused a depletion of myo-inositol and probably other changes in the metabolism of inositol phospholipids in Li(+)-loaded fibres, we conclude that some metabolites of phosphoinositides may play modulation roles in excitation-contraction coupling in frog twitch muscle fibres.