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Guanylin receptor guanylate cyclase (GC-C) peaks in neonatal intestine and is involved in either enterocyte proliferation or chloride secretion. The latter is more potent when GC-C activator guanylin, or its analog Escherichia coli heat-stable enterotoxin (ST), is added to the mucosal rather than serosal side of intestinal monolayers. By using Ussing chambers, we investigated transepithelial ion transport and enterocyte proliferation and their mechanisms in response to the addition of guanylin or ST to the mucosal or serosal side of Caco-2 monolayers and in ileal specimens from neonates. GC-C activation showed a polar pattern of the effects. GC-C mucosal activation resulted in a potent cGMP-chloride secretion activation and in a marginal enterocyte proliferation. Conversely, serosal GC-C activation induced a potent enterocyte proliferation, through MAP kinase ERK 1/2. Finally, the inhibition of ERK1/2 enhanced the Isc increase in response to serosal but not to mucosal ST stimulation, indicating that ERK1/2 also acts as a brake of chloride secretion. These data suggest that the guanylin/GC-C system plays a key role in early postnatal intestinal adaptation exploiting the polar structure of enterocyte.

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

Angiotensin II (AII) receptor binding assays were performed in rat adipocytes from three separate anatomic depots. Fat cells were isolated by collagenase digestion, and plasma membranes were prepared from the epididymal, mesenteric, and retroperitoneal fat depots of male Sprague-Dawley rats at 100 days of age. Binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific in membranes from all depots, identifying a receptor with a similar affinity of approximately 1 nM. Site-associated differences in receptor number were observed, with epididymal and mesenteric fat cell membranes exhibiting significantly more receptors than retroperitoneal fat cells when binding was expressed per unit of membrane protein. When corrected for cell volume, the number of receptors per cell ranked epididymal > retroperitoneal > mesenteric. Inhibitory constants for the peptide agonists AII and AIII and the peptide antagonist [Sar1,Ala8]AII indicated similar affinities in all three depots. Because the receptor has been classified pharmacologically into two subtypes, the AT1 selective antagonist losartan, and the AT2 selective antagonist PD 123,319 were used to classify the adipocyte receptor, indicating an AT1 subtype with an affinity for losartan in the mesenteric and retroperitoneal adipocytes that was significantly greater than the epididymal. Similar studies were performed in adipocyte membranes obtained from human omental and subcutaneous adipose tissue, revealing the presence of an AII receptor in both depots with an affinity of approximately 10 nM for losartan. These data indicate site-specific differences in AII receptor number in fat cell membranes from rats and the existence of human adipocyte AII receptors, suggesting that the adipocyte is significant for the peripheral metabolism of components of the renin-angiotensin system.

CMC-544 is CD22-specific cytotoxic immunoconjugate of calicheamicin that has demonstrated significant anti-tumor activity in phase I clinical trial in B-NHL patients (Blood2005;106:230). In this trial, thrombocytopenia was reported as the most common dose-limiting adverse event. The present non-clinical study attempted to provide insights into the observed thrombocytopenia associated with CMC-544 treatment in B-NHL patients. In vitro, CMC-544 neither bound to nor caused aggregation of platelets in platelet-rich plasma of human or murine origin. When administered to mice, a single dose of CMC-544 resulted in a 50% to 75% reduction in the number of circulating platelets with a nadir on day 3 or 4. The platelet levels returned to normal at the latest by day 12. Similar effects were also observed in cynomolgus macaques with nadirs on day 4 or 5. CMC-544 dose not cross-react with either murine or cynomolgus macaque CD22. Unconjugated anti-CD22 antibody, G5/44 (humanized IgG4 antibody) had no effect on circulating platelets. CMC-544-induced thrombocytopenia in mice was associated with a concomitant reduction in circulating levels of thrombopoietin (TPO) with a nadir on day 4. In contrast, treatment with carboplatin, a cytotoxic chemotherapeutic agent known to cause thrombocytopenia, caused thrombocytopenia in mice but with a nadir on day 11. Unlike that with CMC-544, carboplatin-induced thrombocytopenia was associated with an increase in the levels of circulating TPO. Thus, CMC-544-induced thrombocytopenia appears to be mechanistically and kinetically distinct from that associated with conventional cytotoxic chemotherapeutic agents. In this murine model, pretreatment with human IGIV (Carimune) failed to prevent CMC-544-associated thrombocytopenia. However, subcutaneous pretreatment of mice with oprelvekin (Neumega/rhIL-11; 250 ug/kg) significantly reduced the magnitude of the CMC-544-associated thrombocytopenia. When evaluated in cynomolgus macaques, daily subcutaneous pretreatment with oprelvekin (125 ug/kg) for 5 days completely prevented CMC-544-associated thrombocytopenia and also suppressed elevations in serum hepatic aminotransferases associated with the CMC-544 treatment. Concurrent administration of oprelvekin with CMC-544 failed to prevent the initial thrombocytopenia but did accelerate complete recovery of platelets counts to the pretreatment levels and also suppressed elevations in serum hepatic aminotransferases. These results suggest that thrombocytopenia associated with CMC-544-treatment may in part be due to CMC-544-induced hepatic changes, including decreased TPO production. This study further provides evidence that oprelvekin not only stimulates megakaryocytes to produce platelets but is also hepatoprotective, reducing the degree of CMC-544-mediated hepatotoxicity. We have presented preclinical evidence that the prophylactic use of oprelvekin can ameliorate CMC-544-induced thrombocytopenia and thus, supports the clinical evaluation of oprelvekin administered with CMC-544 to augment its therapeutic benefit in patients with B-non-Hodgkin’s lymphoma.