The motility of gastrointestinal tract is regulated by classical neurotransmitters, neuropeptides, and humoral agents. Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein, Bv8, and a nontoxic protein of mamba snake venom. These human cDNAs encode two secreted proteins of 86 and 81 amino acids. Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach. Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has confirmed the formation of five pairs of disulfide bonds. The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range. The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle. To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle. The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors. Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle. In summary, we have identified two novel endogenous regulators of gastrointestinal motility. The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.

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Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5-to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis. Protease nexin 1 (PN1)1 is a 43-kDa serine protease inhibitor that is a member of the SERPIN superfamily (1). It is an important physiological regulator of thrombin in tissues, and like its plasma counter part, antithrombin III, it is activated by heparin (1, 2). Like other members of the SERPIN family, PN1 is proteolytically attacked by its target protease but arrests proteolytic cleavage resulting in the formation of a 1:1 stoichiometric complex with that protease (1, 2). Only PN1 that is in complex with a protease is internalized and degraded by cells at a significant rate (3). This is presumably due to the formation or unmasking of a receptor binding site in PN1 when in complex with a protease, which is absent in the free SERPIN. One candidate site within the PN1 sequence for this function has recently been identified (4). Mechanistically, this ensures that SERPINs will remain extracellular until they have become part of an inhibitory complex. Once they have formed an inhibitory complex with a protease, the rapid removal of protease-SERPIN complexes is important for two reasons. First, since there is evidence to suggest that protease-SERPIN complexes may not be covalently linked (5), the uninternalized complexes represent a potential source of active protease formed when the complexes dissociate. Second, in the case of one S...

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Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells in culture is related to the somatomedin family of growth regulatory polypeptides.MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CIEF) at concentrations of 10-200 ng/ml. MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is' bound to a specific high molecular weight carrier protein.In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and'DNA synthesis. In addition, purified MCP competitively inhibits the binding of i25I1abeled MSA to these cells. In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses. These results indicate that the inhibitory effect of MCP'is the result of'specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins.