Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit.

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

A host-rotaxane derivatized with carboxylic acids efficiently delivers a highly cationic fluoresceinated peptide.

A cleft-[2]rotaxane (CR2+2-) was derivatized with carboxylic acids to enhance the intracellular delivery of a highly cationic or anionic pentapeptide. CR2+2- delivers the fluorescein (Fl) tagged peptide Fl-KKALR to a greater amount than Fl-QEAVD, and at a higher concentration, a greater amount than Fl-AVWAL. The level of delivery is largely temperature and ATP independent, suggesting that the Fl-peptide.CR2+2- complexes pass through the cellular membrane without requiring active cell-mediated processes. This study shows that selective delivery of peptides is possible by using a suitably derivatized host-rotaxane as the transporter.

Macrophages mediate inflammation-enhanced metastasis of ovarian tumors in mice.

The tumor microenvironment is known to have a profound effect on tumor progression in a highly context-specific manner. We have investigated whether peritoneal inflammation plays a causative role in ovarian tumor metastasis, a poorly understood process. Implantation of human ovarian tumor cells into the ovaries of severe combined immunodeficient mice resulted in peritoneal inflammation that corresponds temporally with tumor cell dissemination from the ovaries. Enhancement of the inflammatory response with thioglycolate accelerated the development of ascites and metastases. Suppression of inflammation with acetyl salicylic acid delayed ascites development and reduced tumor implant formation. A similar prometastatic effect for inflammation was observed when tumor cells were injected directly into the peritoneum of severe combined immunodeficient mice, and in a syngeneic immunocompetent mouse model. Inflammation-modulating treatments did not affect primary tumor development or in vitro tumor cell growth. Depletion of peritoneal macrophages, but not neutrophils or natural killer cells, reduced tumor progression, as assessed by ascites formation and peritoneal metastasis. We conclude that inflammation facilitates ovarian tumor metastasis by a mechanism largely mediated by macrophages, and which may involve stromal vascular endothelial growth factor production. The confirmation of these findings in immunocompetent mice suggests relevance to human disease. Identifying the mechanisms by which macrophages contribute to tumor metastasis may facilitate the development of new therapies specifically targeting immune cell products in the tumor microenvironment.

Determining the binding and intracellular transporting abilities of a host-[3]rotaxane.

The cellular permeability of compounds can be enhanced in the presence of a host-[2]rotaxane (HR). The effective concentration of an HR is limited by the stoichiometry of the complex formation of the HR and the delivered compound. We speculate that a complex forms between the HR and a guest during membrane passage. To further explore the relationship between guest binding and guest delivery and to obtain more efficient delivery devices, we present, in this report, the first example of a cyclophane-[3]rotaxane (Cy3R), which has two wheels and a cyclophane as a blocking group. The properties of Cy3R were compared to a new cyclophane-[2]rotaxane (Cy2R) that has the same cyclophane pocket as Cy3R but only a single wheel. The second wheel of Cy3R can form additional noncovalent bonds, e.g., salt bridges, cation-pi interactions or aromatic-aromatic interactions, with appropriately functionalized guests. We show by flow cytometric analysis that Cy3R transfers Fl-AVWAL (76%) and to a lesser degree Fl-QEAVD (26%) into live cells. The level of Fl-peptide within a cell is concentration dependent and largely temperature and ATP independent, suggesting that a Cy3R.Fl-peptide complex passes through the cellular membrane without requiring active cell-mediated processes. Cy2R, on the other hand, forms weaker complexes and requires a higher concentration to transfer materials into cells. These results demonstrate that the addition of a second wheel on a rotaxane can improve guest binding in various solvents and hence delivery through cellular membranes.

Investigation of the intracellular delivery of fluoresceinated peptides by a host-[2]rotaxane.

The development of methods to transport peptides into cells via a passive mechanism would greatly aid in the development of therapeutic agents. We recently demonstrated that an impermeable fluoresceinated pentapeptide enters the cytoplasm and nucleus of COS 7 cells in the presence of a host-[2]rotaxane by a mechanism that does not depend on an active cell-mediated process. In this report, we further investigate the ability of the host-[2]rotaxane to deliver peptides possessing a wide range of polarities (negatively charged, positively charged, polar, and apolar side chains) into live cells. Only in the presence of the host-[2]rotaxane were the Fl-peptides taken up by COS 7 and ES2 cells. Flow cytometry experiments demonstrated that the level of delivery is largely temperature and adenosine 5'-triphosphate (ATP) independent, and the membranes remain intact. Although the level of transport does depend upon the nature of the side chains, it does not correlate with calculated LogD values, indicating that an additional interaction with the host-[2]rotaxane is modifying the permeability properties of the peptide. The amount of Fl-peptides transported from an aqueous phase into a chloroform phase in the presence of the host-[2]rotaxane correlates with the intensity of cellular fluorescence. Extraction and U-tube studies show that the Fl-peptide can be released from its complex with the host-[2]rotaxane into an aqueous phase, and the host-[2]rotaxane can transport a greater than a stoichiometric amount of an Fl-peptide through a CHCl3 layer. These studies demonstrate the utility of the host-[2]rotaxane in delivering peptides of all polarities across a cell membrane.

Determining the intracellular transport mechanism of a cleft-[2]rotaxane.

Rotaxanes are a class of interlocked compounds that have been extensively investigated for their potential utility as switches or sensors. We recently demonstrated that rotaxanes have further application as agents that transport material into cells. This novel finding prompted our investigation into the mechanism by which rotaxanes are involved in transmembrane transport. Two-dimensional NMR analysis showed that a cleft-containing rotaxane exists in two dominant conformations ("closed" and "open"). To determine the importance of conformational flexibility on the ability of the rotaxanes to bind guests and transport material into cells, the rotaxane was chemically modified to lock it in the closed conformation. Charged guests interact less favorably with the locked rotaxane, as compared to the unmodified rotaxane, both in an aqueous solution and in DMSO. In a chloroform solution, both rotaxanes bind the guests with similar affinities. The locked rotaxane exhibited a reduced capacity to transport a fluoresceinated peptide into cells, whereas the unmodified rotaxane efficiently delivers the peptide. Flow cytometry experiments demonstrated that a high percentage of the cells contained the delivered peptide (89-98%), the level of delivery is concentration dependent, and the rotaxanes and peptide have low toxicity. Cellular uptake of the peptide was largely temperature and ATP independent, suggesting that the rotaxane-peptide complex passes through the cellular membrane without requiring active cell-mediated processes. The results show that the sliding motion of the wheel is necessary for the delivery of materials into cells and can enhance the association of guests. These studies demonstrate the potential for rotaxanes as a new class of mechanical devices that deliver a variety of therapeutic agents into targeted cell populations.