Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Increased 4‐hydroxy‐2‐nonenal‐induced proteasome dysfunction is correlated with cardiac damage in streptozotocin‐injected rats with isoproterenol infusion

Increase in 4‐hydroxy‐2‐nonenal (4HNE) due to oxidative stress has been observed in a variety of cardiac diseases such as diabetic cardiomyopathy. 4HNE exerts a damaging effect in the myocardium by interfering with subcellular organelles like mitochondria by forming adducts. Therefore, we hypothesized that increased 4HNE adduct formation in the heart results in proteasome inactivation in isoproterenol (ISO)‐infused type 1 diabetes mellitus (DM) rats. Eight‐week‐old male Sprague Dawley rats were injected with streptozotocin (STZ, 65 mg kg^?1). The rats were infused with ISO (5 mg kg^?1) for 2 weeks by mini pumps, after 8 weeks of STZ injection. We studied normal control (n = 8) and DM + ISO (n = 10) groups. Cardiac performance was assessed by echocardiography and Millar catheter at the end of the protocol at 20 weeks. Initially, we found an increase in 4HNE adducts in the hearts of the DM + ISO group. There was also a decrease in myocardial proteasomal peptidase (chymotrypsin and trypsin‐like) activity. Increases in cardiomyocyte area (446 ± 32·7 vs 221 ± 10·83) (µm²), per cent area of cardiac fibrosis (7·4 ± 0·7 vs 2·7 ± 0·5) and cardiac dysfunction were also found in DM + ISO (P < 0·05) relative to controls. We also found increased 4HNE adduct formation on proteasomal subunits. Furthermore, reduced aldehyde dehydrogenase 2 activity was observed in the myocardium of the DM + ISO group. Treatment with 4HNE (100 μM) for 4 h on cultured H9c2 cardiomyocytes attenuated proteasome activity. Therefore, we conclude that the 4HNE‐induced decrease in proteasome activity may be involved in the cardiac pathology in STZ‐injected rats infused with ISO. Copyright © 2016 John Wiley & Sons, Ltd.     

Cholecystokinin and tryptic activity in the gut and body of developing Atlantic halibut larvae: evidence for participation in the regulation of protein digestion

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual^?1) and the tryptic activity [μmol arginine‐methyl‐coumarinyl‐7‐amide (MCA) min^?1 individual^?1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2 ± 0·1 and 0·14 ± 0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67 ± 5·58 and 2·71 ± 0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (M_G, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFF M_G represented 5·5% of whole body dry mass (M_w, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min^?1 per mg dry mass (M)] in the gut increased from 2·74 ± 1 ± 98 at 13 DAFF to 5·00 ± 0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M^?1) but no significant differences were found, although the data indicated an increase (from 23·38 ± 11·26 at 13 DAFF to 36·27 ± 8·96 fmol M^?1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube‐feeding appeared to stimulate post‐prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Interactions between gnathiid isopods,cleaner fish and other fishes on Lizard Island,Great Barrier Reef

The rate of emergence of micropredatory gnathiid isopods from the benthos, the proportion of emerging gnathiids potentially eaten by Labroides dimidiatus, and the volume of blood that gnathiids potentially remove from fishes (using gnathiid gut volume) were determined. The abundance (mean ±s.e .) of emerging gnathiids was 41·7 ± 6·9 m^?2 day^?1 and 4552 ± 2632 reef^?1 day^?1 (reefs 91–125 m²). The abundance of emerging gnathiids per fish on the reef was 4·9 ± 0·8 day^?1; but excluding the rarely infested pomacentrid fishes, it was 20·9 ± 3·8 day^?1. The abundance of emerging gnathiids per patch reef was 66 ± 17% of the number of gnathiids that all adult L. dimidiatus per reef eat daily while engaged in cleaning behaviour. If all infesting gnathiids subsequently fed on fish blood, their total gut volume per reef area would be 17·4 ± 5·6 mm³ m^?2 day^?1; and per fish on the reefs, it would be 2·3 ± 0·5 mm^?3 fish^?1 day^?1 and 10·3 ± 3·1 mm³ fish^?1 day^?1 (excluding pomacentrids). The total gut volume of gnathiids infesting caged (137 mm standard length, L_S) and removed from wild (100–150 mm L_S) Hemigymnus melapterus by L. dimidiatus was 26·4 ± 24·6 mm³ day^?1 and 53·0 ± 9·6 mm³ day^?1, respectively. Using H. melapterus (137 mm L_S, 83 g) as a model, gnathiids had the potential to remove, 0·07, 0·32, 0·82 and 1·63% of the total blood volume per day of each fish, excluding pomacentrids, caged H. melapterus and wild H. melapterus, respectively. In contrast, emerging gnathiids had the potential of removing 155% of the total blood volume of Acanthochromis polyacanthus (10·7 mm L_S, 0·038 g) juveniles. That L. dimidiatus eat more gnathiids per reef daily than were sampled with emergence traps suggests that cleaner fishes are an important source of mortality for gnathiids. Although the proportion of the total blood volume of fishes potentially removed by blood‐feeding gnathiids on a daily basis appeared to be low for fishes weighing 83 g, the cumulative effects of repeated infections on the health of such fish remains unknown; attacks on small juvenile fishes, may result in possibly lethal levels of blood loss.     

Chronic stress of rainbow trout Oncorhynchus mykiss at high altitude: a field study

The stress response of Oncorhynchus mykiss in high‐altitude farms in central Mexico was investigated over two seasons: the cool (9·1–13·7° C) dry winter season, and the warmer (14·7–15·9° C), wetter summer season. Fish were subjected to an acute stress test followed by sampling of six physiological variables: blood cortisol, glucose, lactate, total antioxidant capacity, haemoglobin concentration and per cent packed cell volume (V_PC%). Multivariate analyses revealed that lactate and total antioxidant capacity were significantly higher in the summer, when water temperatures were warmer and moderate hypoxia (4·9–5·3 mg l^?1) prevailed. In contrast, plasma cortisol was significantly higher in the winter (mean ± s.e .: 76·7 ± 4·0 ng ml^?1) when temperatures were cooler and dissolved oxygen levels higher (6·05–7·9 mg l^?1), than in the summer (22·7 ± 3·8 ng ml^?1). Haemoglobin concentrations (mg dl^?1) were not significantly different between seasons, but V_PC% was significantly higher in the summer (50%) than in the winter (35%). These results suggest that in summer, effects of high altitude on farmed fish are exacerbated by stresses of high temperatures and hypoxia, resulting in higher blood lactate, increased total antioxidant capacity and elevated V_PC% levels.     

Measurement of blood volume in the elasmobranch fish Scyliorhinus canicula following acute and long‐term salinity transfers

A technique using ⁵¹chromium‐labelled erythrocytes was used to measure blood volume in Scyliorhinus canicula following long‐term and acute salinity transfers. Basal whole‐blood volume was 5·6 ± 0·2 ml 100 g^?1 (mean ±s .e .), this increased (6·3 ± 0·2 ml 100 g^?1) following +14 day acclimation to 80% sea water (SW) and decreased (4·6 ± 0·2 ml 100 g^?1) following acclimation to 120% SW. These changes were shown to be primarily due to changes in plasma volume, with no significant changes in extrapolated red‐cell volume being demonstrated. Blood volume was also measured in the same animals during 10 h acute transfer to 100% SW. Plasma volume in S. canicula during acclimation from 80% SW was significantly reduced (4·5 ± 0·3 ml 100 g^?1) after 6 h of transfer to 100% SW. Blood volume in animals during acclimation from 120% SW was significantly increased (4·8 ± 0·2 ml 100 g^?1) after 4 h of acute transfer. The osmoregulatory implications of these different timeframes during hyposaline and hypersaline transfer are discussed, along with the importance of this in vivo technique as context for in vitro studies with haemo‐dynamic stimuli.     

Zinc and salinity effects on membrane transport in Chara connivens

Pressure-probe measurements showed that the pressure relaxation of internodal cells of the freshwater alga Chara connivens slowed considerably when 1–5 mol m^?3 Zn²⁺, or more especially Zn²⁺ and 75 mol m^?3 NaCl, were present in the medium for periods of 1 h or longer. These results indicate that the water permeability of the Chara membrane is decreased by Zn²⁺, and that this effect is enhanced by 75 mol m^?3 NaCl. Specific values taken after 375 min exposure were: 5 mol m^?3 Zn²⁺ and 75 mol m^?3 NaCl caused the half-time for bulk water movement to increase from 7·8±2·3 to 79·5±5·4s, corresponding to a decrease in the hydraulic conductivity (Lp) from (13·0±3·3) × 10^?7 m s^?1 mPa^?1 to (1·25±0·23) × 10^?7 m s^?1 MPa^?1 (mean±S.D., n= 10). These changes are not seen in the presence of NaCl alone, and to a reduced extent in the presence of 5 mol m^?3Zn²⁺ alone (after 375 min, Lp was (2·4±0·1) × 10^?7 m s^?1 MPa^?1, mean±S.D., n = 6). Ca²⁺ cannot substitute for Zn²⁺, but seems to competitively inhibit Zn²⁺. There was another, kinetically distinct effect of Zn²⁺: the ingress of Na⁺ within 15 min of exposure to 75 mol m^?3 NaCl is halved by the presence of 1–5 mol m^?3 Zn²⁺, although internal osmolality is little changed by Zn²⁺. In spite of this, Zn²⁺ does not exert the long-term protection against NaCl that has been reported for Ca²⁺. Depending on the concentration of Zn²⁺ and the duration of the exposure, the effects on water permeability were fully or partly reversible within 24–48 h. The mechanism of these changes is difficult to identify. One possibility is a zinc-induced restriction of trans-membrane channels to give single-file channels which can be blocked by salt.     

Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

Stimulation of phosphatidylcholine biosynthesis by hemicholinium-3, a potent inhibitor of choline uptake in human leukemic monocyte-like U937 cells

The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [³H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [³H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [³H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.     

COMPARISON OF DEVELOPMENT AND PHOTOSYNTHETIC GROWTH FOR FILAMENT CLUMPS AND REGENERATED MICROPLANTLET CULTURES OF AGARDHIELLA SUBULATA (RHODOPHYTA, GIGARTINALES)

Two axenic, in vitro liquid suspension cultures were established for Agardhiella subulata (C. Agardh) Kraft et Wynne, and their growth characteristics were compared. This study illustrated how reliable routes for the development of suspension cultures of macrophytic red algae of terete thallus morphology can be achieved for biotechnology applications. Undifferentiated filament clumps of 2–8 mm diameter were established by induction of callus-like tissue from thallus explants, and lightly branched microplantlets of 2–10 mm length were established by regeneration of filament clumps. The filament clumps were susceptible to regeneration. Adventitious shoot formation was reliably induced from 40% to 70% of the filament clumps by gentle mixing at 100 rev min^?1 on an orbital shaker. The specific growth rate of the microplantlets was higher than the filament clumps in nonagitated well plate culture (4%–6% per day for microplantlets vs. 2%–3% per day for filament clumps) at 24° C and 8–36 μmol photons·m^?2·s^?1 irradiance (10:14 h LD cycle) when grown on ASP12 artificial seawater medium at pH 8.6–8.9 with 20%–25% per day medium replacement. Oxygen evolution rate vs. irradiance measurements showed that relative to the filament clumps, microplantlets had a higher maximum specific oxygen evolution rate (P_o,max= 0.181 ± 0.035 vs. 0.130 ± 0.023 mmol O₂·g^?1 dry cell mass·h^?1), but comparable respiration rate (Q_o= 0.040 ± 0.013 vs. 0.033 ± 0.017 mmol O₂·g^?1 dry cell mass·h^?1), compensation point (I_c= 3.8 ± 2.4 vs. 5.7 ± 1.2 μmol photons·m^?2·s^?1), and light intensity at 63.2% of saturation (I_k= 17.5 ± 3.9 vs. 14.9 ± 2.6 μmol photons·m^?2·s^?1). The microplantlet culture was more suitable for suspension culture development than the filament clump culture because it was morphologically stable and exhibited higher growth rates.     

Introducing specific antibodies into electropermeabilized cells is a valuable tool for eliminating specific cell functions

A technique is established for the role of intracellular proteins to be eliminated and thereby gives information about their specific role in signal transduction within cells. Rat pancreatic islets as well as INS-1 cells (an insulin secreting cell line) were electrically permeabilized in order to introduce high molecular weight compounds. Optimized conditions were five exposures with 15-s intervals, τ=200 ms, an electric field of 1·36 kV per 0·4 cm in a specific permeabilization buffer at a calculated Ca⁺⁺ concentration of 5×10⁻⁸ M . In electroporation control experiments the spectrophotometrically measured uptake of the cell membrane-impermeable propidium iodide, FITC-labelled dextran (MW∼4000) and FITC-labelled antibodies (MW∼150,000) was established as being 81·5±5·0, 82·7±3·0 and 81·0±1·0 per cent of maximum, respectively. These data were corroborated qualitatively by visualizing microscopically the fluorescence of the FITC-labelled compounds in islets as well as in INS-1 cells. The cells appear to reseal since control experiments indicated a short-lived outflow of lactate dehydrogenase (MW of 140,000 which is similar to that of antibodies) and of insulin for the first 15–20 min. After electroporation the cells were functionally intact, i.e. responded to the stimulus carbachol (CCh). Only 18·0±10·1 per cent of cells had not resealed after 2 h (propidium iodide uptake measured at various time intervals after electroporation). As was shown recently the effect of specific compounds such as CCh and CCK₈ on insulin release was eliminated selectively by antibodies against specific G proteins thus proving this method to be a valuable tool. In conclusion, adding antibodies to electrically permeabilized cells is a valuable tool for eliminating a specific cell function in order to elucidate the specific role of intracellular compounds. This method can probably be used for testing the specific role of other proteins in cell functions. © 1997 John Wiley & Sons, Ltd.