Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts.

The literature suggests that cholesterol and sphingomyelin might be essentially confined to plasma membranes in mammalian cells; however, this premise has thus far escaped a direct test. We explored the issue in three ways. First, we fractionated whole homogenates of cultured human fibroblasts by equilibrium sucrose density gradient centrifugation. We found that the profiles of cholesterol and sphingomyelin were indistinguishable from those of two plasma membrane markers, 5' nucleotidase and [3H]galactose, which was conjugated to the surface of intact cells from an exogenous donor by galactosyltransferase. Second, we determined the relative surface areas of intact cells from their uptake of 1-(4-trimethyl-amino)phenyl-6-phenylhexa-1,3,5-triene, a cationic fluorescent dye which partitions into but does not cross plasma membranes. Relative to human red cell ghosts, the apparent surface area of the fibroblasts was 17,500 microns2/cell while for canine hepatocytes, the value was 11,500 microns2/cell. The relative ratios of cell cholesterol to dye binding (hence, surface area) were quite similar in ghosts, fibroblasts, and liver cells; namely 1.0, 1.12, and 0.67, respectively. Finally, we found that the specific ratios of both cholesterol and sphingomyelin to 5' nucleotidase were only 10% less in gradient-purified plasma membranes than in whole homogenates. Similar results were obtained using an entirely different method of purification: two-phase aqueous partition. The cholesterol and sphingomyelin in fractions rich in other membranes was closely proportional to their 5' nucleotidase content, suggesting that the presence of these lipids reflected contamination by plasma membrane fragments. The 5' nucleotidase/phospholipid ratio in the purified plasma membrane fraction was roughly twice that in whole cells. We conclude that the compartment marked by 5' nucleotidase in cultured human fibroblasts contains approximately 90% of the two named lipids and half the cell phospholipid phosphorus.

Lateral diffusion of lectin receptors in fibroblast membranes as a function of cell shape.

Anchorage-dependent fibroblasts respond to biochemical growth signals only when attached to and spread on a suitable substrate surface. Attachment of fibroblasts initiates a cytoskeletal assembly process that results in the organization of long actin stress fibers and microtubules which may be required for transmembrane signal transduction. Fibroblasts maintained in suspension, however, remain spherical with no apparent stress fibers or lengthy microtubules. Because of the significant differences in cytoskeletal organisation induced by shape modification, and the resulting possible changes in organization and dynamics of membrane receptors, the technique of fluorescence redistribution after photobleaching (FRAP) was employed to examine the lateral mobility of wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors in the plasma membrane of untransformed and Kirsten murine sarcoma virus-transformed Balb/c 3T3 fibroblasts in the spread and spherical state. An examination of FITC-WGA and FITC-sCon A binding to the plasma membrane for both cell lines in a spread or spherical state demonstrated no significant differences in the number of WGA or Con A receptors as a function of shape or transformation. The primary observations from this study are (a) membrane WGA and sCon A receptors in spherical Balb/c 3T3 fibroblasts display mobility 12 times faster than in the spread state, while phospholipid mobility is similar and apparently shape independent, (b) transformed cells in the spread state have WGA and sCon A receptor mobilities similar to those of untransformed cells in the spread state, (c) flat adherent, but not unattached spherical, Balb/c 3T3 fibroblasts are subject to Con A-induced global modulation and (d) transformed cells in the spherical state contain a significant population of cells (approximately 30%) with WGA receptor mobilities faster than those observed in spherical untransformed cells. These observations are discussed in terms of a linked matrix model for membrane protein diffusion.

Clonal selection by fluorescence redistribution after photobleaching (FRAP)--a "fast" lateral mobility fibroblast mutant (E7G1).

Fluorescence redistribution after photobleaching (FRAP) was utilized to select a "fast" lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 +/- 1.6) X 10(-9) cm2/s and (2.7 +/- 2.3) X 10(-9) cm2/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 +/- 1.9 X 10(-9) cm2/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 +/- 0.19) X 10(-9) cm2/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses.

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.

Estrogen action in the mouse uterus: adrenal modulation of receptor levels.

Adrenal regulation of estradiol receptor levels was demonstrated in the ovariectomized mouse uterus. After ovariectomy, cytosol receptor levels initially followed a cyclic pattern, with a periodicity of approximately 5 days and a range of range of 2.25-4.80 x 10(-13) mol estrogen receptor/100 micrograms DNA. Nuclear receptor levels showed marked fluctuations initially, but stabilized after day 15 at approximately 0.5 x 10(-13) mol/100 micrograms DNA. Microsomal receptor levels also changed with time, but at a lower level than the cytosol and nuclear fluctuations. Concurrent cyclic changes in uterine or vaginal histology were not observed. After simultaneous ovariectomy and adrenalectomy, cytosol estradiol receptor levels remained low (less than or equal to 1.5 x 10(-13) mol/100 micrograms DNA) from 5-24 days, with no fluctuations. Nuclear and microsomal receptor levels changed, but to a smaller extent than those in ovariectomized mice. Concurrent histological changes were not observed. The residual cytosolic estrogen receptor remaining in the ovariectomized-adrenalectomized mouse uterus was characterized. Scatchard analysis of saturation binding data showed one class of binding sites with a Ka of 6.56 nm-1 (n = 350 fmol/mg protein). This cytosol receptor sedimented at 8S on low salt sucrose density gradients. Receptor specificity was assessed by competitive equilibrium binding. The rank order of ligands was 17 beta-estradiol = diethylstilbestrol greater than nafoxidine. Progesterone and 5 alpha-dihydrotestosterone showed no appreciable binding activity. After the injection of estradiol (10 micrograms/kg), 80-90% of the total available estrogen receptor translocated to the nuclear compartment. This translocated receptor could stimulate uterine hypertrophy and hyperplasia, as assessed in 1- and 3-day bioassays, and was able to initiate progesterone receptor synthesis. From these results, we conclude that the adrenal can modulate uterine estrogen receptor levels.