Insulin appears to promote microtubule assembly in rat adipocytes. Neither oxytocin nor high concentrations of glucose has this effect. Colchicine inhibits stimulation by insulin of lipid and glycogen synthesis without influencing stimulation by insulin of glucose oxidation. The anabolic effects of oxytocin or high concentrations of glucose are not inhibited by colchicine. The "directive effect" of insulin may involve microtubules.
Abstract These studies were initiated with the objective of isolating epithelial and stromal cells of human prostatic tissue in undamaged state, in order to study the cellular distribution of steroid receptors in benign prostatic hyperplasia (BPH) relative to normal prostate. Initial experiments showed that when BPH tissue immersed in tissue culture media was progressively fragmented by various cutting procedures, epithelial elements were selectively released as clumps of variable size and individual cells, but that a large percentage of these cells were damaged, as evidenced by their failure to exclude trypan blue (TB). These observations suggested that if tissue framentation were carried out under defined conditions that minimize cell damage, BPH subfractions might be obtained containing a large percentage of undamaged cells. To determine conditions of tissue fragmentation which result in maximal recovery of epithelial cells which exclude TB, rat ventral prostate (RVP) was chosen as a model system. Experiments with RVP revealed that maximal yields of such cells were obtained in “large” epithelial clumps (>30 cells per clump) released under the following conditions: (1) chopping the tissue with razor blades in a large volume (2 ml/100 mg RVP) of a Ca 2+ ‐free tissue culture medium (Joklik's‐MEM) containing 1% casein, (2) carrying out the entire fractionation procedure in the cold, and (3) maintaining a 1% casein concentration in the medium during chopping, as well as in subsequent washing procedures, to protect cells from proteolytic activity. In large epithelial clumps, cells in the interior of the clump were not stained by TB but the cells at the periphery of the clump were freely permeable to TB. Single epithelial cells and small epithelial clumps (3–10 cells) released by razor blade fragmentation were also permeable to TB. When large epithelial clumps were incubated at 20°C for 90 min, the clumps disaggregated into smaller clumps and morphologically intact single cells, which did not exclude TB. The residual tissue fragments remaining after chopping contained the bulk of stromal cells plus some epithelial elements. The latter could be removed by gentle rubbing of the fragments on a sieve in the presence of medium. The stromal fraction thus obtained consisted of stromal cells, embedded in mesenchymal matrix, which were not stained by TB and appeared normal when examined histologically by light microscopy. The androgen receptor content per unit DNA (AR/DNA) of epithelial clumps and stromal fractions from castrate (1 day) and normal intact rats were compared to control RVP (coarsely minced and washed, but not further dissociated) using [ 3 H]‐R‐1881 as ligand. In RVP from castrate animals (where AR is unoccupied and cytosolic), the mean AR/DNA ratio in large epithelial clumps was 17% of the control value. However, on a protein basis, AR per milligram cytosolic protein in epithelial clumps was 80% of the unfractioned control. After disaggregation of large epithelial clumps at 20°C, the recovered cells no longer contained detectable levels of AR. The mean AR/DNA ratio in stromal cell fractions from castrate RVP was about half that found in epithelial clump fractions. In RVP from normal intact animals (where AR is occupied with endogenous DHT and predominantly localized in nuclei), AR was measured by a [ 3 H]‐R‐1881 exchange assay. The mean AR/DNA ratio in epithelial clumps from normal RVP was about 45% of the value found in control tissue; in stromal fractions the mean AR/DNA ratio was about 60% of the found in epithelial clumps. Estradiol receptor (ER) in normal intact rats, measured using [ 3 H]‐R‐2858 as ligand was found to be unoccupied, cytosolic, and present in low concentration relative to unoccupied, cytosolic AR in castrate RVP. Highest ER/DNA ratios were found in the stromal cellular fraction with successively lower values in unfractionated RVP and epithelial clumps. Arginase activity per unit DNA in epithelial clumps was about 28% of that found in control tissue. The mean arginase/DNA ratio in stromal fractions was about 80% of the value found in the control and consistently higher than in epithelial clumps. Thus in RVP, arginase appears to be a marker for “viability” of stromal as well as epithelial cells, rather than a specific marker for epithelial cells. The present results demonstrate that AR and ER are both present in epithelial and stromal subfractions of RVP. Since AR/DNA ratios were higher in epighelial clumps than in stromal fractions in both normal and castrate RVP, (despite the greater degree of cell damage in epithelial elements relative to stroma, as suggested by arginase content) this finding indicates that AR concentration is much higher in epithelial than in stromal cells. IN contrast, Er appears to be predominantly localized in stromal cells.
Estrogen and androgen receptors have been investigated in rat ventral prostate epithelium and stroma. High speed supernatants were prepared from unfractionated or fractionated prostates. Cytosols from intact rats were incubated with 2 nM 3H-estradiol (E2) in presence of 80 nM dihydrotestosterone (DHT), and cytosols from 1 day castrated rats were incubated with 2 nM 3H-DHT, at 0 C for 4 h. They were submitted to ultracentrifugation on glycerol-Tris gradients. The amounts of hormones bound to the saturable 8S binding components were determined on a comparative basis. The values for E2-receptor in intact rats were 2.3, 15.4 and 5.9 fmol/mg cytosol protein in unfractionated prostate, stroma and epithelium,respectively. The corresponding values for DHT-binding were 31.9, 17.2 and 29.2 fmol/mg protein. In addition, Scatchard analysis of saturable E2 and DHT binding, using the protamine precipitation technique, essentially confirmed the results of glycerol gradients, and led to the conclusion that, contrary to androgen receptor, the major part of estradiol receptor is localized in stroma.
An adenyl cyclase preparation derived from epithelial cells of the urinary bladder of the toad, Bufo marinus , is described. This cyclase preparation is specifically stimulated by neurohypophyseal hormones and various synthetic analogs which evoke a hydroosmotic response in the intact bladder. The relative stimulatory effects of these compounds have been compared on the cyclase preparation and in the intact bladder. The peptide concentrations required for half-maximal stimulation (affinity) in the cell-free and intact systems were parallel; however the magnitude of stimulation produced by saturating concentrations of peptides did not correlate. Furthermore, it was found that peptide analogs which inhibit the hydroosmotic effect of [8-arginine]-vasopressin on the intact bladder also inhibit the stimulation of the toad bladder cyclase preparation by vasopressin. Prostaglandin E 1 , mercaptans, and disulfides, which inhibit the hormone-induced hydroosmotic response of the intact bladder, did not antagonize the stimulation of the toad bladder cyclase preparation by vasopressin.
The effect of insulin administration upon D-xylose-1-C14 penetration into the diaphragm and gastrocnemius muscles of functionally nephrectomized normal, hypophysectomized, and adrenalectomized rats has been examined. It was found in all groups that after the administration of tracer amounts of D-xylose, this sugar enters the cell water of diaphragm to a greater extent than in gastrocnemius muscle, both in the presence and absence of exogenous insulin. Insulin increases the apparent intracellular distribution of D-xylose in both muscles in all three types of rats. After insulin administration, the intracellular concentration of D-xylose in diaphragm muscle was estimated to be about two times greater than D-xylose concentration in plasma; D-xylose accumulation was not observed in gastrocnemius muscle of insulin-treated rats. Intracellular accumulation of D-xylose occurs in diaphragm of insulin-treated rats at plasma concentrations of D-xylose ranging from 4 to 2200 µg/ml; however, a "saturation" phenomenon appears to be operative, since intracellular distribution declines as plasma D-xylose concentration is increased within this range. A decline in intracellular D-xylose distribution also occurs in gastrocnemius as plasma D-xylose is increased, suggesting that entry into this muscle as well does not exhibit the characteristics of a simple diffusion process. The significance of these in vivo observations is briefly discussed in relation to widely accepted assumptions concerning sugar permeability in muscle.
A method for assaying corticosteroid activity based on the protection against the lethal action of bacterial endotoxin has been developed. Linear relationships between survival and dose of endotoxin were observed when a mixture of endotoxin and steroid were given intracardially to adrenalectomized rats. pThe relative activities of corticosteroids in protecting against endotoxins are different from their apparent anti-inflammatory activities.
