Morphine Stimulates Mesangial Cell TNF-α and Nitrite Production

Background: Intravenous opiate abusers are susceptible to develop heroin and HIV-associated nephropathies; however, the role of opiates in the development of these kidney lesions is not clear. Patients with opiate addiction are prone to recurrent infections. Methods: The effect of morphine was studied on the generation of TNF- with or without LPS (lipopolysaccharide) by cultured mouse mesangial cells. In addition, the effect of morphine was evaluated on mesangial cell nitrite production. To evaluate the role of opiate receptors, we studied the effect of naloxone and naltrexone on mesangial cell TNF- and nitrite production. To determine the role of TNF- on mesangial cell nitrite production, we examined the effect of anti-TNF- antibody on morphine-induced nitrite production. Assay of TNF- and nitrite production was carried by ELISA and Griess method respectively. Results: Morphine alone did not enhance the generation of TNF- by mesangial cells, however, an enhanced (P < 0.001) TNF- production was observed when mesangial cells were first treated with morphine for 18 h and then activated further with LPS. Maximum release of TNF- was seen at a concentration of 10^–12 M of morphine. Opiate receptor antagonists (naloxone and naltrexone) inhibited the effect of morphine. Morphine also amplified (P < 0.0002) the effect of LPS on mesangial cell nitrite production. Anti-TNF- antibody attenuated morphine induced nitrite generation. Conclusion: We conclude that morphine stimulates the generation of TNF- by LPS-activated mesangial cells. This effect of morphine seems to be opiate receptor mediated and has a downstream effect in the form of mesangial cell nitrite generation. The present in vitro study provides the basis for a hypothesis that morphine may be playing a role in the development of heroin and HIV-associated nephropathies.     

Association of Phenotypic Changes in B Cell Lymphocytes and Plasma Viral Load in Human Immunodeficiency Virus-Infected Patients

Many B cell abnormalities have been reported in human immunodeficiency virus (HIV)-infected patients, including changes in the expression of , , and CD22 molecules on the cell surface. Phenotypic changes in these markers on B cells isolated from HIV-seropositive patients with high or low levels of plasma viremia were measured. The phenotypic changes in B cells isolated from such patients were compared with the markers on B cells isolated from HIV-seronegative individuals using three-color flow cytometry. HIV patients showed a reduction in the proportion of mature B cells isolated from peripheral blood mononuclear cells compared with B cells isolated from HIV-seronegative individuals. An increase in the proportion of B cells expressing both and molecules on the cell surface was also seen in association with high-HIV plasma viremia. A low plasma viral load was accompanied by a reduction in the proportion of B cells expressing both and molecules to a level comparable to those seen in HIV-seronegative individuals. HIV-seropositive individuals demonstrated an increase in the proportion of committed B cells, as indicated by an increase in the proportion of B cells expressing molecules. This observation may explain the poor humoral response of HIV seropositive patients to neo-antigens. Our results demonstrate that phenotypic changes indicative of in vivo B cell activation and an increase in immature cells are associated with HIV infection, particularly with a high plasma viral load. Phenotypic changes in B cell markers may correlate with functional deficits of B cells.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Morphine stimulates superoxide formation by glomerular mesangial cells

Focal glomerulosclerosis is the predominant glomerular lesion in heroin addicts. We studied whether morphine, a metabolite of heroin, could directly affect the formation of superoxide by glomerular mesangial cells. Mesangial cells preincubated with morphine (10^–8 M) showed a higher (P<0.001) production of superoxide when compared to control cells (control) 401±21 vs. morphine 610±41 nM/mg protein/h). This effect of morphine on mesangial cells was dose dependent. Naloxone, an opiate antagonist, attenuated morphine-induced formation of Superoxide by mesangial cells [control, 317±4; morphine (10^–8 M), 573±9; and naloxone (10^–8 M) + morphine (10^–8 M), 333±6 nM/mg protein/h]. We conclude that morphine enhances formation of superoxide by mesangial cells and this effect of morphine seems to be mediated through opiate receptors. Since superoxide has been demonstrated to cause mesangiolysis, we propose that morphine may be playing a role in the induction of mesangial injury in patients with opiate abuse.This work was supported by National Institute of Health Grant R01-DA-06753.     

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (P_i) from the breakdown of extracellular ATP. Enzyme activity involved Ca²⁺/Mg²⁺-dependent and Ca²⁺/Mg²⁺-independent dephosphorylation of ATP. Ca²⁺/Mg²⁺-dependent ecto-ATPase was active over a limited range of 0.01–1.0 mM ATP, while Ca²⁺/Mg²⁺-independent ATPase activity was active over a range of 0.1–30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0–9.0), but increased in a relatively linear manner with: (1) time of reaction (0–90 min), (2) number of cells (1–20 oocytes), and (3) temperature (10–37°C). Ecto-ATPase activity was unaffected by ouabain (100 M), sodium azide (100 M), and oligomycin (5 g/ml) (as inhibitors of endo-ATPases) and -glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP,-methylene ATP, ,-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P₂-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca²⁺/Mg²⁺-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide,serum proteins,and modulators of signal transduction

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gramnegative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055 B5, 10 ng/ml-1g/ml) was a weak stimulus for generation of superoxide anion (O ₂ ^– ) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4–1.0% vol/vol, equivalent to 128–320g protein/ml), O ₂ ^– generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25–1.0g/ ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O ₂ ^– generation was observed. Stimulation of macrophages for generation of O ₂ ^– either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor herbimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12M), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5g/ml protein). Essentially complete inhibition of O ₂ ^– synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1g/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256g/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O ₂ ^– . Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca⁺⁺-dependent, but do not relay on G-protein-mediated signaling.     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells

The elementary K⁺ conductance activated by the cAMP or the Ca²⁺ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl^–-free conditions, an inwardly rectifying whole-cell K⁺ current was evoked by either forskolin 10 (mol/l) or acetylcholine 1 (mol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8±2.5 pS (n=26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K⁺ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mol/l free Ca²⁺ at the inner side of the membrane. This channel was blocked by Ba²⁺ (5 mol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca²⁺ with and EC₅₀ of 0.25 mol/l and strongly reduced by intracellular MgATP with an IC₅₀ of 600 mol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K⁺ channel was also recorded, with similar characterictics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K⁺ channel regulated by intracellular Ca²⁺ and phosphorylation supports the main K⁺ current activated by secretory agonists in normal cystic fibrosis cell lines. This conductance possibly represents the major pathway for K⁺ recycling at the basolateral membrane during transepithelial fluid secretion.     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.     

Stimulation of protein kinase C activity may increase microvascular permeability to colloidal carbon viaα-isoenzyme

The vasculature of the isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution in vitro. Various phorbol-related compounds that are known to have different affinities for the protein kinase C (PKC) isoenzymes, and bradykinin (BK), were tested for their ability to cause the microvascular endothelium to become permeable to injected colloidal carbon (CC). Phorbol 12,13-dibutyrate (PDB), 12-deoxyphorbol 13-phenylacetate (DOPPA), thymeleatoxin (TMX), and resiniferatoxin (RFX), each at a concentration of 1M, were found to increase permeability. Pretreatment with the PKC inhibitor Ro 31–8220 (1M) significantly reduced the response to all of these compounds. Indomethacin (1M), on the other hand, reduced only the effect of RFX. 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA) (1M) and BK (10M) did not increase CC leakage. These results suggest that the Ca²⁺-dependent PKC-isoenzyme was involved in the increase in endothelial permeability. BK does not appear to stimulate PKC activity in this experimental situation.     

Local vesicle populations in rat superior cervical ganglia and the vesicle hypothesis

Summary The local vesicle population (LVP), namely the population of synaptic vesicles lying in a zone 0.25 m wide adjacent to the presynaptic membrane, has been measured at synapses of rat superior cervical ganglia fixed for electron microscopy following experiments performed during a 60 min periodin vitro. The mean LVP of 78 synapses in three unstimulated control ganglia was 122.0 ± s.e. 5·9 vesicles m^-2. Ganglia fixed 1 min after 3 min 10 Hz tetani of the preganglionic nerve had a significantly (P<0.001) higher mean LVP (158.0 ± s.e. 5·0). Replacement of the Ca²⁺ of the bathing medium by Mg²⁺ for the final 30 min did not affect the mean LVP of unstimulated ganglia but prevented the increase following preganglionic stimulation. When a bathing medium with Mg²⁺ replaced by Ca²⁺, or when a drug such as caffeine, morphine or adrenaline was present for the final 30 min of the experimental period, preganglionic nerve stimulation reduced the mean LVP below the mean value in paired unstimulated ganglia.The findings are discussed in the light of recent ideas of transmitter physiology and it is concluded that they are compatible with the concept that synaptic vesicles contain, or are associated with, the synaptic transmitter.     

Ionic regulation of human basophil releasability. I. Inhibitory effect of copper

The effects of copper (CuSO₄ and CuCl₂) onin vitro histamine release from human basophils stimulated by anti-IgE and Ca²⁺ ionophore A23187 were evaluated. Both CuSO₄ and CuCl₂ caused a dose-related inhibition of histamine release, which was more pronounced on anti-IgE-than on Ca²⁺ ionophore-induced histamine release. The concentration which produced 50% inhibition of anti-IgE-induced histamine release was 1.3 M for CuSO₄ and 1.5 M for CuCl₂; the maximal inhibition of Ca²⁺ ionophore-induced histamine release was 33% for CuCl₂ (4 M) and 51% for CuSO₄ (16 M). The inhibitory effect on anti-IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before IgE-induced histamine release persisted also when extracellular Cu²⁺ was removed by cell washing before stimulation, whereas no inhibition of Ca²⁺ ionophore-induced histamine release was found when extracellular Cu²⁺ was removed. The activity of Cu²⁺ was independent of any effects of deuterium oxide and colchicine, two agents known to interact with microtubules. Increased extracellular Ca²⁺ concentrations reduced the inhibitory effect of CuCl₂ on Ca²⁺ ionophore-induced histamine release, and Schild plot analysis demonstrated that Cu²⁺ ions are competitive antagonists of Ca²⁺ ions.These results indicate that Cu²⁺ ions in the micromolar range down-regulate anti-IgE- and Ca²⁺ ionophore-induced histamine release. Since Cu²⁺ concentration in human plasma is in the micromolar range (30 M with 10–30% of free Cu²⁺), it is conceivable that Cu²⁺ ions contribute to thein vivo regulation of histamine release from human basophils.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

Effect of praziquantel on the free-living stages ofSchistosoma mansoni

Summary The effect of the new schistosomicide praziquantel (2-cyclohexyl-carbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1a]isoquinolin-4-one) on the miracidia and cercariae ofSchistosoma mansoni was investigated.In vivo praziquantel inhibits hatching of miracidia for 24 h after administration of 500 mg/kg to infected mice. In vitro a concentration of 10 g/ml inhibits subsequent hatching in drug-free water. Free swimming miracidia are rapidly killed by 1 g/ml. Even 0.01 g/ml is still partially effective.In a solution of 0.03 g/ml cercariae lose their ability to swim within 10 min. This effect is reversible in drug-free water. Morphological damage to cercariae incubated in 0.1 g/ml is clearly evident. However, cercariae are fully infective when given subcutaneously to mice after a 3-h incubation period. Incubation in 1 g/ml reduces the infection rate by 80%. A 2-h incubation in 0.1 g/ml almost completely inhibits the percutaneous infection through the abdominal skin. The number of cercariae that develop to schistosomules is reduced by more than 90%. After a 2-h incubation in a concentration of 0.01 g/ml the swimming ability of cercariae is impaired in such a way that the number of cercariae penetrating in the tail immersion test and developing to schistosomules is reduced by half.Praziquantel is a more potent protective agent than the molluscicides copper sulphate, sodium pentachlorophenate and Bayluscide^® or cadmium and zinc ions.

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Zusammenfassung Es wurde der Einfluß des neuen Schistosomenmittels Praziquantel (2-Cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1 a]isochinolin-4-on) auf die Miracidien und Cercarien vonSchistosoma mansoni untersucht.In vivo verhindert Praziquatel das Schlüpfen von Miracidien aus Eiern für 24 h nach einer Behandlung infizierter Mäuse mit 500 mg/kg. Nach Inkubation von Eiern in 10 g/ml schlüpfen anschließend keine Miracidien in präparatfreiem Wasser. Frei schwimmende Miracidien werden durch 1 g/ml schnell abgetötet und noch 0,01 g/ml haben eine deutliche Wirkung.Cercarien werden in vitro durch 0,03 g/ml innerhalb von 10 min schwimmunfähig. Dieser Effekt ist in präparatfreiem Wasser reversibel. Nach 3stündiger Vorbehandlung in 0,1 g/ml sind die Cercarien deutlich morphologisch geschädigt. Trotzdem sind sie noch normal entwicklungsfähig, wenn sie Mäusen subcutan injiziert werden. Nach Inkubation in 1 g/ml ist der Infektionserfolg allerdings um 80% reduziert. Nach nur 2stündiger Inkubation in 0,1 g/ml ist die Zahl der Cercarien, die sich nach percutaner Infektion durch die Bauchhaut der Maus zu Schistosomulae entwickelt, um mehr als 90% reduziert. Nach 2stündiger Inkubation in nur 0,01 g/ml ist das Schwimmvermögen der Cercarien so beeinträchtigt, daß sich nach percutaner Schwanzinfektion nur noch halb so viele zu Schistosomulae entwickeln wie in der Kontrolle.Praziquantel ist in seiner infektionsverhindernden Wirkung den Molluskiziden Kupfersulfat, Pentachlorphenolnatrium und Bayluscid^® sowie auch Cadmium- und Zinkionen überlegen.

     

Intracellular calcium in primary cultures of rat renal inner medullary collecting duct cells during variations of extracellular osmolality

There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Ca_i under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110±4 nmol/l; n=119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563±87 nmol/l (n=7; P<0.01), followed by a decrease over 4–6 min to twice the initial values. The calcium increase was smaller (260±14 nmol/l; n=5; P<0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 mol/l), D-600 (40 mol/l), diltiazem (40 mol/l), and nifedipine (40 mol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La³⁺) and gadolinium (Gd³⁺) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 mol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/ or voltage dependent) remains to be determined. The transient increase in Ca_i in turn may trigger inorganic and organic osmolyte fluxes observed previously.     

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on muscarinic receptor-induced Ca2+ mobilization in a human salivary epithelial cell line

We have investigated the effect of W-7, a calmodulin (CaM) antagonist, on Ca²⁺ mobilization in a human salivary epithelial cell line, HSG-PA, after muscarinic receptor stimulation. In a medium containing 1.5 mmol/l Ca²⁺, W-7 reduced both the maximum peak increase in cytosolic Ca²⁺ ([Ca²⁺]_i) which follows stimulation by carbachol (Cch, 100 mol/l) and the sustained nature of the response. Using an experimental approach which allows separate visualization of the intracellular Ca²⁺ release and extracellular Ca²⁺ entry phases, W-7 was shown preferentially to inhibit Ca²⁺ release. At 100 mol/l W-7, Cch-induced Ca²⁺ release was completely inhibited, but Cch-induced Ca²⁺ entry was partially (40%) maintained. This W-7 residual Ca²⁺ entry response was abolished when cells were depolarized with high K⁺ or gramicidin D. W-7 also substantially inhibited Cch-induced inositol trisphosphate (IP₃) production (75%). W-5, a less potent CaM antagonist than W-7, had markedly smaller effects on Cch-induced Ca²⁺ mobilization and IP₃ formation. W-7 (100 mol/ l) completely blocked (comparable to 10 mol/l atropine) the binding of the muscarinic antagonist [³H] quinuclidinyl benzilate (QNB) to muscarinic receptors on cell membranes, whereas Cch (at 100 mol/l) had minimal effects on ligand binding. W-7 and W-5 were equipotent in their ability to inhibit [³H] QNB binding. These results suggest that W-7 reduces Ca²⁺ mobilization in HSG-PA cells by a mechanism which likely involves the antagonism of a CaM regulatory step(s) but may also involve at least a partial blockade of the muscarinic receptor. In addition, in the presence of W-7, Ca²⁺ entry can occur via a receptor-operated Ca²⁺ pathway which is modulated by membrane potential.     

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the K_ATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 M) and ADP (100 M) reversibly increased the channel activity. In the presence of internal Mg²⁺ (1 mM), low concentrations of ADP (<300 M) enhanced channel activity and high concentrations of ADP (>300 M) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg²⁺, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [S] (100 M), which behaved as a weak GDP agonist in the presence of Mg²⁺, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K⁺ channel opener pinacidil was found to activate the K_ATP channel but only in the presence of internal GDP, ADP and GDP [S]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the K_ATP channel.     

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of α2-adrenergic and A1-adenosine agonists,insensitive to pertussis toxin and dependent on extracellular calcium

The accumulation of cyclic adenosine 3,5-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A₁ agonist of adenosine (-)-N ⁶-(R-phenylisopropyl) adenosine (PIA), an ₂-adrenergic agonist clonidine (CLO), and prostaglandin E₂ (PGE₂). The negative regulation elicited by PGE₂ was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 M PGE₂ with either 0.1 M PIA or 1 M CLO led to an inhibition of the response to AVP (80.0±3.5%, SEM, N=7 and 92.6±0.8%, N=5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE₂ in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 g/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE₂. PGE₂-induced inhibition was prevented in a calcium-free medium [0 Ca²⁺+0.1 mM [ethylene-bis (oxyethylenenitrilo)] tetraacetate (EGTA)]: values were 67.0 ±2.1% and 5.8±8.7% (± SEM) in 2 mM Ca²⁺ and 0 Ca²⁺ medium, respectively, N=7. When applied to Fura-2-loaded OMCD, 0.3 M PGE₂ increased intracellular calcium concentration ([Ca²⁺]_i) with a peak phase (in 2 mM or 0 Ca²⁺ medium) followed by a plateau phase (observed only in 2 mM Ca²⁺ medium). It is concluded that: (1) in the rat OMCD, PGE₂, PIA and CLO act on the same AVP-sensitive cell, (2) PGE₂ induces a cumulative inhibition on the cAMP level when combined with other inhibitors by a mechanism insensitive to pertussis toxin, (3) the presence of extracellular calcium is a prerequisite condition to observe PGE₂-induced inhibition, and (4) the inhibition by PGE₂ might be linked to its capacity of increasing [Ca²⁺]_i.