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Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.

The purpose of this study was to examine and quantify hepatic uptake and degradation of gastrin and cholecystokinin peptides. Rat livers were perfused in situ, without recirculation, with Krebs-Ringer bicarbonate (pH 7.4) containing 10% bovine erythrocytes, gassed with 95% O2-5% CO2. Gastrin and cholecystokinin peptide fragments of various lengths were injected into a portal vein via a sidearm syringe for over 1 min, and hepatic venous effluent was collected every minute for 20 min. After injection of 125I-labeled gastrin peptides more than eight amino acids in length, greater than 95% radioactivity appeared in the hepatic venous effluent, all of which was intact peptide, as determined by immunoprecipitation, trichloroacetic acid precipitation, and Sephadex gel chromatography. Decreasing the chain length to eight or less amino acids resulted in progressive increases in hepatic uptake and degradation of gastrin fragments. Injected cholecystokinin peptides greater than seven amino acids in length traversed the liver intact, whereas smaller cholecystokinin peptides were cleared by the liver. The liver eliminated greater than 90% of the biologically active carboxyl-terminal tetrapeptide amide common to gastrin and cholecystokinin. The results of this study indicate that gastrin and cholecystokinin peptides longer than seven amino acids traverse the liver without significant degradation, however, smaller peptides are progressively cleared during hepatic transit. Therefore, if small gastrin and cholecystokinin peptides from antral and intestinal mucosa are released and reach the portal venous circulation, hepatic inactivation would appear to prevent them from reaching the systemic circulation, precluding their significant contribution to gastric acid secretion and other physiologic functions.

The ability of a purified sea anemone (Stichodactyla helianthus) protein cytolysin to interact with a variety of interfaces was investigated by means of the Wilhemy plate method. At the air:water and lipid:water interfaces, the toxin lowered the surface pressure most readily as the aqueous phase pH increased towards the isoelectric point (9.8) of the toxin. The affinity of the toxin for both the phospholipid:water and the oil:water interfaces was much greater than for the air:water interface. Although the toxin had previously been found to avidly bind to sphingomyelin-containing phospholipid dispersions and bilayers, it failed to display any preferential interaction with a sphingomyelin monolayer relative to one of dipalmitoylphosphatidyl-choline under identical conditions, even when the monolayers were maintained at 40 dynes/cm, a pressure considered to produce phospholipid packing densities similar to those observed in cell membranes. Unlike many other membrane-active protein cytolysins, the ability of Stichodactyla cytolysin to penetrate these phospholipid monolayers was not affected by the initial surface pressure over the range 0-32 dynes/cm. However, at 40 dynes/cm initial packing pressure, the surface pressure generated by the cytolysin was similarly reduced in both sphingomyelin and dipalmitoylphosphatidylcholine monolayers. Our results suggest that the protein cytolysin initially binds reversibly to cell and artificial bilayer membranes in a non-specific manner; sphingomyelin domains in the bilayer then provide optimal conditions for insertion into the membrane and subsequent assembly of a stable multimeric complex which functions as an ion channel.

The present studies were directed toward examining the effects of gastrin-releasing peptide (GRP) on acid secretion and on beta-adrenergic-stimulated gastrin and somatostatin release using the isolated vascularly perfused rat stomach. Including pentagastrin in perfusion buffer increased acid output from 2.2 +/- 0.4 mueq H+/h during control perfusion to 18.8 +/- 1.8 mueq H+/h (P less than 0.01). No significant changes in acid secretion were detected when either GRP or specific antibodies to GRP were included in perfusate in the absence or presence of pentagastrin. Inclusion of 10(-9) M isoproterenol in the perfusate did not change acid output with respect to control; however, gastrin and somatostatin release into the portal venous effluent was significantly enhanced. Peak gastrin and somatostatin concentrations observed at 15 min were 753 +/- 43% (P less than 0.001) and 345 +/- 43% (P less than 0.01), respectively, of basal levels. When antibodies to GRP were included in perfusate containing isoproterenol, gastrin and somatostatin release into the portal venous effluent was significantly inhibited. The results of these studies indicate that GRP does not affect basal or pentagastrin-stimulated gastric acid secretion in the isolated perfused rat stomach. However, under the conditions of these experiments, beta-adrenergic stimulation of gastrin and somatostatin release appears to be mediated, at least in part, through GRP.

Stichodactyla helianthus cytolysin III, a 17 kDa basic polypeptide isolated from a Caribbean sea anemone, is one of the most potent hemolysins yet found in a living organism. This toxin has been reported to form new ion channels in artificial lipid bilayer membranes. The ability of this toxin to attack cell membranes is greatly enhanced by the presence of sphingomyelin. In order to investigate the mechanism by which the cytolysin causes cell lysis, we have prepared a highly active [3H]cytolysin derivative by reductive methylation with sodium cyanoborohydride and [3H]formaldehyde. A dimethylated toxin derivative was used to investigate the basis for the differential lytic activity of this polypeptide upon erythrocytes from six mammalian species. Using both direct [3H]toxin binding and indirect (Thron method) binding techniques, we found that the interspecies differences are due to variable membrane susceptibilities toward the bound toxin, rather than to differences in membrane affinity for the toxin. Similarly, we showed the enhanced lytic activity of the toxin for rat erythrocytes at elevated pH to be caused by enhanced activity of the bound toxin.

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.

In order to examine hepatic clearance of gastrointestinal regulatory peptides, rat livers were perfused in situ, and radiolabelled somatostatin (S-14, S-28), gastrin-releasing peptide (GRP-14, GRP-27), and vasoactive intestinal peptide (VIP) were injected into the portal vein and hepatic venous effluent was collected. S-14 and S-28 were not affected significantly by hepatic transit: 91.6 +/- 2.8% (SEM) of S-14 and 95.9 +/- 2.2% of S-28 were recovered, and neither peptide was degraded by hepatic transit, as determined by immunoprecipitation and gel chromatography. GRP-14 and GRP-27 were also not affected by hepatic transit: 91.5 +/- 1.6% of GRP-14 and 94.4 +/- 2.4% of GRP-27 were recovered intact. In contrast, when radiolabelled VIP was infused into the portal vein, 56.7 +/- 7.4% of injected labelled VIP appeared in the hepatic venous effluent, of which only 33.5 +/- 1.2% was intact peptide. Results of these studies indicate that enteric VIP released into the splanchnic/portal circulation is cleared by hepatic transit. However, somatostatin and GRP peptides appear to traverse the liver intact and could potentially produce systemic biological effects.

The present studies were directed toward examining the effect of somatostatin on gastrin release and acid secretion by the isolated vascularly perfused rat stomach. Rat stomachs were perfused in situ with Krebs-Ringer bicarbonate buffer containing 10% ovine erythrocytes and gassed with 95% O2-5% CO2. Inclusion of pentagastrin in the perfusion buffer increased acid output from 2.2 +/- 0.4 microEq H+/h during control perfusion to 18.8 +/- 1.8 microEq H+/h (p less than 0.01). In order to determine the effect of somatostatin on acid secretion and gastrin release, specific antibodies to somatostatin were included in the perfusate. Inclusion of antibodies to somatostatin in the buffer without pentagastrin did not significantly enhance acid output; however, gastrin concentration in the portal venous effluent increased from 15.1 +/- 2.0 to 25.2 +/- 5.2 pg/ml at 45 min (p less than 0.05). When antibodies to somatostatin were perfused in the presence of pentagastrin, acid output increased by 32% to 24.9 +/- 1.2 microEq H+/h (p less than 0.05); however, no change in gastrin concentration over basal was detected in the portal effluent. Results of these studies indicate that the capacity of the isolated rat stomach to secrete acid permits direct assessment of factors involved in the regulation of acid secretion. Under the conditions of these experiments, gastric somatostatin inhibits basal gastrin release and directly inhibits pentagastrin-stimulated acid secretion without affecting gastrin release.

A novel method of synchronizing monolayer tissue culture cells is described. By limiting the period of attachment of trypsinized cells and the subsequent removal of unattached cells a G1 population of cells is isolated. Evaluation of the degree of synchrony has been carried out by measuring the labeling index and incorporation of [3H]thymidine into DNA. Further conformation of synchrony, as well as a comparison with synchrony by isoleucine deprivation, was obtained by flow cytometry. The expected peak in DNA synthesis rate following limited attachment was observed. This peak becomes more prominent and shifts to earlier times with shorter attachment intervals. The synchronization method described is simple, rapid, yields a substantial number of cells, and is applicable to many cell lines.