Expression of sperm Ca2+-activated K+ channels in Xenopus oocytes and their modulation by extracellular ATP.

Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels involved has been precluded by the small size of the sperm, especially in mammalian species. In the present study sperm ion channels were expressed in Xenopus oocytes by injection of RNAs of spermatogenic cells isolated from the rat testes. The RNA-injected oocytes responded to ATP, a factor known to regulate sperm activation, with the activation of an outwardly rectifying whole-cell current which was dependent on K+ concentrations and inhibitable by K+ channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP-induced current could be mimicked by a Ca2+ ionophore but suppressed by a Ca2+ chelator applied intracellularly, indicating a Ca2+ dependence of the current. Single-channel measurements on RNA-injected oocytes revealed channels of large conductance which could be blocked by CTX and TEA. Co-injection of germ cell RNAs with the antisense RNA for a mouse gene encoding slowpoke 'Maxi' Ca2+-activated K+ channels resulted in significant reduction of the ATP- and ionomycin-induced current. The expression of the 'Maxi' Ca2+-activated K+ channels in sperm collected from the rat epididymis was also confirmed by Western blot analysis. These results suggest that sperm possess Ca2+-activated K+ channels which may be involved in the process of sperm activation.

Functional expression of a Ca2+-activated K+ channel in Xenopus oocytes injected with RNAs from the rat testis.

The present study investigated the feasibility of using Xenopus oocytes to express sperm ion channel by injection of RNAs extracted from the rat testis. The RNA-injected oocytes expressed an outwardly rectifying current which was dependent on K+ concentration and inhibitable by K+ channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The Ca2+ ionophore, ionomycin, could also stimulate current activation with similar current characteristics in the RNA-injected oocytes, suggesting the expression of a Ca2+-activated K+ channel. Immunolocalization indicated predominant Ca2+-activated K+ channel immunoreactivity associated with spermatogenic cells. Reverse transcriptase-polymerase chain reaction studies confirmed the expression of the Ca2+-activated K+ channel mRNA in isolated spermatogenic cells. Our results suggest that ion channels and/or receptors of spermatogenic cells could be investigated using the Xenopus oocyte as an expression system. The present study also suggests that sperm may possess a Ca2+-activated K+ channel which has been implicated in the process of sperm activation and gamete interaction.

Cation and anion channels in rat and human spermatozoa.

Ionic fluxes are thought to be important in the initiating process of gamete interaction such as acrosome reaction. Different populations of ion channels in rat and human spermatozoa were investigated using the planar lipid bilayer technique. Membrane proteins were isolated from rat and human sperm and inserted into lipid bilayer via fusion. We observed K(+) selective and Na(+)-selective channels, as well as divalent permeable cation channels in membrane preparations from rat sperm K+ channels, which were sensitive to the K+ channel blocker, tetraethylammonium (0.1 mM), exhibited a mean single channel conductance of 24 pS. Whereas, larger conductance, 109 pS, was found to be associated with Na+ channels. Low conductance anion channel, 15 pS, was also observed when permeant cations in the bathing solutions were substituted with N-methyl-D-glucamine leaving Cl- as the major permeant ion species. This channel exhibited a slower channel open and closed kinetics when compared to other cation channels. Both cation and anion channels with characteristics similar to that found in rat sperm were also observed in preparations from human sperm. The variety in the types of ion channels observed in rat and human spermatozoa suggests that ion channels may play different roles in sperm physiology and gamete interaction.

[Synthesis and antifungal activity of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols].

Twenty-three 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols were synthesized and evaluated for antifungal activities in vitro. The synthetic methods of the intermediates are also reported. The compounds belong to two kinds: sulfones and sulfides. All of the compounds are new. The results of preliminary antifungal test showed that most of the sulfide derivatives exhibited potent activities against six kinds of common pathogenic fungi such as Microsporum lanosum, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Cladosporium carrionii, and Saccharomyces torulopsis in vitro. Compounds IVe, f,i,j showed equal or more potent activities when compared with ketoconazole and were markedly superior to fluconazole. The sulfone derivatives showed lower antifungal activity.

In vitro culture of Doritaenopsis: comparison between formation of the hyperhydric protocorm-like-body (PLB) and the normal PLB.

Characteristics of the hyperhydric protocormlike-bodies (hPLBs) and the normal PLBs (nPLBs) of Doritaenopsis are morphologically and ontogenetically compared. The hPLBs have a translucent and turgid appearance which is due to a lack of air volume in the intercellular spaces. The hPLBs have a lower capacity of shoot formation but a higher capacity of differentiation of new PLBs than the nPLBs. The new PLBs derived from the hPLBs can be recovered from hyperhydricity using a medium containing potato juice. This suggests the possibility that hPLBs can be used for the in vitro propagation of orchid plants.

Adrenergic receptors on cultured rat epididymal cells: regulation of Cl- conductances.

Adrenergic regulation of epididymal Cl- currents was studied by the whole-cell patch-clamp technique using various alpha- and beta-receptor agonists and antagonists in primary cultured rat cauda epididymal cells. Cl- currents could be activated with varying frequency by noradrenaline (primarily alpha- and beta 1-adrenoceptor-selective agonist, 1-5 microM), isoprenaline (nonselective beta-adrenoceptor agonist, 5 microM), salbutamol (beta 2-adrenoceptor-selective agonist, 2 microM), and phenylephrine (alpha 1-adrenoceptor-selective agonist, 1-2 microM). Noradrenaline alone elicited Cl- current activation in 85% of the cells examined. In the presence of phentolamine (nonselective alpha-adrenoceptor antagonist, 15 microM), noradrenaline elicited Cl- current activation in 63% of the cells examined, whereas noradrenaline-induced activation was observed in 33% of the cells examined in the presence of both atenolol and butoxamine (beta 1- and beta 2-adrenoceptor antagonists, respectively, 10 microM). In 27% of single cells examined, a second current activation in response to salbutamol was observed after the first response to phenylephrine. When the order of stimuli was reversed, dual activation was also observed in 22% of the single cells examined, indicating the presence of both alpha- and beta-adrenoceptors in single epididymal cells. Profiles of time- and voltage-dependent Cl- current upon activation by different adrenoceptor agonists exhibited characteristics similar to those previously reported for Ca2+ and cAMP-activated Cl- currents, suggesting that regulation of epididymal Cl- conductances could be mediated by different adrenoceptor subtypes involving Ca2+ and cAMP as intracellular second messengers.

[Protective effect of captopril on cultured rat myocardial cells with anoxia and reoxygenation injury].

The effect of captopril (Cap) on electric activity of cultured rat myocardial cells under anoxia and reoxygenation was studied with standard microelectrode techniques. Results showed that anoxic solution caused lowerings of MDP, APA, and Vmax, and a shortening of APD50. All myocytes revealed multiform arrhythmias, and most cells stopped beating within 30 min, while only 40% of the cells exhibited arrhythmias but none stopped beating in the presence of 40 mg.L-1 under the same condition. During reoxygenation, most cells resumed beating in 10 min but some of these cells stopped beating again. The electric activities in rebeating cells during reoxygenation for 30 min were lower than those in normoxic cells. Cap (40 mg.L-1)-treated cells rebeat quickly after reoxygenation and no cell stopped beating any more, with parameters higher than those in untreated cells. These results demonstrate that Cap yields some beneficial effects on preventing anoxia and reoxygenation injury in cultured rat myocardial cells.

[Effect of Astragalus membranaceus on electrophysiological activities of acute experimental Coxsackie B3 viral myocarditis in mice].

A murine model for observing the effect of Astragalus membranaceus (AM) on electrophysiological activity of the right ventricular myocardium was developed in 4 week-old male BALB/c mice infected with Coxsackie B3 virus (CB3V). The conventional microelectrode technique and real-time microcomputer data processor system was used. The survival rate in infected-AM treated mice was significantly higher and the percentage of abnormal action potential was much lower than those in control mice (P < 0.05 and < 0.01 respectively). Some abnormal electrophysiological parameters, such as APA, OS and Vmax in infected myocardium were found to be improved by AM treatment. Single dose of AM did not show beneficial effect in murine myocardium infected with CB3V. The results suggested that AM might be valuable in the prevention and treatment of acute myocarditis involving Coxsackie B3 virus.

[Macrological identification of original and commercial Chinese drug gusuibu (Rhizoma drynariae)].

Twelve original species and 7 commercial species of Chinese drug Gusuibu were identified and discussed based on macrological observation and comparison. The survey of 65 samples collected from 23 provinces throughout the country showed that at the present time the major species of commercial Gusuibu is derived from the rhizomes of Drynaria fortunei, which makes up more than 70 percent of the total samples.

Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells.

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.

Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells.

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.

Electrophysiological and pathological observations on experimental coxsackie B-3 viral myocarditis in mice.

Electrophysiological action of right ventricular myocardium examined by standard intracellular microelectrode technique and real-time microcomputer data processor system and histological and ultrastructural changes of myocardium in BALB/c mice infected with coxsackie B-3 virus from 3 days to 9 months were observed. It was found that electrophysiologic parameters of action potential changed very quickly at the early stage (3 days to 1 month) of the disease. Those abnormalities became most apparent by the 5-30th day, and 7 patterns of abnormal action potential occurred frequently within the same period. These changes were basically parallel to the myocardial lesions. At the late stage (3-9 months) the electrophysiological parameters were nearly normal, while the myocardial lesions decreased gradually. However, the abnormal patterns of action potential were still detected, even though they were improved gradually. The results suggest that myocardial damages caused by viral infection may lead to changes of cardiac electric action, which may be one of the factors in arrhythmias in the episode of viral myocarditis.

Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

[Influence of dexamethasone on electrical activities in cultured rat beating myocardial cells infected with Coxsackie B-2 virus].

The effects of dexamethasone (Dex) on electrical activities in cultured rat beating myocardial cells infected with 100 TCID-50 Coxsackie B-2 virus (CB2V) was evaluated by conventional intracellular microelectrode technique. The frequency began to increase, the beating % decreased, and multiform arrhythmias were shown in the infected group 24 h post-challenge. Meanwhile, the cytopathic effect (CPE) appeared rapidly from 1+ to 3+. In the infected and Dex-treated group, the beating % was higher and the arrhythmias and CPE were less than in the infected group at the same intervals. The numbers of non-beating cells increased parallel to the incubation time in the infected group. Decreases of maximal diastolic potential (MDP), maximal upstroke rate (Vmax), overshoot (OS) and action potential amplitude (APA), and abbreviation of action potential duration (APD50 and APD100) in infected and Dex-treated group were less than those in control group during 24-96 h post-challenge. Premature beats, tachycardia, bradycardia and fibrillation occurred in the early stages after infection. It is surmised that steroids can probably save the lives of patients with severe myocarditis if Dex was supplemented.

Effect of astragalus membranaceus on electric activities of cultured rat beating heart cells infected with Coxsackie B-2 virus.

Astragalus membranaceus (AM) which has a protective effect on rat beating heart cells infected experimentally with Coxsackie B-2 virus was evaluated on the basis of changes in morphologic and electric activity of the cells. Rhythm, beating frequency, beating percentage, cardiac cellular damage and cytopathic effects (CPE) were monitored every 24 h after challenge; electric activities parameters were measured by conventional intracellular microelectrode technique. Significant protective effects were demonstrated when AM was given in the early period of infection. The results suggest that AM should be valuable in preventing and treating acute myocarditis caused by Coxsackie B virus.

[Effect of Astragalus membranaceus on electrical activities of coxsackie B-2 virus-infected rat myocardial cells in culture].

Beating Myocardial cell cultures of neonatal rats were prepared in vitro and infected with coxsackie B-2 virus. The cells were evaluated in the post-infected period for changes in beating percentage and cytopathic effect (CPE), alterations in the electrical activities by standard microelectrode techniques, and the protective effect of Astragalus membranaceus (AM) on coxsackie B-2 virus-infected neonatal rat myocardial cell cultures was observed. The beating percentage began to decrease in the infected group at 24 hr and only 27.9 +/- 18.6% was beating at 96 hr after virus challenge, premature beats, tachycardia and fibrillation occurred commonly during the experiment. Meanwhile the CPE appeared rapidly from 1+-3+ at the same interval. Resting potential, action potential amplitude, duration and rate of uptake were shown a significant decrease through 24-96 hr (P less than 0.01). In contrast, the beating and electrical activities were nearly normal and less CPE was shown in myocardial cells treated with AM 1 hr after virus challenge through 24-96 hr (P less than 0.05). The results suggest that AM may be valuable in prophylaxis and treatment of acute coxsackie B-2 virus caused myocarditis.