Caveolae require intact VAMP for targeted transport in vascular endothelium.

Caveolae appear to function in vesicular trafficking of specific molecular cargo into and across vascular endothelial and other cells. They contain the molecular machinery for docking and fusion, similar to other vesicular trafficking systems, yet the mechanisms mediating ligand internalization and targeted intracellular transport by caveolae remain unclear. Using immunoelectron microscopy, we show that caveolae in the microvascular endothelium of rat lung express vesicle-associated membrane protein (VAMP)-2 (also called synaptobrevin) on their cytoplasmic surface. Immunofluorescence studies of cholera toxin B (CTB)-FITC internalization in toxin-treated cells demonstrate that intact VAMP-2 is necessary for the efficient trafficking of caveolar ligands. The CTB subunit binds preferentially to GM1 in caveolae, and N-ethylmaleimide treatment drastically inhibits the intracellular accumulation of CTB. The cleavage of caveolar VAMP-2 with VAMP-specific neurotoxins (botulinum D and F but not A) significantly inhibits CTB endocytosis and targeted intracellular accumulation in cultured endothelial cells. This impairment of caveolae-mediated trafficking provides evidence that caveolae require intact VAMP-2 for efficient targeted delivery via vesicle docking with target organelles.

In situ flow activates endothelial nitric oxide synthase in luminal caveolae of endothelium with rapid caveolin dissociation and calmodulin association.

Acute changes in pressure or shear stress induce the rapid release of nitric oxide (NO) from the vascular endothelium resulting in vasodilation. Endothelial nitric oxide synthase (eNOS) regulates this flow-induced NO secretion. The subcellular location of flow-induced eNOS activity in the endothelium in vivo as well as the mechanisms by which hemodynamic forces regulate eNOS activity are unknown. The luminal cell surface of the endothelium, which is directly exposed to circulating blood stressors, has been examined for eNOS expression and functional activity. Immunoelectron microscopy of rat lung tissue shows eNOS labeling on the endothelial cell surface primarily within caveolae. Subcellular fractionation to purify luminal endothelial cell plasma membranes and their caveolae directly from rat lungs reveals that eNOS is not only concentrated but also enzymatically active in caveolae. Increasing vascular flow and pressure in situ rapidly activates caveolar eNOS with apparent eNOS dissociation from caveolin and association with calmodulin. Hemodynamic forces resulting from increased flow appear to transmit through caveolae to release eNOS from its inhibitory association with caveolin, apparently to allow more complete activation by calmodulin and other possible effectors. These data demonstrate a physiological relevant mechanotransduction event directly in caveolae at the luminal endothelial cell surface. Caveolae may serve as flow-sensing organelles with the necessary molecular machinery to transduce rapidly, mechanical stimuli and thereby regulate eNOS activity.

Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium.

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a "pinching off" or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239-242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.

Pharmacokinetics and tissue distribution of cisplatin and conjugates of cisplatin with carboxymethyldextran and A5B7 monoclonal antibody in CD1 mice.

The plasma disposition kinetics and tissue distribution of platinum was evaluated following intravenous bolus administration to CD1 immune-competent mice of cisplatin, cisplatin conjugated to anti-CEA monoclonal antibody A5B7 via a carboxymethyl dextran (CMdextran) carrier molecule, and cisplatin coupled to the CMdextran in the absence of antibody. In addition, the in vivo characteristics of 125I-labeled A5B7 were compared with and without conjugation to CMdextran. Conjugation of cisplatin [clearance (CL = 0.62 mL/min/g, volume of distribution at steady-state (Vdss) = 16 mL/g] to CMdextran restricted its tissue distribution (Vdss = 0.43 mL/g) and reduced its systemic clearance (CL = 0.055 mL/min/g). Subsequent conjugation of the complex to A5B7 further reduced both its distribution (Vdss = 0.20 mL/g) and clearance (CL = 0.016 mL/min/g). Clearance of A5B7 (CL = 0.002 mL/min/g) was increased by conjugation to CMdextran (CL = 0.014 mL/min/g); tissue distribution was unchanged. A5B7-CMdextran-cisplatin was relatively stable in plasma and other tissues, except the liver. The extent of distribution of platinum into tissues (lung, liver, muscle, kidney) was markedly influenced by conjugation, with the influence being greatest for unmodified cisplatin and least for the A5B7-CMdextran conjugate. However, the time courses of tissue distribution, expressed in mean residence time scales, were similar, implying a common mechanism controlling tissue uptake.

Role of GTP hydrolysis in fission of caveolae directly from plasma membranes.

Caveolae are specialized invaginated cell surface microdomains of undefined function. A cell-free system that reconstituted fission of caveolae from lung endothelial plasma membranes was developed. Addition of cytosol and the hydrolysis of guanosine triphosphate (GTP) induced caveolar fission. The budded caveolae were isolated as vesicles rich in caveolin and the sialoglycolipid GM1 but not glycosyl-phosphatidylinositol (GPI)-anchored proteins. These vesicles contained the molecular machinery for endocytosis and transcytosis. In permeabilized endothelial cells, GTP stimulated, whereas GTPgammaS prevented, caveolar budding and endocytosis of the cholera toxin B chain to endosomes. Thus, caveolae may bud to form discrete carrier vesicles that participate in membrane trafficking.

Separation of caveolae from associated microdomains of GPI-anchored proteins.

In situ coating of the surface of endothelial cells in rat lung with cationic colloidal silica particles was used to separate caveolae from detergent-insoluble membranes rich in glycosyl phosphatidylinositol (GPI)-anchored proteins but devoid of caveolin. Immunogold electron microscopy showed that ganglioside GM1-enriched caveolae associated with an annular plasmalemmal domain enriched in GPI-anchored proteins. The purified caveolae contained molecular components required for regulated transport, including various lipid-anchored signaling molecules. Such specialized distinct microdomains may exist separately or together in the plasma membrane to organize signaling molecules and to process surface-bound ligands differentially.

Clobazam and norclobazam quantitation in serum by capillary gas chromatography with electron-capture detection.

We have developed a simple, rapid method for determination of the antiepileptic drug clobazam and its major active metabolite, norclobazam in serum. Serum (200 microL) made alkaline with sodium borate buffer is extracted with toluene. After evaporation of the organic layer and reconstitution with toluene, the extract is analyzed within 5 min by capillary gas chromatography (Hewlett Packard HP 5890A GC; 63Ni electron-capture detector; HP-5 column). Linear calibrations for clobazam and norclobazam (clobazam: r = 0.999; norclobazam: r = 0.997) have permitted automated integrator calculation of results. Imprecision and accuracy were evaluated using an in-house control. Intra- and interassay coefficients of variation (CV) were 6.2% (n = 10) and 9.8% (n = 21), respectively for clobazam and 5.7% (n = 10) and 6.3% (n = 21), respectively for norclobazam. The mean concentrations for clobazam and norclobazam in the control were 3.5 mumol/L and 8.7 mumol/L respectively, representing 106% and 100% of the weighed-in values. Simplicity, specificity, sensitivity, and rapidity are attributes that make this assay suitable for monitoring of clobazam and norclobazam in serum.

Antibody to a 63 kilodalton insect protein in ankylosing spondylitis.

Ankylosing spondylitis (AS) is associated with antibodies to a heat shock puff on drosophila chromosomes. This observation was investigated by immunoblotting using extracts of the Schneider insect cell line and HeLa cells, before and after heat shock. An insect protein of 63 kilodaltons (but no equivalent human protein) was recognised by 21 (46%) of 46 serum samples from patients with AS, one of two patients with Reiter's syndrome, four (7%) of 60 patients with systemic lupus erythematosus, and two (4%) of 50 control subjects, but not by serum samples from patients with rheumatoid arthritis (RA). Previous heat shock did not appear to affect the strength of reaction, but ML-30, a monoclonal antibody to the mycobacterial 65 kilodalton heat shock protein (hsp65), also recognised an insect protein of 63 kilodaltons by immunoblotting. Antibodies to recombinant mycobacterial hsp65 were measured by enzyme linked immunosorbent assay (ELISA) in serum samples from patients with AS and RA. IgA binding to hsp65 was increased in 41% of AS and 19% of RA serum samples, but there was no correlation with detection of antibody to the insect 63 kilodalton protein.

The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin.

Covalent linkage of the A chain of ricin to the LICR-LOND-Fib75 monoclonal antibody produced an immunotoxin, Fib75-SS-ricin A, which demonstrated immunospecific toxicity to human bladder carcinoma cells in tissue culture (Forrester et al., 1984). The present studies have shown that ricin B chain potentiates the toxicity of the immunotoxin by two orders of magnitude and also significantly increases the rate of protein synthesis inhibition. Using immunoelectron microscopy, the receptor-mediated endocytosis and intracellular routing of the immunotoxin was studied with and without ricin B chain treatment after immunolocalisation of the conjugate. Fib75-SS-ricin A was internalised by the EJ cells predominantly in uncoated pits and vesicles and directed to the endosomes. Some degradation of the complex appeared to take place in multivesicular endosomes at early timepoints and 24 h after internalisation, most of the immunotoxin was found in lysosomes. Some ricin A chain epitopes were detected in Golgi vesicles. Cells treated with immunotoxin and ricin B chain endocytosed the complex predominantly in coated pits and coated vesicles. Using pre-embedding immunoperoxidase techniques, ricin chains were found in the whole Golgi complex and most of the conjugate escaped lysosomal degradation. Internalised immunotoxin was recycled back to the plasma membrane in an active form associated with vesicles which appeared to be derived predominantly from multivesicular endosomes. A similar mode of recycling has recently been reported (McIntosh et al., 1990) for ricin holotoxin in the same cell line. These observations may explain the potentiating effect of toxin B chains in the antibody-directed targeting of toxin A chains.

An ineffective monoclonal antibody-ricin A chain conjugate is converted to a tumouricidal agent in vivo by subsequent systemic administration of ricin B chain.

An immunotoxin comprising a tumour-specific monoclonal antibody (11/160) coupled to ricin A chain, although inactive in in vitro cytotoxicity assays against HSNtc sarcoma target cells, was found to be capable of significant tumouricidal activity in syngeneic rats if potentiated by ricin B chain. The 11/160-ricin A, when bound to tumour cells prior to their inoculation, led to a slight inhibition of tumour growth s.c. compared with untreated sarcoma cells or those coated with antibody alone. However, all tumours in these groups developed progressively (69/69), whereas in those rats receiving 15 micrograms or 150 micrograms ricin B chain i.v. 5 min after tumour cell inoculation, the 'take rate' was reduced to 75% and 30% respectively, and significantly longer latent periods were evident for those tumours which did develop. Ricin B chain similarly inhibited, in a dose-dependent manner, the lung colonisation potential of 11/160-ricin A coated HSNtc cells. No effects were obtained if the B chain treatment followed inoculation of untreated or antibody-coated cells, suggesting that systemically administered B chain is capable of gaining access to and activating antibody-ricin A chain conjugates bound to the surface of syngeneic sarcoma cells in lung or subcutaneous sites. Tumour inhibition was obtained in some instances with intervals of up to 24 h between inoculation of conjugate-coated tumour cells and B chain. Experiments are in progress to determine if such potentiation may be feasible in a therapeutic rather than a prophylactic setting using this syngeneic solid tumour system.

The carriage and delivery of substances to lymphatic tissues by recirculating lymphocytes. I. The concentration of ricin in lymphocyte traffic areas.

Thoracic duct lymphocytes (TDL) were loaded in vitro with ricin before intravenous injection into syngeneic rats. TDL that had been incubated at 10 micrograms of ricin/5 X 10(7) cells/ml migrated from the blood into the spleen and lymph nodes (LN) according to the physiological pattern, and TDL incubated at 10 times that concentration were only slightly impaired in their ability to enter LN. The transfer of cells to recipients with thoracic duct fistulae indicated that very few ricin-treated lymphocytes left the LN to recirculate back to lymph. Most of the ricin-loaded lymphocytes died within the lymphatic tissues, probably between 7 and 15 hr after injection. The ricin toxicity was transferred locally, causing selective damage to the cell population within the traffic areas of the lymphatic tissues without disrupting the tissue architecture. This pattern of intensive cell destruction was not seen after a lethal dose of free ricin, which caused more diffuse and less severe damage to the spleen and LN, proving that lymphocytes are effective carriers of ricin. The surviving host lymphocytes were distributed abnormally, presumably because of the obvious damage to small blood vessels in LN and elsewhere. Lymphocytes accumulated especially in the red pulp of the spleen. Although the method described has drawbacks, it might be developed in order to concentrate ricin in the vicinity of neoplastic cells in diffuse lymphomas and leukaemias.

Transfer of ricin toxicity by spleen cells.

The ability of spleen cells to internalise ricin and release it in a form capable of killing an untreated cell population has been studied. Potential donor cells were incubated with a range of ricin concentrations and the amount of ricin subsequently released was found to be related to the ricin concentration of the primary incubation. Ricin release was detected either by using radiolabelled toxin or by measuring its cytotoxic effects. Cells which, after incubation with ricin, were washed with lactose or anti-ricin IgG had a slightly reduced ability to transfer the toxin to other cells, suggesting that much of the ricin had been internalised by the donor cells. The presence of 100 mM lactose in the incubation medium was unable to inhibit the uptake of ricin and did not prevent the release of competent toxin. Cells carrying the toxic material were lethal to animals and in vitro the released toxin was as susceptible to inactivation by anti-ricin IgG as was free ricin.

Delivery of ricin and abrin A-chains to human carcinoma cells in culture following covalent linkage to monoclonal antibody LICR-LOND-Fib 75.

With the object of generating specific cytotoxic agents, we have prepared covalent conjugates of the A-chains of ricin and of abrin with monoclonal antibody LICR-LOND-Fib 75 and investigated their toxicity toward a human tumor cell line in culture. Both conjugates proved to be potent cytotoxins toward cells carrying the appropriate antigen. The agent containing abrin A-chain was toxic at a significantly lower concentration and exerted its maximum effect more rapidly than the one containing ricin A-chain. Inclusion of chloroquine in the incubation medium significantly enhanced the toxic action of both conjugates without loss of immunospecificity. Because of the widespread occurrence on human tumor cell lines of the antigen recognized by Fib 75, these conjugates, particularly the one containing abrin A-chain, may find application in freeing human bone marrow ex vivo of infiltrated tumor cells prior to reinfusion as an autograft.

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent.

We report the conversion of a non-cytotoxic antibody-ricin A chain conjugate to one displaying specific cytotoxic effects comparable with that of native ricin, by the addition of ricin B chain as a second stage reagent. The results suggest that this conversion is achieved by the association of the added B chain with the A chain of the conjugate, and not through a primary binding of B chain at the cell surface.

Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro.

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable in inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/ cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.