Ravindra K. Pathak

Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle.

Lactate and pyruvate cross cell membranes via a monocarboxylate transporter (MCT) with well-defined properties but undefined molecular structure. We report the cloning of a cDNA encoding MCT1, a monocarboxylate transporter whose properties resemble those of the erythrocyte MCT, including proton symport, trans acceleration, and sensitivity to alpha-cyanocinnammates. A Phe to Cys substitution in MCT1 converts it to Mev, a mevalonate transporter. MCT1 is abundant in erythrocytes, cardiac muscle, and basolateral intestinal epithelium. In skeletal muscle it is restricted to mitochondria-rich myocytes. As sperm traverse the epididymis, MCT1 switches from sperm to epithelial cells. MCT1 is present at low levels in liver, suggesting another MCT in this tissue. By exporting lactate from intestine and erythrocytes, MCT1 participates in the Cori cycle. It also participates in novel pathways of monocarboxylate metabolism in muscle and sperm.

Expression of the VLDL Receptor in Endothelial Cells

In this article we describe the cellular distribution of the very low density lipoprotein receptor (VLDLR), a transmembrane protein that is expressed at high concentrations in skeletal muscle, heart, adipose tissue, and brain but in only trace amounts in the liver. Indirect immunofluorescence localization studies were performed in murine and bovine tissues using a rabbit polyclonal anti-human VLDLR antibody. Immunoreactive VLDLR protein was detected in the endothelium of capillaries and small arterioles but not in veins or venules of bovine skeletal muscle, heart, ovary, and brain. In the liver, there was intense staining of the capillaries and arterioles that supply the capsule and hepatic vessels but no staining of the sinusoidal surfaces. We failed to detect any signal from nonendothelial cells in the liver or peripheral organs. The VLDLR was also expressed at high levels on the endothelial surface of bovine coronary arteries; in contrast, little or no staining was seen in aortic endothelium. Antibody staining of cultured bovine coronary artery endothelial cells demonstrated punctate cell-surface staining, as well as staining of large and small cytoplasmic vesicles. This tissue and cell pattern of expression suggests that the VLDLR plays a role in the transport of VLDL or another plasma constituent from the vascular compartment to adjacent tissues.

Early effects of PP60v‐src kinase activation on caveolae

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat‐1 cell expressing a temperature sensitive pp60v‐src kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24–48 h after cells were shifted to the permissive temperature, a set of caveolae‐specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin‐1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin‐1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine‐phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60v‐src kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype. J. Cell. Biochem. 71:524–535, 1998.

Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling.

We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.

Histidine-rich calcium binding protein, a sarcoplasmic reticulum protein of striated muscle, is also abundant in arteriolar smooth muscle cells

SummaryHistidine-rich calcium binding protein (HRC) is a luminal sarcoplasmic reticulum protein abundant in skeletal and cardiac muscle. Using immunofluorescence to examine non-muscle tissues, we now show that HRC is also abundant in the smooth muscle cells lining the walls of small arteries and arterioles. Arterioles that contain only one or two layers of smooth muscle cells are very brightly stained while small muscular arteries demonstrate a lesser degree of immunoreactivity only in cells just adjacent to the lumen of the vessel. In contrast, visceral smooth muscle cells from the gastrointestinal and genitourinary tracts show no HRC immunofluorescence. We also examined the subcellular distribution of HRC in arteriolar smooth muscle by immunoelectron microscopy. HRC was found in electron-dense vesicles beneath the plasma membrane, in small electron-lucent vesicles and in the nuclear envelope, suggesting a location within a calcium-sequestering compartment. These findings suggest that HRC plays a role in sarcoplasmic reticulum function that is unique to striated and arteriolar smooth muscle.