Critical role of the endogenous cannabinoid system in mouse pup suckling and growth.

Delta9-tetrahydrocannabinol, the active principle in marijuana, is a cannabinoid receptor agonist. Both the crude drug and delta9-tetrahydrocannabinol have been used as appetite promoters. The endogenous cannabinoid, arachidonoyl ethanolamide (anandamide), likewise a cannabinoid receptor agonist, has been shown to have the same effect. In contrast, the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (SR141716A) reduces food intake. Here, we report that administration of SR141716A to newly born mouse pups (either a single administration on postnatal day 1, or daily for a week as of postnatal day 2) had a devastating effect on milk ingestion and growth. The first 24 h after birth appeared the most critical for the growth stunting effect of SR141716A. Death followed within 4-8 days. Co-administration of delta9-tetrahydrocannabinol almost fully reversed the effect of the antagonist in the week-long regimen. Co-administration of 2-arachidonoyl glycerol, an endocannabinoid, with 2-palmitoyl glycerol and 2-linoleoyl glycerol, which enhance 2-arachidonoyl glycerol potency, resulted in a significant delay in mortality rates caused by the antagonist. We conclude that the endocannabinoid system plays a vital role in milk suckling, and hence in growth and development during the early stages of mouse life.

Thrombin receptor overexpression in malignant and physiological invasion processes.

Although the involvement of soluble and matrix-immobilized proteases in tumor cell invasion and metastasis is well recognized, the role of proteolytically activated cell surface receptors has not been elucidated. We report here that thrombin receptor, a member of the protease-activated receptor family, is preferentially expressed in highly metastatic human breast carcinoma cell lines and breast carcinoma biopsy specimens. Introduction of thrombin receptor antisense cDNA considerably inhibited the invasion of metastatic breast carcinoma cells in culture through a reconstituted basement membrane. During placental implantation of the human embryo, thrombin receptor is transiently expressed in the invading cytotrophoblasts. These results emphasize the involvement of thrombin receptor in cell invasion associated with tumor progression and normal embryonic development.

Specific involvement of glypican in thrombin adhesive properties.

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.

The involvement of thrombin RGD in metastasis: characterization of a cryptic adhesive site.

Adhesive interactions between cells and the subendothelial extracellular matrix take place at several stages during tumor progression and metastasis. We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which can be exposed in the presence of low concentrations of plasmin and cell-associated heparan sulfate proteoglycan. Thus, thrombin may act as a matrix-adhesive molecule via activation of the alpha v beta 3 integrin. We have now identified a 31 amino acid fragment as the minimal thrombin-generated cleavage product, which contains an active RGD site, following gel filtration analysis on FPLC Superdex 75 column. The role of membrane-associated heparan sulfate in thrombin conversion to an adhesive protein was demonstrated by using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Incubation of both thrombin and a low concentration of plasmin on the surface of wild type CHO cells resulted in a typical digestion cleavage profile upon gel filtration. No cleavage products were observed when thrombin and a suboptimal plasmin concentration were incubated on monolayers of CHO cell mutants lacking heparan sulfate. Next, we examined the possible role of the thrombin RGD site during the progression of tumor development and metastasis. Toward this, we tested murine melanoma cells expressing low (B16-F1 cells) and high (B16-BL6 cells) lung colonization potentials in cell adhesion assays in vitro. Differential adherence capability of the cells was observed: while high attachment levels of B16-BL6 cells were obtained, the low metastatic B16-F1 cells did not adhere to thrombin RGD. Antibodies raised against the RGD site in thrombin specifically recognized thrombin digested with plasmin, but were unable to interact with native thrombin or prothrombin and inhibited potently B16-BL6 melanoma cell adhesion. Furthermore, the antibodies failed to recognize RGD in other adhesive plasma proteins such as vitronectin, fibrinogen, or fibronectin. Provided that the RGD-containing fragments of thrombin are widely distributed throughout the vascular system, they may have a significant role during tumor progression and dislodgement of metastatic cells. The development of RGD mimetics and/or specific antibodies might thus be applied to inhibit a critical step in metastatic spread.