The countryside or the city: Which environment is better for the honeybee?

For a number of years, the decline of honeybee (Apis mellifera) in North America and Europe has been the subject of much debate. Among the many factors proposed by hundreds of studies to explain this phenomenon is the hypothesis that agricultural activities using pesticides contribute to the weakness of bee colonies. Moreover, while urban beekeeping is presently booming in several cities, we do not know if this environment is more beneficial for bees than the typical, rural area. In the summer of 2018, we sampled honeybees (foragers and larvae) in rural (Laurentians) and urban (city of Montreal) areas and compared them using the following biomarkers: carotenoids, retinoids, α-tocopherol, metallothionein-like proteins (MTLPs), lipid peroxidation, triglycerides, acetylcholinesterase activity (AChE) and proteins. Pesticides, pharmaceuticals and personal care products (PPCPs) and metals were also quantified in honeybees' tissues. Our result revealed that, globally, urban foragers had higher levels of insecticides and PPCPs and that metals were in greater concentrations in urban larvae. Compared to rural foragers, urban foragers had higher concentrations of MTLPs, triglycerides, protein and AChE activity. The multifactorial analysis confirmed that insecticides, some metals and PPCPs were the most influential components in the contaminant‒biomarker relationships for both foragers and larvae.

Capacitative calcium entry and proliferation of human osteoblast-like MG-63 cells.

UNLABELLED: Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca2+) control in numerous cellular functions; however, involvement of capacitative Ca2+ entry (CCE) in proliferation of bone cells is less well investigated. OBJECTIVES: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. MATERIALS AND METHODS: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. RESULTS: Intracellular Ca2+ store depletion by thapsigargin induced CCE in MG-63 cells; this was characterized by a rapid transient increase of intracellular Ca2+ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet-derived growth factor. Inhibitors of store-operated Ca2+ channels (2-APB and SKF-96365) prevented CCE, while voltage-dependent Ca2+ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage-dependent Ca2+ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2-APB and SKF-96395, inhibited it. Cell cycle analysis showed that 2-APB and SKF-96395 lengthen the S and G2/M phases, which would account for the reduction in cell proliferation. CONCLUSIONS: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation.

Reciprocal inhibition of Cd and Ca uptake in isolated head kidney cells of rainbow trout (Oncorhynchus mykiss).

Some environmental pollutants, including cadmium (Cd) and zinc (Zn), can act as endocrine disruptors in fish, either in vivo or through a direct action on steroidogenic cells, as has been demonstrated in vitro. We have previously characterized Cd uptake in head kidney (homologue of mammalian adrenal) cells of rainbow trout (Oncorhynchus mykiss) and have provided evidence for a Cd/Ca interaction. Here, we pursued our investigation of metal competition for uptake. Our results show that inorganic speciation conditions favour Cd uptake with optimal level of accumulation for Cd2+ compared to chlorocomplexes (CdCl(n)(2-n)). Calcium uptake was studied for the first time in the fish head kidney cells and Ca was found to be less efficiently accumulated compared to Cd. A specific saturable mechanism of transport was characterized for Ca uptake but voltage-gated or La-sensitive cationic channels are unlikely to contribute appreciably. A concentration-dependent reciprocal inhibition was observed between Ca and Cd, whereas, Zn proved to inhibit Cd uptake exclusively. Additive inhibitory effect on Cd uptake was obtained with co-exposure to Ca and Zn. We conclude that Cd, but not Zn, may decrease Ca availability to the head kidney tissue. Also, Zn may partially protect against Cd toxicity but Zn would not protect against Cd-induced perturbation of Ca homeostasis.

Cadmium-induced apoptosis in rat hepatocytes does not necessarily involve caspase-dependent pathways.

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. Depending on the exposure conditions, Cd may cause necrosis or apoptosis. Oxidative stress is believed to participate in Cd toxicity but the molecular signaling responsible for Cd-induced apoptosis in non-malignant liver cells still needs to be clarified. Therefore we have studied apoptosis in primary cultures of rat hepatocytes incubated with low levels of Cd for short exposure times. Studies of nuclear morphology, chromatin condensation, and oligonucleosomal DNA fragmentation demonstrate that 1-5 microM Cd induces apoptosis as early as 6-12 h with minor effects on MTT activity. A concomitant time- and concentration-dependent increase in caspase-9 and -3 activities was observed, whereas Cd did not affect caspase-8 activity as much, suggesting a minor role of the death-receptor pathway. Significant release of cytochrome c into the cytosol demonstrated the involvement of a mitochondrial-dependent apoptotic pathway. However, cell pre-treatment with caspase inhibitors (Z-VAD-fmk or Ac-DEVD-CHO) did not prevent apoptosis. Increases in the cytosolic levels of the mitochondrial apoptosis-inducing factor (AIF) were also observed: kinetics of cytochrome c and AIF release were similar. These results show that Cd-induced apoptosis in rat hepatocytes is time- and concentration-dependent. The early apoptotic events involved mitochondrial-dependent pathways but not necessarily caspase-dependent signaling.

Multiple effects of mercury on cell volume regulation, plasma membrane permeability, and thiol content in the human intestinal cell line Caco-2.

In a previous study, we characterized Cd-Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation value of 0.3 micromol/L (203)Hg was measured in the presence of 100 micromol/L unlabeled Hg in the serum-free exposure medium without modification in the initial uptake rate. This phenomenon was eliminated at 4 degrees C. Mercury induced an increase in tritiated water and [(3)H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 micromol/L Hg and 2 mmol/L N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects. Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport. During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and consequently cell capacity.

Reciprocal inhibition of Cd and Pb sulfocomplexes for uptake in Caco-2 cells.

Cadmium-lead interactions for uptake were studied in the TC7 clone of human enterocytic-like Caco-2 cells as a function of inorganic metal speciation. We have previously shown that Cd uptake in these cells involves both the free cation Cd2+ and chlorocomplex (CdCln(2-n)) species. Here we show 1.9 times higher uptake levels for 109CdCln(2-n) compared to 210PbCln(2-n). Reciprocal inhibitions of chlorocomplexes were observed with a much higher inhibitory effect of Cd compared to Pb. Replacing Cl- by NO3- increased both the level of aquo ion 109Cd2+ and 109Cd accumulation. In contrast, higher levels of 210Pb2+ did not favor 210Pb uptake. For both metals, higher uptake data were recorded in the presence of SO4(2-), leading to sulfocomplex formation, compared with Cl-. Reciprocal inhibitions were minimal at high-cation levels but were significant and comparable in the presence of sulfo-complexes. We conclude that, in addition to Cd2+ (but not Pb2+), sulfocomplexes of both metals would preferentially be taken up compared to chlorocomplexes. NRAMP2 is not involved in Pb2+ uptake, and the NRAMP2-mediated Cd2+ uptake is insensitive to Pb. Uptake of Pb chlorocomplexes could involve specific mechanisms but of very low affinity, whereas uptake of Pb sulfocomplexes occurs with high affinity.

Cadmium uptake in isolated adrenocortical cells of rainbow trout and yellow perch.

Cadmium uptake was studied in isolated adrenocortical cells of rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens) to test the hypothesis that the greater sensitivity of trout cells to Cd-induced disruption of cortisol secretion observed in previous studies is correlated to higher level of metal accumulation. There was no evidence for interspecies differences in accumulation level, and a specific transport mechanism of similar affinity has been characterized in both fish species. However, inhibition of Cd uptake by calcium was observed in rainbow trout exclusively. The free metal ion Cd(2+) and chlorocomplexes CdCl(n)(2-n) both contribute to Cd accumulation with different level of contribution between fish species. We conclude that interspecies differences in sensitivity to Cd endocrine disrupting effect are not necessarily related to different levels of metal accumulation but would rather be linked to transport pathways and metal speciation. Cadmium/calcium competition for uptake could be a determinant of the early Cd-induced impaired cortisol secretion in trout but not perch cells.

Cadmium uptake in rat hepatocytes in relation to speciation and to complexation with metallothionein and albumin.

Cadmium (Cd) uptake has been studied in primary cultures of rat hepatocytes focusing on the impact of inorganic and organic speciation. Uptake time-course studies over a 60-min exposure to 0.3 microM (109)Cd revealed a zero-time uptake and a slower process of accumulation which proceeds within minutes. (109)Cd uptake showed saturation kinetics (K(m) = 3.5 +/- 0.8 microM), and was highly sensitive to inhibition by Zn and Hg. There was no evidence for sensitivity to the external pH nor for any preferential transport of the free cation Cd(2+) over CdCl(n) (2-n) chloro-complexes. According to the assumption that only inorganic metal species are available, metal uptake decreased upon albumin (BSA) addition to the exposure media. In contrast, higher levels of (109)Cd accumulation were obtained under optimal conditions for Cd complexation by MT. Comparison among uptake data obtained under inorganic and organic conditions revealed that Cd-MT would be taken up 0.4 times as rapidly as Cd(inorg). We conclude that uptake of Cd in rat hepatocytes involves specific transport mechanism(s) subjected to Zn or Hg interactions. Uptake of inorganic Cd is not proportional to the levels of free Cd(2+) and does not involve the divalent cation transporter DCT1 nor the co-transporter Fe(2+)-H(+) NRAMP2. We found Cd-MT but not Cd-BSA to be available for the liver cells, and have estimated a binding affinity four orders of magnitude higher for Cd complexation with MT compared to BSA; MT may have a significant role in Cd delivery to the liver.

Cadmium-induced mitochondrial membrane-potential dissipation does not necessarily require cytosolic oxidative stress: studies using rhodamine-123 fluorescence unquenching.

The impact of cadmium on the cellular redox state and mitochondrial membrane potential (psi(m)) has been studied by monitoring dichlorofluorescein (DCF), CMXRos (dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine), and Rh-123 fluorescence in 5-day-old TC7 cells, a highly differentiated clone of the human intestinal Caco-2 cell line. Flow cytometry analyses, using DCFH oxidation to DCF, clearly revealed that a 30-min incubation to 50 microM cadmium (Cd) is sufficient to induce reactive oxygen species (ROS) formation; this effect was completely eliminated by the presence of 50 mM mannitol for the 30-min incubation period, but mannitol only partially scavenged ROS for the longer period of time studied. Imaging studies using fluorescence video microscopy revealed a parallel increase in (DCF) fluorescence in the nuclear and cytoplasmic regions as soon as Cd was added to the exposure medium. Flow cytometry analyses monitoring CMXRos fluorescence clearly showed that Cd also leads to psi(m) disruption, but, contrary to what was observed for ROS formation, mannitol was completely inefficient in preventing this effect. Further investigation using fluorescence microscopy and Rh-123 fluorescence unquenching revealed that although mannitol did not protect against Cd-induced dissipation of psi(m), it considerably delayed the process. We found that Rh-123 unquenching, occurring during probe redistribution, is a suitable tool to monitor the decrease of psi(m). We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH(*), as well as the loss of psi(m). However, psi(m) dissipation does not necessarily require cellular OH(*) and may occur in the absence of apparent oxidative injury.

Cadmium transport through type II alveolar cell monolayers: contribution of transcellular and paracellular pathways in the rat ATII and the human A549 cells.

Cadmium (CD) transport in alveolar type II (ATII) cells has been studied using two in vitro models widely used to investigate lung function: primary cultures of rat ATII cells and the human cell line A549. Nonlinear regression analyses of the uptake time-course of (109)Cd revealed: a zero-time accumulation, a fast process of accumulation which proceeds within minutes, and a much slower process which takes hours. This three-step mechanism was characterized by different parameter values under dishes-or filter-growth conditions. A higher initial uptake rate (v(i)) and equilibrium accumulation (A(max)) of (109)Cd were found in the rat ATII cells; these differences were not related to a higher level of adsorption onto the external surface of the cell membrane. Specific transport systems of similar capacity but different affinity (threefold higher in rat cells) were characterized. A significant transepithelial transport of (109)Cd, with similar P(coeff) in both cell models, could not be exclusively related to cellular metal release. Results on 3H-mannitol permeability together with (109)Cd efflux data strongly suggest a greater contribution of the paracellular pathways in Cd transport through A549 cell monolayers. These differences in transport properties between the two lung cell models may modify the dose-response curve for Cd toxicity.

Evidence for cadmium uptake through Nramp2: metal speciation studies with Caco-2 cells.

The specific uptake of 0.3 microM (109)Cd by the TC7 clone of the human enterocytic-like Caco-2 cells increased 4-fold as the pH(out) was lowered from 7.5 to 5.5; the stimulatory effect of acidic media being more pronounced when the level of the free ion (109)Cd(2+), relative to total (109)Cd, was increased. The initial uptake rate was 12-fold higher under conditions, optimizing (109)Cd(2+) accumulation over that of (109)CdCl(2-n)(n) (NO(-)(3)/pH(out) 5.5); a saturable system of transport has been characterized (K(m) = 1.1 +/- 0.1 microM, V(max) = 87 +/- 3 pmol/3 min/mg protein). An excess of Fe(2+) failed to affect (109)Cd uptake when the pH(out) was 7.4, whereas a strong inhibition was observed under NO(-)(3)/pH(out) 5.5 conditions. In contrast, the maximal inhibitory effect of Zn(2+) was observed under Cl(-)/pH(out) 7.4 conditions. This results strongly suggest that Fe(2+) may compete with Cd(2+) for Nramp2, whereas Zn and CdCl(2-n)(n) compete for another system of transport that has yet to be identified.

Cadmium uptake by Caco-2 cells: effects of Cd complexation by chloride, glutathione, and phytochelatins.

Short-term cadmium uptake by the highly differentiated TC7 clone of enterocytic-like Caco-2 cells was studied as a function of Cd speciation. For low metal concentrations and with a constant free [Cd(2+)] = 43 nM, initial uptake rates of (109)Cd increased linearly as a function of increasing concentration of chlorocomplexes (Sigma[(109)CdCl(2-n)(n)]) over the range from 0 to 250 nM. When normalized as a function of the metal concentration, the absorption rate for the chlorocomplexes was less than that estimated for uptake of the free Cd(2+) cation. Metal absorption decreased upon organic ligand addition in the exposure media, but much less than predicted from the assumption that only inorganic metal species would be transported. Under exposure conditions where the concentration of each of the inorganic species was kept constant, (109)Cd uptake increased with increasing concentrations of cadmium glutathione ((109)Cd-GSH) or phytochelatin ((109)Cd-hmPC(3)) complexes. A specific system of very high affinity but low capacity has been characterized for (109)Cd-GSH transport, whereas accumulation data increased linearly with (109)Cd-hmPC(3) up to 6 microM. Comparison among uptake data for 0.3 microM inorganic (109)Cd, (109)Cd-GSH, or (109)Cd-hmPC(3) yields the following accumulation ratios: Cd-GSH/Cd(inorg) = 0.2; Cd-hmPC(3)/Cd(inorg) = 0.5. These results clearly show that Cd(2+) is not the exclusive metal species participating in Cd absorption, though, for comparable Cd concentrations, its contribution to transport would be more important than that of other species. Cadmium bound to thiol-containing peptides may be absorbed via transport systems that differ from those involved in absorption of the inorganic metal species.

Evidence for an intracellular barrier to cadmium transport through Caco-2 cell monolayers.

109Cd transport was studied in the highly differentiated TC7 clone of the enterocytic-like Caco-2 cells grown on filters. Accumulation curves for 0.3 microM 109Cd over 12 h from the apical (AP) or the basal (BL) sides revealed a three-step mechanism involving: 1) a zero-time accumulation Ao; 2) a fast process Af(t1/2 < or = 10 min); and 3) a slow process of uptake As (5 h < or = t1/2 < or = 10 h) responsible for the major cellular levels of 109Cd. The relative contribution of adsorption to total accumulation is greater for short exposure times (< or = 35%), but is no longer significant after the exposure times needed to reach equilibrium. Transepithelial transport was less than 4% of the cellular level at 12 h. A negligible but specific binding onto the BL surface of the filters was characterized. Saturable systems of accumulation with comparable affinities (Km = 2.5+/-0.5 and 5.4+/-0.4 microM) but distinct capacities (Vmax = 8.9+/-1.2 and 312+/-22 pmol/min/mg protein) were identified at the AP and BL cell membranes, respectively. Efflux studies revealed that Cd accumulation is only partially reversible, with an exclusive metal release at the same side. A 2-h exposure on both sides simultaneously failed to demonstrate any competition for cellular accumulation: uptake was additive relative to AP and BL uptake values. These data suggest that Af leads to an accumulation of loosely bound Cd, whereas As represents irreversible intracellular binding processes. We conclude that Cd transport occurs exclusively by a transcellular route and that saturation of the intracellular high-capacity binding sites is the rate-limiting step in Cd absorption.

Caco-2 cell line used as an in vitro model to study cadmium accumulation in intestinal epithelial cells.

109Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 microM 109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t(1/2) = 17.3 +/- 1.3 min) with an apparent in-to-out distribution ratio (alphae) of 11.6 +/- 0.8. The amplitude of the rapid phase (U0) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable alphae values were observed at equilibrium. In both clones, the t(1/2) and alphae values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, 109Cd uptake at 1 min (U1) was unaffected by Ca concentrations four order of magnitude in excess, but both U0 and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U0 disappeared when EDTA was present in the wash solutions. U1 showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (Km = 3.8 +/- 0.7 microM). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of 109Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption.

Triiodothyronine stimulates the expression of sucrase-isomaltase in Caco-2 cells cultured in serum-free medium.

In a previous study we have shown that triiodothyronine (T3) added to a serum-free medium supplemented with insulin, transferrin, and selenous acid (ITS) can stimulate Caco-2 cell differentiation. In this study we have focused on the effects of T3 on sucrase activity. The results obtained demonstrate that T3 (50 nM) does not change Caco-2 cell proliferation but enhances sucrase activity from 50 to 80%. Similar increases were observed whether or not insulin was present in the culture medium, showing that there was no synergistic effect between T3 and insulin on sucrase activity. Moreover, T3 acts specifically during the differentiation period since addition of T3 to the defined TS medium before confluency is reached does not stimulate sucrase activity. Sucrase kinetic parameters were evaluated for the first time in Caco-2 cells under various culture conditions. The presence of a single enzyme was verified, with a Km of about 7 mM and a Vmax around 20 nmol of substrate hydrolyzed min-1 mg-1 of protein. Our results showed that T3 did not change the enzyme's affinity for sucrose but doubled the Vmax. Moreover, immunoblotting using anti-sucrase-isomaltase (SI) antibodies revealed an approximately twofold increase in the relative amount of SI immunoreactive protein in T3-stimulated cells compared to untreated cells. Results obtained by both Northern hybridization and RT-PCR amplification showed a significant increase in SI mRNA contents. These results suggest that T3 acts primarily on sucrase expression at the mRNA level.

Ornithine decarboxylase activity is associated with proliferation but not with T3-induced differentiation of Caco-2 cells.

Ornithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, spermine) concentrations were measured in parallel in enterocyte-like Caco-2 cells maintained under various culture conditions. ODC activity was maximal at the beginning of the exponential growth phase, decreasing dramatically thereafter to a negligible level at confluency (day 9). Kinetic studies performed on day 3 revealed the presence of a single enzyme with a Km around 200 microM and a Vmax of about 2 nmol CO2 released/h/mg protein. Similar values were obtained in both serum-supplemented and transferrin/selenium (TS)-defined culture media, indicating that ODC kinetic parameters are not affected by any factors present in serum. Polyamine concentrations were maximal on day 5. By day 9, they returned to initial levels and remained at these fairly high values until day 21. Since we have previously shown (Jumarie and Malo, 1994, in Vitro Cell. Dev. Biol., 30A:753-760) that triiodothyronine (T3) stimulates differentiation but not proliferation of Caco-2 cells maintained in TS-defined medium, we investigated if it induces differentiation by a polyamine-dependent mechanism. Short- and long-term measurements revealed similar ODC activity and polyamine levels whether T3 was present or not in the culture medium. These results clearly demonstrate that polyamine synthesis is more likely to be associated with Caco-2 cell proliferation, and that the T3 effect on Caco-2 cell differentiation does not involve polyamine biosynthesis. Moreover, our data show that ODC activity is not solely regulated by intracellular polyamine concentration.

Alkaline phosphatase and peptidase activities in Caco-2 cells: differential response to triiodothyronine.

Caco-2 cell human colon adenocarcinoma cell line was used to study the hormonal regulation of small intestinal epithelial cell differentiation. We had previously shown that insulin-transferrin-selenium and triiodothyronine (5 x 10(-8) M)-supplemented medium can best replace serum after 2 days of culture for both the maintenance and differentiation of Caco-2 cells. The present study demonstrates that precoating petri dishes with complete serum allows the growth and differentiation of Caco-2 cells seeded directly in serum-free medium. On the other hand, precoating with dialyzed serum inhibits alkaline phosphatase and dipeptidyl-dipeptidase IV activities by more than 50%. The results obtained with complete serum-precoated culture plates indicate that there is no synergy between insulin and triiodothyronine because cells maintained in transferrin-selenium and triiodothyronine-supplemented medium, with or without insulin, express comparable enzyme activities. Moreover, large increases in alkaline phosphatase and dipeptidyl-dipeptidase IV activities were observed when triiodothyronine was added to the culture medium by the time confluency was reached. In contrast, gamma-glutamyltransferase was lowered to a greater extent when triiodothyronine was present from the beginning of culture. These findings show that triiodothyronine preferentially stimulates alkaline phosphatase and dipeptidyl-dipeptidase IV activities during the differentiation period whereas it selectively inhibits gamma-glutamyltransferase during the proliferation phase. Triiodothyronine acts in a dose-dependent manner.

Polarized distribution of neutral endopeptidase 24.11 at the cell surface of cultured human intestinal epithelial Caco-2 cells.

The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth, and expresses a number of brush-border-membrane-associated hydrolases typical of a differentiated phenotype. Among these are alkaline phosphatase, dipeptidyl peptidase IV and sucrase-isomaltase (sucrase, EC 3.2.1.48). Neutral endopeptidase 24.11 [EC 3.4.24.11, neprilysin (NEP)] is another abundant protease of normal enterocytes but its presence in Caco-2 cells has not been fully documented yet. In this paper, we show that Caco-2 cell extracts hydrolyse tritiated [D-Ala2Leu5]enkephalin with a Km of 180 microM, very close to the value obtained for the NEP present in the rabbit kidney (118 microM). Western-blot analysis of brush-border membranes purified from post-confluent cells revealed a protein with an apparent molecular mass of 94000 Da similar to that of the rabbit kidney NEP. The amount of enzyme in cell extracts increased as a function of the age of the culture, indicating that NEP expression is correlated with the degree of cell differentiation as is also the case for sucrase and dipeptidylpeptidase IV (DPP-IV). Binding of a radiolabelled antibody to Caco-2 cell monolayers grown on semi-permeable filters indicated that 95% of NEP molecules present at the cell surface are on the apical side. Immunocytochemical and flow cytometric analysis of intact and permeabilized cells were also used to investigate the presence of NEP and DPP-IV at the surface of Caco-2 cells. Whereas DPP-IV staining appeared to be homogeneous throughout the entire cell population, NEP-related fluorescence exhibited a bimodal distribution which indicates an uneven expression of the protein at the cell surface. Permeabilization of monolayers with saponin before staining restored a labelling pattern for NEP similar to the one obtained for DPP-IV. This suggests that although DPP-IV and NEP follow similar patterns of expression when enzymic activities are measured on whole-cell extracts, targeting of these brush-border proteins to the cell surface appears to be regulated in different ways.

Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro.

Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.