The antimetastatic drug NAMI-A potentiates the phenylephrine-induced contraction of aortic smooth muscle cells and induces a transient increase in systolic blood pressure.

The ruthenium-based drug imidazolium trans-imidazoledimethylsulphoxidetetrachlorido ruthenate (NAMI-A) is a novel antitumour drug under clinical evaluation. In this study, NAMI-A is tested on aortic rings in vitro and on the systolic blood pressure in vivo with the aim of evaluating its effects on smooth muscle cells and, more in general, on the vascular system. Pre-incubation of aortic rings with 10 µM NAMI-A for 10 min potentiates the contraction induced by phenylephrine (PE). The reduction of the B max value of [(3)H]-prazosin bound to NAMI-A-treated aortic rings and the ability of NAMI-A to displace [(3)H]-prazosin and [(3)H]-IP3 binding by 25 and 42%, respectively, suggest the involvement of α1-adrenoceptor in mediating the effects on smooth muscle cells. NAMI-A also decreases the number of maximal sites of [(3)H]-prazosin bound to kidney membrane preparation from 34 to 24 fmol/mg proteins. A single i.p. dose (105 mg/kg) or a repeated treatment for 6 consecutive days (17 mg/kg/day) in Wistar rats increases the systolic blood pressure, respectively, 1 h and 3 days after treatment, and the responsiveness of rat aortic rings to PE. Atomic absorption spectroscopy confirms the presence of ruthenium in the aortic rings excised from the treated rats. These findings suggest monitoring the cardiovascular parameters when the drug is used in humans for treating cancer patients, particularly if the drug is associated with chemicals that are potentially active at the cardiovascular level.

Long noncoding RNA GAS5: a novel marker involved in glucocorticoid response.

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation.

Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells.

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.

Mast cells and their secretory granules are angiogenic in the chick embryo chorioallantoic membrane.

BACKGROUND: Many data suggest that the density of mast cells is highly correlated with the extent of both normal and pathological angiogenesis. OBJECTIVE: In this study we have compared in an in vivo assay, the chick embryo chorioallantoic membrane, the angiogenic potential of mast cell suspensions isolated from rats, degranulated mast cells and their secretory granules. METHODS: Gelatin sponges adsorbed with cell suspensions of rat mast cells, degranulated mast cells and their secretory granules were implanted on the top of the chorioallantoic membrane at day 8 of incubation. At day 12 the angiogenic response was evaluated macroscopically, microscopically and by a morphometric method of 'point counting'. RESULTS: Isolated mast cells and their secretory granules, but not degranulated mast cells, induced an angiogenic response in the chorioallantoic membrane. The addition of antifibroblast growth factor-2 or antivascular endothelial growth factor antibodies reduced the angiogenic response of both mast cells and their secretory granules by 50% and 30%, respectively. CONCLUSION: These data support the evidence that the angiogenic properties of mast cells depend on the angiogenic molecules contained in their secretory granules and indicate that fibroblast growth factor-2 and vascular endothelial growth factor are the angiogenic cytokines primarily and perhaps synergistically responsible for this vasoproliferative activity.

Characterization of multidrug transporters in a normal renal tubular cell line resistant to doxorubicin. Multidrug transporters in the LLC-PK(1) cell line and its resistant counterpart.

LLC-PK(1) is a proximal tubular cell line derived from normal pig kidney which has a structure and function similar to those of renal proximal tubular cells and which expresses baseline levels of P-glycoprotein. We isolated by drug selection a doxorubicin-resistant cell line (LLC-PK(1)/ADR) that exhibited a multidrug-resistant phenotype; this cell line was characterized by reduced intracellular drug concentrations, an increased drug extrusion, and increased expression of a 170-kDa P-glycoprotein detected by Western blot analysis with monoclonal antibody C219. In addition, an increased expression of MDR1 mRNA was seen by reverse transcriptase-polymerase chain reaction. These results suggest that it is possible to induce the overexpression of P-glycoprotein by chronic treatment with doxorubicin in a normal cell line that physiologically expresses low levels of this protein. This multi-resistant cell line could provide an interesting model for studying the role of P-glycoprotein and the consequence of its induction in a normal tissue.

Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function.

Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.

Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart.

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

Biochemical and microscopic evidence for the internalization of drug-containing mast cell granules by macrophages and smooth muscle cells.

During mast cell degranulation the soluble component of the granule is released into extracellular fluid, whereas two neutral proteases and heparin proteoglycans form the extracellular granule remnants. These structures are negatively charged and bind with high affinity LDL and other basic molecules. In this study we show that granule remnants expelled into extracellular fluid are able to bind the aminoglycoside antibiotic gentamicin and the anticancer agent doxorubicin in a dose-dependent manner. In addition, granule remnants loaded with the two basic substances are subsequently phagocytosed by macrophages. Indeed, when cells are incubated for 24 h with 1 mg/ml gentamicin, the intracellular concentration of the drug, which in basal conditions is extremely low, increases significantly in the presence of degranulating mast cells (from 5.1 +/- 1.0 to 25.4 +/- 2.5 microg/mg protein) and a good correlation between histamine release and gentamicin uptake is evident. The antineoplastic agent doxorubicin can penetrate cells by passive diffusion; however, when mast cells are added to macrophage monolayer, incubated for 30 min with 50 microM of the antineoplastic agent, a significant increase in intracellular doxorubicin concentration is observed (from 3.5 +/- 0.2 to 4.7 +/- 0.2 microg/mg protein). Internalization of granule remnants carrying gentamicin or doxorubicin is also evident in smooth muscle cells of the synthetic phenotype. In particular, when smooth muscle cells are incubated for 24 h with 1 mg/ml gentamicin, addition of isolated granules increases the uptake from 2.4 +/- 0.2 to 4.8 +/- 0.4 microg/mg protein. Similar results are obtained in smooth muscle cells incubated for 4 h with doxorubicin 50 microM (from 3.3 +/- 0.2 to 4.8 +/- 0.5 microg/mg protein). Data are confirmed by microscopic experiments by means of fluorescence microscopy and electron microscopic studies. The study demonstrates that basic substances can enter phagocytic cells when loaded to granule remnants. The phenomenon can be of particular interest for substances like the aminoglycosides that do not cross biological membranes; indeed, the storage of these antibiotics in phagocytic cells could have important consequences on their antibacterial activity in vivo. Macrophages and smooth muscle cells can also act as a reservoir for doxorubicin. High concentrations of the antineoplastic agent in these cells could be responsible for toxicity, as well as play an important role in the transport of the drug to tumor cells.

Kinetics of doxorubicin handling in the LLC-PK1 kidney epithelial cell line is mediated by both vesicle formation and P-glycoprotein drug transport.

The subcellular distribution of doxorubicin was evaluated in living non-fixed LLC-PK1 cells, which maintain the structural and functional characteristics of the kidney proximal tubule epithelium and also express P-glycoprotein. After 10 min incubation, doxorubicin fluorescence was detectable in the nucleus. The intensity of nuclear fluorescence progressively increased, reaching the maximum at the end of the first hour. Then, the nuclear signal started to decrease and, at 2 h, doxorubicin fluorescence disappeared almost completely from the cell nucleus. Cytoplasmic fluorescent vesicles first appeared in the perinuclear region after 10 min doxorubicin exposure and increased in number and size over a period of 2 h. From 2 to 5 h, fluorescent vesicles moved unidirectionally to the cell periphery. Disappearance of doxorubicin punctate fluorescence in LLC-PK1 cells treated with methylamine or monensin demonstrated that drug accumulation occurred inside acidic compartments. In addition, the cytoplasmic pattern of doxorubicin fluorescence was very similar to that observed upon exposure to the acidotropic tracer LysoSensor Blue. Involvement of P-glycoprotein in doxorubicin handling by LLC-PK1 cells was suggested by modified intracellular doxorubicin distribution after cell incubation with verapamil and vinblastine. Moreover, the fluorescent P-glycoprotein substrate Bodipy FL Verapamil was shown to accumulate in LLC-PK1 cells in a manner that is quite similar to that observed for doxorubicin. P-glycoprotein expression was evaluated by immunoblot using the JSB-1 and C219 monoclonal antibodies. Immunofluorescence analysis was performed using the JSB-1 monoclonal antibody. P-glycoprotein immuno-reactivity was found both on the plasma membrane and intracytoplasmically in a perinuclear position. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that MDR1 gene was expressed. This study indicates that a rapid intracellular redistribution accompanies the process of doxorubicin uptake by LLC-PK1 cells. Although these cells are non-tumour cells derived from the normal epithelium of the proximal renal tubule, they display a model of doxorubicin redistribution which is characteristic of doxorubicin-resistant tumour cells.

Endocytosis of gentamicin in a proximal tubular renal cell line.

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 degrees C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.

Adriamycin induces exocytosis in rat and beige mouse peritoneal mast cells: an ultrastructural, morphometric and biochemical study.

The process of exocytosis was studied in rat and beige mouse peritoneal mast cells stimulated by adriamycin (ADR) at 37 degrees C and 22 degrees C. ADR induces a non cytotoxic histamine release that is followed by a significant uptake of the drug. Examination was performed by transmission electron microscopy and, at the same time, histamine release and ADR uptake were measured by spectrofluorimetry. ADR accumulation in mast cells was investigated also by fluorescence microscopy. In rat mast cells stimulated at 37 degrees C, the secretory process developed abruptly and was virtually complete after 30 sec. Electron microscopy showed rapid intracytoplasmic channel formation and extrusion of secretory granules; spectrofluorimetry revealed a massive release of histamine and rapid uptake of ADR. In addition, fluorescence microscopy showed mast cells exhibiting an intense orange-yellow fluorescent signal localized at the secretory granules. At 22 degrees C, rat mast cells showed alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane. Histamine release and ADR uptake proceeded less quickly than at 37 degrees C. Quantitative analysis of rat mast cell ultrastructure demonstrated that histamine release induced by ADR stimulation was achieved by sequential exocytosis. This process presents both morphological and biochemical affinities with the exocytosis induced by basic secretagogues such as compound 48/80. In beige mouse mast cells the process of exocytosis progressed more slowly and was completed after 20 min at 37 degrees C. By electron microscopy, the cytoplasm presented a rigid structure due to abundance of actin-like fibrils. Granule fusion was an uncommon feature and exocytosis was mostly the result of single granule opening to the cell exterior without extrusion of granule matrices.

Transport of doxorubicin in mast cell granules and the effect of the calcium antagonist verapamil.

P-glycoprotein has been identified in mast cells stabilized in culture as well as in rat peritoneal mast cells, and is primarily concentrated on the granular membrane. This study aimed to define the role of this protein in the transport and accumulation of doxorubicin in mast cell granules and in its histamine releasing effect. The reverting agent verapamil, that is a substrate for P-glycoprotein, inhibited doxorubicin uptake in intact mast cells in a dose and time dependent manner, but had no effect on the exocytotic action of the antineoplastic drug. Doxorubicin was also concentrated in granules with intact membranes and the uptake was dependent on temperature and showed a trend for saturation. Verapamil and vinblastine, another substrate for P-glycoprotein, significantly reduced doxorubicin concentrations in intact granules. Similar results were obtained with the metabolic inhibitors sodium metavanadate, N-ethylmaleimide, and sodium azide, whereas ouabain, an inhibitor of sodium-potassium ATPase, was without effect. Doxorubicin was taken also up in granule remnants, consisting of a proteoglycan matrix without membrane, that are extruded from mast cells upon stimulation. However, the uptake was not dependent on temperature and was not modified by P-glycoprotein substrates or metabolic inhibitors. Rat peritoneal mast cells were examined for the expression of P-glycoprotein at the protein level with C219 monoclonal antibody, using Western blot, confirming that P-glycoprotein was expressed in mast cells. These data suggest the presence of a P-glycoprotein active in the transport of doxorubicin, in mast cell granules.

Effect of low temperature on mast cell exocytosis. Early cytoplasmic vesicle formation and the role of cytoskeleton.

Mast cells stimulated with adriamycin at 4 degrees C underwent a unique exocytotic reaction. Rat peritoneal cells including mast cells were stimulated in vitro with adriamycin (100 micrograms/ml) for 0, 10, 30 or 60 sec and observed by transmission electron microscopy. Early changes could be observed after 10 sec stimulation and consisted in an approximately 5-fold increase (p < 0.001) of 0.05-0.2 micron diameter cytoplasmic vesicles. The Golgi apparatus showed signs of activation and vacuolization. From 10 to 30 sec, cytoplasmic vesicles fused with the perigranular membranes and with the membranes of developing secretory channels. At 60 sec, the number of vesicles and vacuoles diminished to nearly two-fold starting levels. The exocytotic reaction characteristically resulted in the formation of enormously dilated granular cavities. The secretory process appeared incomplete; after 60 sec, in fact, maximal histamine release was 20% and exocytosis could be found in approximately 30% of mast cells. Pre-incubation with vinblastine followed by adriamycin stimulation at 37 degrees C determined a dose-dependent inhibition of histamine release which was accompanied by the ultrastructural appearance of numerous 0.05-0.5 micron cytoplasmic vesicles and by signs of inhibited exocytosis. Our results support the concept that hyperstability of the cortical cytoskeleton coupled with microtubule perturbation would be responsible for the depressed pattern of mast cell exocytosis observed at 4 degrees C. Although stimulation at 4 degrees C induces a paradoxal secretory process, we believe that this approach may represent a useful model for understanding some basic mechanisms of exocytosis in mast cells.

Handling of doxorubicin by the LLC-PK1 kidney epithelial cell line.

The characteristics of doxorubicin handling have been studied in the cultured kidney epithelial cell line LLC-PK1, which has structure and function similar to those of renal tubular cells and expresses P-glycoprotein. The uptake of doxorubicin by LLC-PK1 cells was time dependent, reaching a steady state at about 4 hr, and reduced at low temperature; the initial uptake was saturable. The efflux of doxorubicin from LLC-PK1 cells was also temperature dependent but, even at 37 degrees C, a significant percentage of the drug remained associated with the cells after 180 min, which suggests a strong cellular binding, and the fluorescence microscopy revealed that the drug was concentrated in intracellular organelles. Substances that are substrates for P-glycoprotein, such as verapamil, vinblastine, vincristine and quinidine, significantly increased doxorubicin concentrations in LLC-PK1 cells. Similar results were obtained with the metabolic inhibitors sodium metavanadate and 2,4-dinitrophenol. On the other hand, the uptake was not affected by the classic organic cation transport drugs cimetidine, decynium 22 or decynium 24, nor by the organic anion drug probenecid. These results indicate that, in LLC-PK1 cells, doxorubicin enters by passive diffusion, is trapped in intracellular organelles and then is extruded from cells by a mechanism that probably involves P-glycoprotein. On the contrary, substances that interfere with the renal organic cation or anion secretory system have no effect on doxorubicin net accumulation in these cells.

Stimulation of rat peritoneal mast cells induces phagocytosis of adriamycin by rat peritoneal macrophages.

Rat and beige mouse peritoneal mast cells, induced to exocytose with the antineoplastic agent adriamycin, extrude their granule remnants in the extracellular medium. These granules are loaded with the fluorescent drug adriamycin and exhibit intense yellow-reddish fluorescent staining. Granules extruded from mast cells were ultimately phagocytosed and could be observed inside the macrophages by fluorescence microscopy. All stages of the internalization process could be followed by electron microscopy. Granules adhering to the cell surface of macrophages were first embraced by short superficial projections, then enveloped by deep surface infoldings, and finally engulfed into the macrophage cytoplasm. Phagocytosis occurred exclusively in macrophages; granules were observed also on the surface of eosinophils and lymphocytes, but never inside these cells. The concentrations of adriamycin in macrophages, measured by spectrofluorimetry, were significantly higher when these cells were incubated with adriamycin and granule remnants in comparison with adriamycin alone. Preincubation with the endocytosis inhibitor cytochalasin B significantly reduced the granule mediated adriamycin uptake. As a consequence of the phagocytosis of adriamycin loaded mast cell granules, macrophages can concentrate the antineoplastic drug. These cells act as reservoirs of adriamycin and could have an important role in both the antitumor and toxic effects of the drug.

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation.

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studies showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators.

Adriamycin-induced histamine release from heart tissue in vitro.

It has been proven that the anthracyclines induce an important, noncytotoxic histamine release from rat peritoneal mast cells. As mast cells derived from different tissues exhibit marked heterogeneity, the effect of Adriamycin in comparison with other antineoplastic agents was tested on fragments of the right heart auricle, which contain a great number of mast cells. In this experimental model, Adriamycin induced a dose-dependent histamine release that was significantly limited by the antiexocytotic drug sodium cromoglycate. The antineoplastic agents cisplatin and 5-fluorouracil, in contrast, did not provoke any comparable histamine release. In the formulation employed in clinical settings, paclitaxel was also capable of inducing a histamine release comparable with that of Adriamycin; the exocytotic activity, however, was also evident when the tissue fragments were treated with Cremophor EL alone, without the addition of paclitaxel, whereas treatment of samples with paclitaxel dissolved in ethanol did not induce any releasing action. These data thus suggest that the secretory activity should be ascribed to the solvent Cremophor EL and not to paclitaxel. The release of histamine induced by paclitaxel in Cremophor EL/ethanol was also limited by sodium cromoglycate. These results again indicate that histamine release from mast cells derived not only from the peritoneal cavity but also from the cardiac tissue could play a role in the cardiotoxicity of anthracyclines and of paclitaxel in the clinically employed formulation.

Binding of aminoglycoside antibiotics by degranulating mast cells.

The aminoglycoside antibiotics are polycationic at physiological pH and hence do not enter most cells. Indeed, when intact mast cells were incubated with gentamicin or tobramycin, extremely low concentrations of the two drugs could be measured. In contrast, when a controlled, specific degranulation of mast cells was induced by treating the cells with compound 48/80, Adriamycin or concanavalin A, a dose-dependent histamine release was observed and a significant increase of the intracellular concentrations of the aminoglycosides resulted. The uptake of gentamicin and tobramycin was not dependent on the type of stimulus, but was proportional to the quantity of histamine release. The aminoglycoside binding to granular material obtained from sonicated cells was similar to that of degranulating mast cells. These data suggest the occurrence of ionic interactions between the positively charged groups of the aminoglycosides and the negatively charged groups of granular matrix that are exposed as a consequence of exocytosis. The significant of this phenomenon is at present unclear but studies are in progress to clarify the fate of the aminoglycosides bound to mast cells.

Optimised method for determination of ticlopidine in serum by high performance liquid chromatography.

In this study we verified whether high performance liquid chromatography with UV detection (HPLC-UV) could be optimised and hence become a useful method for measuring ticlopidine concentrations in patient blood. The extraction step was improved by adding isoamylic alcohol in the extraction mixture, resulting in a better recovery. Moreover, the extraction efficiency was consistently ameliorated by evaporation to dryness of the organic extract; we could therefore utilise a lower (213 nm) wavelength, corresponding to the maximum of absorption, achieving a better sensitivity. With these technical improvements, the limit of quantitation is 0.02 microgram/ml, and the limit of detection is 0.01 microgram/ml, hence comparable to levels obtained with gas-liquid chromatography with nitrogen detection (GC-NPD). In addition, HPLC method, if compared with GC-NPD, is simpler and can be easily used for routine determinations of a lot of serum samples.

Effect of paclitaxel and Cremophor EL on mast cell histamine secretion and their interaction with adriamycin.

The effect of paclitaxel and of its solvent Cremophor EL, a polyoxyethylated castor oil on histamine release in rat peritoneal mast cells has been tested. Paclitaxel dissolved in Cremophor EL/ethanol and Cremophor EL alone induced a moderate histamine release; this secretory activity is provoked by the solvent Cremophor EL but is so scanty that it seems most unlikely to be responsible of the severe hypersensitivity reactions frequently caused by this drug. Since in a number of protocols paclitaxel is used in combination with anthracyclines, and since anthracyclines are well known histamine releasing agents, the effect of the combination of these substances was also studied. Cremophor EL did not increase the exocytotic activity of adriamycin; on the contrary, the release of histamine induced by this anthracycline was significantly limited. This is an interesting observation since it has been suggested that anthracycline-induced histamine release is involved in the pathogenesis of the cardiotoxicity caused by these drugs. Low concentrations of paclitaxel in Cremophor EL and Cremophor EL alone inhibited adriamycin uptake into the granules of mast cells, a process mediated by an active transport system, recently identified with the P-170 glycoprotein pump.