Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1ß.

BACKGROUND: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in eukaryotic cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, the deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1ß was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins. RESULTS: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1ß were shown to produce caveolin-1ß, in its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1ß is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blot and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1ß oligomerization for vesicle formation. CONCLUSIONS: Lactococcus lactis cells can heterologously overexpress caveolin-1ß, generating caveolin-1ß enriched intracellular neo-formed vesicles. These vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.

Protective effects of the phytogenic feed additive "comfort" on growth performance via modulation of hypothalamic feeding- and drinking-related neuropeptides in cyclic heat-stressed broilers.

Identification of alternatives to antibiotics in livestock and poultry is necessary. Fueled by consumer preferences, phytogenic feed additives are increasingly used in the food system; however, their mode of action is not well defined. Here, we used broiler chickens, in which appetite and feeding behavior regulation are controlled by complex mechanisms, to determine the effect of the phytogenic feed additive "comfort" (PFA-C) as well as its underlying molecular mechanisms on growth performance in heat-stressed broiler chickens. Heat stress significantly increased birds' core body temperature, water intake, and the hypothalamic expression of heat shock protein (HSP) 70, whereas it decreased feed intake, BW, and woody breast incidence. Phytogenic feed additive "comfort" supplementation downregulated the hypothalamic expression of HSP70, reduced core body temperature, increased feed and water intake, and improved BW in HS broilers. At molecular levels, the effect of PFA-C on growth performance seemed to be mediated by modulation of hypothalamic expression of melanocortin receptor 2, arginine vasopressin, aquaporin 2, and sodium and potassium-transporting ATPase subunit beta 1 polypeptides. In summary, PFA-C supplementation ameliorates heat stress productivity losses via a potential cytoprotective effect, reduction of hypothalamic intracellular stress, and modulation of hypothalamic feeding- and drinking-related polypeptide expression.

Processing evaluation of random bred broiler populations and a common ancestor at 55 days under chronic heat stress conditions.

As a result of genetic selection, the modern broiler is more efficient, higher yielding, and faster growing than the bird of the 1950s. Unfortunately, as a result of improvement in growth rate, the modern broiler has the potential to struggle under heat stress conditions. The present study evaluates 3 different random bred populations and a common ancestor under both a thermal neutral and heat stress conditions after a 54-D grow-out period. The lines used in this study included the Athens Canadian Random Bred (ACRB), a 1995 Random Bred (95RAN), a 2015 Random Bred (MRB), and a Junglefowl (JF). Male chicks (n = 150/line) were placed by line in environmentally controlled chambers. An 8-h daily cyclic heat stress (36°C) was applied to half of the chambers beginning on day 28 (HS) and lasting until processing at day 55, while the remaining chambers remained thermal neutral (TN) at 26°C. Dock weights and carcass weights were lower in the HS-95RAN and HS-MRB, compared to their TN counterparts, while the ACRB and JF had no difference in dock and carcass weights regardless of environmental condition. The MRB line had the highest breast yield (27.79%) while the JF (12.79%) and ACRB (12.42%) had the lowest. The 95RAN line had the highest abdominal fat percentage (2.83%) while the MRB line had the lowest moisture uptake during chill. The HS exposure lowered overall breast yield and breast pH at 15 min and 4 h postmortem but did not have an impact on color (L∗) or 24 h breast drip loss. The MRB was scored for both woody breast and white striping. The TN-MRB group had a higher incidence of moderate and severe woody breast and white striping than the HS-MRB group. Based on the results of this study, it appears that HS has a greater negative impact on the higher yielding lines (MRB and 95RAN) than the ACRB and JF and that clear line differences exist between the random bred lines and their common ancestor.

Divergent selection for relative breast yield at 4 D posthatch and the effect on embryonic and early posthatch development.

Genetic selections for growth promotion in poultry have been highly successful in improving growth, yield, and feed conversion in the modern broiler. These selections have focused on the use of hypertrophy, the increase of muscle fiber size to improve growth. Muscle growth however is not limited solely to hypertrophy but is largely attributable to both hypertrophy and hyperplasia, the increase in muscle fiber number. As muscle fiber size has been theorized to reach an eventual physiological limit, it was determined to develop a novel method of selection focusing on hyperplasia. Divergent selection for 4-day relative breast yield (BY4) was chosen as it is believed to occur at point at which muscle cell number per gram is maximized and satellite cell activity is higher than later in life. Using a random bred control population, divergent selection was undergone for BY4. The 2 broiler lines divergently selected for BY4 are noted as the high and low BY4 lines, respectively (high 4-day breast yield and low 4-day breast yield). Heritability estimates for selection of 4-day breast percentage in the upward and downward directions were 0.63 and 0.44, respectively. Divergent selection resulted in clear divergence in BY4 and shows promise in utilizing BY4 to promote broiler growth and body composition.

The association of cognitive distortions and the type of gambling in problematic and disordered gambling.

BACKGROUND: Previous studies have shown that particular types of gambling are related to the development of gambling-related problems. Further, gambling-related cognitive distortions contribute to the development of disordered gambling. The aim of the present study is to compare different gambling types with respect to cognitive distortions and the development of disordered gambling. METHODS: Based on a proactively screened sample of vocational school students (N = 6718), 309 students were selected to undergo an in-depth interview. We assessed the Gamblers-Belief-Questionnaire (GBQ) to measure gambling-related cognitive distortions and the Stinchfield questionnaire for assessing gambling-related problems. Associations between cognitive distortions, gambling-related symptoms, and types of gambling were analysed using logistic regression analyses. RESULTS: Higher scores on the GBQ subscale "belief in luck/perseverance" led to a significantly higher chance to be classified as a person with Gambling Disorder (Conditional Odds Ratio (COR) = 1.05, Confidence Interval (CI) = 1.02-1.08) as well as problematic gambling (COR = 1.04, CI = 1.01-1.06). Higher scores on the subscale "illusion of control" were also associated with problematic gambling (COR = 1.04, CI = 1.00-1.08). The multivariate analyses of the gambling types identified only sports betting as a predictor for problematic gambling (COR = 1.91, CI = 1.05-3.49). When controlling for cognitive distortions, sports betting was not significant anymore. With respect to disordered gambling, gambling on electronic gambling machines (EGMs) turned out to be a risk factor besides cognitive distortions (COR = 2.59, CI = 1.04-6.49). DISCUSSION: The present study confirmed the high relevance of cognitive distortions for problematic and disordered gambling especially for sports betting and gambling on EGMs. Preventive measures and psychotherapy should take these relationships into account.

Differential expression of water channel- and noncoding RNA biogenesis-related genes in three lines of chickens under a short-term water restriction.

Genetic selection for high growth rate has resulted in tremendous changes not only in feed efficiency, but also in water consumption between modern broilers and their ancestor jungle fowl (JF). However molecular mechanisms involved in water homeostasis are still not well defined. This study aimed, therefore, to determine the effect of short-term water restriction on the expression of water channel- and noncoding RNA biogenesis-related genes in the kidney and whole blood of JF, broiler population from the 1990s (RB1995), and modern broiler population developed in 2015 (ARB2015). Body weight-matched birds from each population were subjected to water restriction (WR) for 3 h or had ad libitum access to water in a 3 × 2 factorial design. The expression of target genes was determined by real-time quantitative PCR. WR significantly reduced body weight in RB1995, but not in JF or ARB2015. In the kidney, WR up-regulated the expression of AQP2 in all chicken populations, AQP3 in the RB1995, and ATP1B1 in JF and ARB2015. However, it down-regulated the expression of AQP4 in ARB2015 but had no effect on AVP expression. The expression of RNase III family enzymes also was altered by WR in a population-dependent manner, with DICER1 being down-regulated in JF and RB1995, Drosha was decreased in RB1995, and ARG2 was up-regulated in ARB2015. The expression of DGCR8 and TRBP1 was not affected by WR in any population; however, DGCR8 mRNA levels were significantly lower in RB1995 and ARB2015 compared to JF under both conditions. TRBP1 gene expression was significantly lower in RB1995 and ARB2015 compared to JF under WR conditions. In the blood, the expression of these genes also was altered by WR, but with different patterns than the kidney. The mRNA abundances of AQP, AVP, DICER1, DGCR8, AGO2, and TRBP1 were significantly decreased by WR in RB1995. However, the expression of AQP2, AVP, DGCR8, and TRBP1 was increased in WR-ARB2015 compared to the control. In the JF, there was no difference in the expression of these genes except for a significant up-regulation of TRBP1 in WR compared to the control group. To the best of our knowledge, this is the first report showing that water channels and the RNase III enzymes are differentially regulated by WR in a population-dependent manner, which may be due to differential postnatal growth and maturation. Their expression in the circulation could open new vistas for identification of new molecular signatures involved in adaptation to water-deprivation stress.

Interaction of macrocyclic lactones with the multidrug transporters: the bases of the pharmacokinetics of lipid-like drugs.

Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.

P-glycoprotein and 'lipid rafts': some ambiguous mutual relationships (floating on them, building them or meeting them by chance?).

P-glycoprotein (P-gp) is an active membrane transporter responsible for cell detoxification against numerous amphiphilic compounds, leading to multidrug resistance in tumor cells. It displays entangled connections with its membrane environment since it recognizes its substrates within the cytosolic leaflet and it also translocates some endogenous lipids to the exoplasmic leaflet. Regarding its relationships with membrane microdomains, 'lipid rafts', a literature analysis concludes that (i) P-gp also exists in rafts and non-raft membrane domains, depending on the cell considered, the experimental conditions and the method used to test it; (ii) cholesterol has a positive influence on P-gp function, and this may be a direct effect of the free cholesterol present in membrane or an indirect effect mediated by the cholesterol-enriched microdomains; (iii) when present in rafts, P-gp interacts with protein partners regulating its activity; (iv) P-gp is a lipid translocase that handles the raft-constituting lipids with particular efficiency, and it also influences membrane trafficking in the cell.

The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway.

Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB.

Treatment of murine transplanted subcutaneous tumors using systemic drug administration.

In vitro data have shown an increased cytotoxic drug uptake into electropermeabilized cells in suspension, leading to a marked cytotoxicity increase (1). Preclinical experiments were required to demonstrate the in vivo applicability of these observations. Obviously, the most convenient laboratory animal model to test new antitumor treatments is the mouse. Indeed, there exist many tumors of different histological types which can be transplanted in mice, either in immunocompetent mice in the case of syngeneic tumors or in immunodepressed mice in the case of allogeneic or xenogeneic tumors. From a practical point of view, mice have the advantage to be rather cheap and to allow a large number of experiments. Moreover, murine tumors are generally easy to transplant, grow rapidly, and can be conveniently followed for their evolution, at least in the case of subcutaneous tumors. Finally, murine subcutaneous tumors are well adapted to test the antitumor effects of electrochemotherapy since they allow the use of a rather simple material to conveniently apply transcutaneous permeabilizing electric pulses.

Treatment of multiple spontaneous breast tumors in mice using electrochemotherapy.

Electrochemotherapy has been developed on the basis of in vitro data showing the huge increase of bleomycin cytotoxicity observed after cell electropermeabilization. Electrochemotherapy has been proven to be highly efficient as a local antitumor treatment on transplanted tumors (1). Indeed, electrochemotherapy resulted in high rates of tumor responses and even cures in preclinical trials on different experimental murine tumors (2). Treatment efficacy results from the local application of adequate electric pulses able to permeabilize the tumor cells located in the tissue volume crossed by the electric field, which allows the bleomycin present in tumor interstitial fluids to enter these cells and to kill them (3). Since the electric pulse delivery is designed to only reversibly permeabilize the tumor cells, electrochemotherapy is almost devoid of any side effects, and it can be safely proposed as a new antitumor treatment to be used in human clinics. Actually, a phase I-II clinical trial of electrochemotherapy has already given very satisfactory results on cutaneous tumors (4).

Treatment of spontaneous soft tissue sarcomas in cat.

Preclinical experiments performed on mice clearly showed that electrochemotherapy can efficiently treat subcutaneous tumors of different histological types (1-4). However, the possibility of relevant clinical applications for electrochemotherapy requires the demonstration of its efficacy as a treatment of tumors larger than those encountered in mice. This necessitates the use of tumors growing in animals that are larger than mice. Since models for such tumors are rare and expensive, a convenient possibility is to test electrochemotherapy on spontaneous tumors that veterinarians have to treat in clinical presentations. Among them, cat soft-tissue sarcomas constitute a set of closely related tumors (fibrosarcoma of grade I or of grade II, and malignant fibrohistiocytoma) which are well suited for electrochemotherapy trials. Indeed, they are subcutaneous tumors, which always escape the conventional surgical treatment after a more or less long time, but which have only a local development for a long period of the disease evolution. Thus this carcinologic situation provides the possibility of testing the local efficiency of electrochemotherapy on animals having a good health status and bearing tumors for which no further conventional therapy can be proposed (5).

Treatment of liver tumors in rabbit.

Electrochemotherapy has been proven to be efficient for the treatment of subcutaneous tumors of different histological types in mice, rats and cats (1-3). Electrochemotherapy is essentially a local treatment. Indeed, it is based on the potentiation of the cytotoxicity of a low-permeant drug, typically bleomycin, by tumor cell permeabilization using brief and intense electric pulses. In the tissues exposed to such electric pulses, the so-called cell electropermeabilization is obtained only in the volume crossed by an electric field of intensity higher than a threshold value (4). The existence of this threshold for tissue permeabilization implies that the potentiation of the cytotoxic drug by the applied electric pulses is limited to the space roughly comprised between the electrodes. In the case of rather small and prominent cutaneous or subcutaneous tumors, it is easy to place two external electrodes on each side of the tumor nodule to be treated and to transcutaneously deliver the permeabilizing electric pulses. For thick and for deep subcutaneous tumors as well as for tumors located in internal organs, this electrode configuration is not appropriate because the electric pulses have to be delivered directly to the tumor tissues. Externally placed electrodes simply do not supply electric fields that extend deep enough for these situations. Intratumoral electric pulse delivery can, however, be achieved using needle-electrodes that are inserted into the tumor volume. We have designed an applicator constituted by multiple parallel and equidistant needles in order to avoid the delivery of very high-voltage electric pulses. A specifically manufactured generator ensures the multiplexed supplying of the electric current to each pair of closest needle electrodes. Thus, the entire tumor volume is divided in small units which are separately permeabilized by the different pairs of closest electrodes.

The basis of electrochemotherapy.

Antitumor electrochemotherapy is a treatment of solid tumors which combines a cytotoxic nonpermeant drug, like bleomycin, with locally delivered permeabilizing electric pulses (1-3). More generally, a new form of vectorization is achieved by the combination of nonpermeant molecules with intracellular targets and of a physical perturbation that locally permeabilizes the cells. This vectorization does not require chemical, biochemical or biological modifications of the targeted compound, since the modification is performed on the target cells. A very convenient way to transiently permeabilize the cells is the use of appropriate electric pulses (short and intense squarewave electric pulses) that are not cytotoxic by themselves (1). These electric pulses reversibly permeabilize the electropulsed cells. Consequently, they allow increased drug delivery inside cells, particularly in the case of drugs for which the plasma membrane is a barrier that limits their access inside the cell [termed nonpermeant drugs] (4). As illustrated in the various protocols reported in this volume, electrochemotherapy using bleomycin is efficient to eradicate subcutaneously transplanted and spontaneous small tumors in mice and rats as well as experimental internal tumors transplanted in rat brain or in rabbit or rat liver. All the clinical trials (3,5-10) confirm the efficacy of this new therapeutical approach based on an original way to deliver nonpermeant cytotoxic drugs.

Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

Mechanisms of electrochemotherapy.

Electrochemotherapy is a new therapeutic approach providing delivery into cell interiors of nonpermeant drugs with intracellular targets. It is based on the local application of short and intense electric pulses that transiently permeabilize cells in tissues. To date, its main application has been the treatment of tumor nodules when the electric pulses are associated with nonpermeant drugs having high intrinsic cytotoxicity. The most convenient drug is bleomycin, a currently used anticancer drug, but cytotoxicity of cisplatin is also increased in vivo by means of this original drug delivery approach. The efficacy of this new method is based on the following mechanisms: (i) electropermeabilization of cells in tissues; (ii) use of nonpermeant drugs having a high intrinsic cytotoxicity; (iii) existence of vascular effects due to the permeabilizing electric pulses; (iv) complementary role of the host's immune system. Preclinical trials have shown the efficacy of this new therapeutic modality in various tumor models. Clinical trials are in progress, demonstrating its feasibility in humans as well as the interest of the method.

Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs.

P-glycoprotein (P-gp) is an active, ATP-dependent plasma membrane transporter which is responsible for the expulsion of various cytotoxic drugs with different chemical structures out of resistant (MDR) cells. It is also capable of transporting a number of other amphiphilic molecules, the so-called MDR-reversing agents, which belong to a very broad variety of chemical families. Moreover, P-gp can also play a role in steroid secretion and cellular detoxification by transporting various other substrates. In this review, we address the problem of the multiple recognition by P-gp of such a large number of amphiphilic molecules. This is both (i) from a basic viewpoint in order to discuss the underlying molecular mechanisms explaining how the general rule of substrate-enzyme specificity can be violated, and (ii) from a more applied pharmacological viewpoint to show in detail how the interaction of various drugs with P-gp leads to important consequences in terms of the relative effects of these drugs in the anticancer chemotherapy context, as well as for their pharmacokinetic distributions in the whole organism, rationalizing possible adverse drug reactions. In particular, we will present evidence that, independently of the technique used, the mutual interactions between P-gp transport substrates cannot always be reduced to simple competitive effects.

Multidrug resistance transporter P-glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents.

P-Glycoprotein, the plasma membrane protein responsible for the multidrug resistance of some tumour cells, is an active transporter of a number of structurally unrelated hydrophobic drugs. We have characterized the modulation of its ATPase activity by a multidrug-resistance-related cytotoxic drug, vinblastine, and different multidrug-resistance-reversing agents, verapamil and the dihydropyridines nicardipine, nimodipine, nitrendipine, nifedipine and azidopine. P-Glycoprotein ATPase activity was measured by using native membrane vesicles containing large amounts of P-glycoprotein, prepared from the highly multidrug-resistant lung fibroblasts DC-3F/ADX. P-Glycoprotein ATPase is activated by verapamil and by nicardipine but not by vinblastine. Among the five dihydropyridines tested, the higher the hydrophobicity, the higher was the activation factor with respect to the basal activity and the lower was the half-maximal activating concentration. The vinblastine-specific binding on P-glycoprotein is reported by the inhibitions of the verapamil- and the nicardipine-stimulated ATPase. These inhibitions are purely competitive, which means that the bindings of vinblastine and verapamil, or vinblastine and nicardipine, on P-glycoprotein are mutually exclusive. In contrast, verapamil and nicardipine display mutually non-competitive interactions. This demonstrates the existence of two distinct specific sites for these two P-glycoprotein modulators on which they can bind simultaneously and separately to the vinblastine site. The nicardipine-stimulated ATPase activity in the presence of the other dihydropyridines shows mixed-type inhibitions. These dihydropyridines have thus different binding sites that interact mutually to decrease their respective, separately determined affinities. This could be due to steric constraints between sites close to each other. This is supported by the observation that vinblastine binding is not mutually exclusive with nifedipine or nitrendipine binding, whereas it is mutually exclusive with nicardipine. Moreover, verapamil binding also interacts with the five dihydropyridines by mixed inhibitions, with different destabilization factors. On the whole our enzymic data show that P-glycoprotein has distinct but interacting binding sites for various modulators of its ATPase function.