Effect of thrombin on proliferation, contraction and prostaglandin production of rat glomerular mesangial cells in culture.

The effect of thrombin on mesangial cell function was investigated. Thrombin caused a dose-dependent increase in [3H] thymidine incorporation (EC50 = 0.36 +/- 0.09 U/ml), intracellular calcium [(Ca++)i] mobilization (EC50 = 1.9 +/- 0.5 U/ml) and prostaglandin E2 (PGE2) production (EC50 = 0.25 +/- 0.02 U/ml) in rat glomerular mesangial cells. These effects were blocked by the thrombin inhibitor, hirudin (KB = 10.4 +/- 0.2 nM). The role of (Ca++)i mobilization and arachidonate metabolism in thrombin-stimulated proliferation was tested by the addition of the calcium channel blocker, nifedipine, and the cyclooxygenase inhibitor, indomethacin, to mesangial cell cultures. Indomethacin, at doses that completely inhibited the thrombin-mediated production of PGE2, had no significant effect on proliferation. The Ca++ channel blocker, nifedipine, inhibited both PGE2 production and [3H] thymidine incorporation in a dose-dependent fashion, but only at concentrations considered nonspecific. In addition to its effects on PGE2, thymidine incorporation and Ca++ mobilization, thrombin caused mesangial cell contraction as determined by a substrate distortion technique. This effect was not inhibited by indomethacin. These results indicate that thrombin can alter mesangial cell function in vitro.     

A mechanism for the stimulation by inorganic mercury of [3H] thymidine incorporation into DNA in cultured Molt-4F cells

Mercuric chloride at a narrow range of concentration (2 to 2.5 X 10(-5)M) facilitated [3H]thymidine incorporation into acid-insoluble material (DNA fraction) of cultured human T lymphoid cells, Molt-4F, after 72-hr culture with the metal. This effect by mercury was observed in spite of the decrease in growth rate and DNA contents of the cells. Thymidine kinase activity in Molt-4F cells treated with 2 X 10(-5)M mercury decreased to 50 to 60% of the control activity. The stimulation of [3H]thymidine incorporation into the cells by mercury, therefore, might be independent of the increase in thymidine kinase activity. 3H-Thymidine incorporation by the control cells decreased as culture time passed. In contrast to the control, [3H]thymidine incorporation by mercury-treated cells increased until 72-hr culture. [3H]Thymidine uptake by the control cells after 24, 48, or 72-hr culture increased until 20 min of incubation period, but thereafter no increase in the uptake was observed until 60 min. On the other hand, [3H]thymidine uptake by the cells treated with mercury for 24 to 72 hr increased linearly until 60 min of incubation period. These results seemed to indicate that the mercury stimulation of [3H]thymidine incorporation might be attributable not to the actual increase of DNA synthesis but to the suppression of the culture time-dependent decrease in the incorporation by the control cells.     

Rapid, radiolabeled macrophage culture method for detection of dapsone-resistant Mycobacterium leprae.

Mycobacterium leprae cells extracted from the skin biopsies of 14 bacilliferous lepromatous patients were maintained in human-murine macrophage cultures for 3 weeks in the presence of [3H]thymidine and DDS (4,4'-diaminodiphenyl sulfone). All cultures except one containing freshly extracted viable bacilli showed significant incorporation of [3H]thymidine as compared to control cultures containing heat-killed bacilli of the corresponding strain. Six susceptible strains of M. leprae obtained from untreated, freshly diagnosed patients showed significant inhibition of the uptake of the radiolabel in the presence of 3 and 10 ng of DDS per ml per culture. Eight strains of M. leprae obtained from patients clinically suspected of DDS resistance were tested in a similar manner. These strains were also concurrently inoculated in the footpads of mice given orally 10(-2), 10(-3), and 10(-4) g of DDS per 100 g of body weight for 9 months. Concordant results were obtained by both methods: five strains were found to be resistant, one was susceptible, and one was partially resistant. Strain VIII did not incorporate [3H]thymidine in the macrophage cultures and proved to be resistant in the mouse footpad. The macrophage culture system provides a sensitive, rapid screening method for the early diagnosis of DDS resistance.     

Threshold Methotrexate Concentration for In Vivo Inhibition of DNA Synthesis in Normal and Tumorous Target Tissues

The suppression of DNA synthesis in host and tumor tissues by methotrexate has been monitored in mice by determining the in vivo incorporation of tritium-labeled deoxyuridine ([(3)H]UdR) into DNA. The duration of inhibition of [(3)H]UdR incorporation in normal tissues was related to the dose of methotrexate and was a direct function of plasma drug concentration. [(3)H]UdR incorporation recovered to 50% of pretreatment levels in bone marrow when plasma methotrexate concentration was 10(-8) M or less, irrespective of the dose administered, while 50% recovery of DNA synthesis in intestinal epithelium was not observed until plasma methotrexate levels were 5 x 10(-9) M or less. Ascitic L1210 leukemia cells did not fully return to pretreatment levels of [(3)H]UdR incorporation at any time, although a partial recovery of incorporation was noted at methotrexate ascitic fluid concentrations of approximately 10(-8) M.Methotrexate did not suppress the incorporation of tritium-labeled thymidine ([(3)H]TdR) into bone marrow and duodenal mucosa, confirming the specificity of its action in inhibiting thymidylate synthesis in host tissues. In the ascites tumor a gradual decline in [(3)H]TdR incorporation was seen after methotrexate, indicating that the tumor tissue depression of [(3)H]UdR incorporation is not solely due to inhibition of thymidylate synthesis.These studies indicate that host tissues are inhibited by extremely low concentrations of methotrexate, and indicate the importance of the slow final phase (t((1/2))=12 h) of drug elimination from plasma in producing a prolonged exposure of sensitive host tissues to inhibitory drug concentrations.     

Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products.

Prostaglandin E2 (PGE2) and several other prostaglandins synthesized by colon suppress the proliferative activity of colonic epithelium. However, bile salts stimulate colonic epithelial proliferation despite the actions of bile salts to enhance the release of arachidonate and consequent colonic synthesis of PGE2. The current study was conducted to assess whether bile salt-induced increases in colonic formation of arachidonate metabolites other than PGE2 were linked to the stimulation of the proliferative activity of colonic epithelium. Within 10 min of addition, deoxycholate markedly stimulated the in vitro release of [14C]arachidonate from prelabeled rat colon. When given in vivo by intracolonic instillation deoxycholate (10 mumol) increased colonic accumulation of immunoreactive prostaglandin E (PGE), thromboxane B2 (TXB2), and the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) by two to fourfold over control in 30 min. This effect of intracolonic deoxycholate was followed by a ninefold increase in mucosal ornithine decarboxylase activity (4 h), and a subsequent two to threefold increase in [3H]thymidine [( 3H]Thd) incorporation into DNA of either mucosal scrapings or isolated pools of proliferative colonic epithelial cells (24 h). Intracolonic instillation of indomethacin (50 mumol) suppressed to low or undetectable levels both basal colonic accumulation of PGE and TXB2 and the increases in each parameter induced by subsequent instillation of deoxycholate. By contrast, indomethacin enhanced accumulation of 12-HETE in both control colons and those subsequently exposed to deoxycholate. The increases in 12-HETE induced by indomethacin alone were correlated with stimulation of mucosal ornithine decarboxylase activity and [3H]Thd incorporation into mucosal DNA. Indomethacin also enhanced the increases in these parameters induced by deoxycholate. Intracolonic instillation of phenidone (25-100 mumol) suppressed accumulation of PGE, TXB2, and 12-HETE in control colons and the increases in these parameters induced by a subsequent instillation of deoxycholate. Phenidone alone did not alter mucosal ornithine decarboxylase activity or [3H]thymidine incorporation into mucosal DNA. However, phenidone suppressed or abolished increases in these parameters induced by a subsequent instillation of deoxycholate. 4-(2-[IH-imidazol-1-yl]ethoxy) benzoic acid hydrochloride UK 37,248, which selectively reduced colonic TXB2 to undetectable levels without altering PGE or 12-HETE, had no effect on control or deoxycholate-induced increases in mucosal ornithine decarboxylase activity or [3H]Thd incorporation into DNA. Neither indomethacin nor phenidone altered the increases in [(14)C]arachidonate release induced in vitro by deoxycholate. Chenodeoxycholate and cholate also stimulated [(14)C]arachidonate release from colon in vitro within 10 min, and increased colonic 12-HETE (30 min) and mucosal ornithine decarboxylase activity (4 h) upon intracolonic installation. Prior installation of phenidone inhibited the increases in both 12-HETE and ornithine decarboxylase activity induced by these bile salts. The results support a role for bile salt-induced increases in colonic accumulation of lipoxygenase products, as reflected by 12-HETE, in the subsequent stimulation of the proliferative activity of colonic epithelium.     

Separate receptors for prostacyclin and prostaglandin E2 on human gel-filtered platelets

Human gel-filtered platelets (GFP) and radiolabeled prostacyclin (PGI2), prostaglandin (PG) E2 and PGE1 were used to ascertain whether PGI2 and PGs of the E series share a common receptor or have their own specific receptors on platelets. Attention was given to ensuring the proper experimental conditions to compensate for the rapid half-life of PGI2 at physiologic pH. Specific [3H] PGI2 binding to GFP was maximal at 5 min and pH 7.45. Scatchard analysis indicated a single class of binding sites with an apparent KD of 4.52 X 10(-8) M and 1130 sites per platelet. Approximately 90% of specifically bound [3H]PGI2 could be dissociated by excess unlabeled PGI2 by 5 min. The IC50 for PGI2 was 66 nM. By 5 min, PGE1 and PGE2 were only 7.17 and 0.03%, respectively, as potent inhibitors of binding. Maximal specific binding of either [3H]PGE2 or [3H]PGE1 to GFP occurred by 60 min. During 60-min incubations with [3H]PGE2, the IC50 values for PGE2 and PGE1 were 3 and 6 nM, respectively. When [3H]PGE1 was used, the IC50 values for PGE1 and PGE2 were 30 and 10 nM, respectively. To examine PGI2 competition for [3H] PGE2 and [3H]PGE1 binding sites, 5-min incubation periods were used. PGI2 was only 0.38% as potent an inhibitor of [3H]PGE2 compared to PGE2 and only 30% as potent an inhibitor of [3H] PGE1 compared to PGE1. Scatchard analysis of the 60-min competition experiments using [3H]PGE2 and [3H]PGE1 and the homologous unlabeled ligand yielded curvilinear plots in both instances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin.

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.     

Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis.

To understand the relationship between the proliferation of epidermis and its arachidonic acid metabolism, we studied human keratinocytes grown in vitro at confluent or nonconfluent densities. Keratinocyte cultures incubated with [14C]arachidonic acid synthesized prostaglandin (PG)E2 PGD2, PGF2 alpha, and small quantities of 6-keto-F1 alpha. Nonconfluent cultures, however, synthesized fourfold more PGE2 than did confluent cultures. When proliferation was studied using [3H]thymidine incorporation into DNA, it was found that this increased synthesis of PGE2 was accompanied by a fourfold increase in the rate of proliferation. When PGE2 synthesis was inhibited by indomethacin, the rate of proliferation of nonconfluent cultures was decreased 40%, while the rate of proliferation of confluent cultures was unchanged. Addition of 1 ng/ml of PGE2, but not PGF2 alpha, PGD2, or a stable analog of PGI2 to the indomethacin-treated nonconfluent cultures restored the initial rate of proliferation. These results suggest that PGE2 is a growth-promoting autocoid for epidermis. The synthesis of PGE2 by epidermis may be enhanced in wound healing and disease states where epidermal continuity is disrupted.     

4-Quinolone drugs affect cell cycle progression and function of human lymphocytes in vitro.

Most antibacterial agents do not affect human lymphocyte function, but a few are inhibitory. In contrast, a pronounced increase in the incorporation of [3H]thymidine in the presence of 4-quinolones was observed in these studies. The uptake of [3H]thymidine into DNA (trichloroacetic acid precipitable) was significantly increased in phytohemagglutinin-stimulated human lymphocytes when they were exposed to eight new 4-quinolone derivatives, ciprofloxacin, norfloxacin, ofloxacin, A-56619, A-56620, amifloxacin, enoxacin, and pefloxacin, at 1.6 to 6.25 micrograms/ml for 5 days. Four less antibacterially active 4-quinolones (nalidixic acid, cinoxacin, flumequine, and pipemidic acid) stimulated [3H]thymidine incorporation only at higher concentrations or not at all. Kinetic studies showed that incorporation of [3H]thymidine was not affected or slightly inhibited by ciprofloxacin 2 days after phytohemagglutinin stimulation but was increased on days 3 to 6. The total incorporation of [3H]thymidine from day 1 to day 6 after phytohemagglutinin stimulation was increased by 42 to 45% at 5 to 20 micrograms of ciprofloxacin per ml. Increased [3H]thymidine incorporation was also seen when human lymphocytes were stimulated with mitogens other than phytohemagglutinin. Ciprofloxacin added at the start of the culture had a more pronounced effect on [3H]thymidine incorporation than when added later. In spite of the apparent increase in DNA synthesis, lymphocyte growth was inhibited by 20 micrograms of ciprofloxacin per ml, and cell cycle analysis showed that ciprofloxacin inhibited progression through the cell cycle. In addition, immunoglobulin secretion by human lymphocytes stimulated by pokeweed mitogen for Epstein-Barr virus was inhibited by approximately 50% at 5 micrograms of ciprofloxacin per ml. These results suggest that the 4-quinolone drugs may also affect eucaryotic cell function in vitro, but additional studies are needed to establish an in vivo relevance.     

Human lymphocyte thiopurine methyltransferase pharmacogenetics: effect of phenotype on 6-mercaptopurine-induced inhibition of mitogen stimulation

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of 6-mercaptopurine (6-MP) and other thiopurine drugs. The level of TPMT activity in human tissue is controlled by a common genetic polymorphism. Our experiments were performed to study the relationship between TPMT phenotype and the effect of 6-MP on [3H]thymidine ([3H]TdR) incorporation into mitogen-stimulated human peripheral blood lymphocytes. Lymphocytes were isolated from blood samples obtained from 12 subjects, four each with each of the three classes of TPMT phenotype. The effect of 6-MP concentration on [3H]TdR incorporation was determined in the presence of two mitogens, phytohemagglutinin at concentrations of 1 and 10 micrograms/ml and concanavalin A at concentrations of 1 and 5 micrograms/ml. ED50 values for 6-MP inhibition of [3H]TdR incorporation into lymphocytes from subjects who genetically lacked TPMT activity were uniformly higher than were ED50 values for lymphocytes from subjects with intermediate or high enzyme activities. However, in the presence of either mitogen, ED50 values decreased as the level of stimulation increased. Therefore, to make it possible to account for degree of mitogen stimulation, the effect of 6-MP on [3H]TdR incorporation was studied in the presence of a series of phytohemagglutinin concentrations from 1 to 10 micrograms/ml. Lymphocytes from subjects who genetically lacked TPMT activity had significantly higher Ki values (1.37 +/- 0.340 microM, mean +/- S.E.M., n = 3) for inhibition of [3H]TdR incorporation by 6-MP than did lymphocytes from subjects with intermediate or high enzyme activities (0.529 +/- 0.068 and 0.327 +/- 0.064 microM, respectively, P less than .05 for both comparisons, n = 3 in both cases).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced Effects of Prostaglandin E1 and Dibutyryl Cyclic AMP upon Human Lymphocytes in the Presence of Cortisol

The combined effects of cortisol and agents acting through a cyclic AMP-mediated mechanism have been studied in cultures of highly purified human peripheral lymphocytes. Incubation with prostaglandin E(1) (PGE(1)), dibutyryl cyclic AMP, or cortisol results in a concentration-dependent inhibition of [(3)H]-thymidine incorporation by both unstimulated and phytohemagglutinin (PHA)-stimulated lymphocytes, and PHA-induced morphologic transformation is prevented. When cortisol and PGE(1) (or dibutyryl cyclic AMP) are added together to lymphocyte cultures, enhanced inhibitory effects are observed.Incubation of unstimulated or PHA-stimulated lymphocytes with PGE(1) results in an elevation of intracellular cyclic AMP levels within 20 min. The concentration of cyclic AMP gradually returns to base-line levels over a 1-6 h period of time. Cortisol alone does not significantly alter cyclic AMP concentrations. However, incubation with PGE(1) in the presence of cortisol results in a greater stimulation of intracellular cyclic AMP levels than that observed with PGE(1) alone. These findings suggest that cortisol may act synergistically with PGE(1) to elevate lymphocyte cyclic AMP levels and to regulate [(3)H]thymidine incorporation and transformation.     

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.     

Potentiation of bleomycin cytotoxicity in Saccharomyces cerevisiae.

Lesions introduced into cellular DNAs prelabeled with [2-14C]thymidine or [6-3H]thymidine, as well as cell killing, were inhibited by the presence of EDTA during 20-min reactions of Saccharomyces cerevisiae cells with the low-molecular-weight bleomycin family of anticancer antibiotics. In contrast, the level of killing by low concentrations of bleomycin was higher among cells which had grown for three generations in defined synthetic complete medium supplemented with ferrous sulfate than among cells grown without iron supplementation. In S. cerevisiae, the uptake of iron is facilitated by a plasma membrane ferric reductase activity and a high-affinity (Km = 5 x 10(-6) M) ferrous uptake system. Lethal effects of 1.3 x 10(-6) M bleomycin increased approximately 50% with 10(-5) M Fe(II), nearly twofold with 10(-4) M Fe(II), and 2.8 times with 10(-3) M Fe(II). Thus, iron preloading is a new experimental approach to increasing and studying the effects of the glycopeptides on cellular DNAs and other cellular targets. This approach could also be used for studying and better understanding DNA repair genes and could serve as a model for studies of redox active chemicals in biological systems.     

Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation.

Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.     

Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium.

The role of local prostaglandin (PG) synthesis in the modulation of the proliferative activity of colonic epithelium was examined in rat colon. Experimental rats were given either indomethacin (5 mg/kg s.c. every 8 h for three doses) or aspirin (0.5 g/100 g diet for 3 d). In rats treated with indomethacin or aspirin, the incorporation of [3H]thymidine (dThd) into DNA in vivo was increased approximately twofold over control in mucosal scrapings from distal colon, and approximately threefold over control in the proliferating pool of epithelial cells isolated from distal colon. [3H]dThd incorporation into DNA was also examined ex vivo immediately after distal colonic resection. It was approximately twofold higher in mucosa of colonic segments (1-h incubation) from rats treated with indomethacin or aspirin in vivo, compared with corresponding values of segments from control rats. Immunoreactive (i) prostaglandin E (PGE), the dominant PG product of colon segment incubates by high-performance liquid chromatography analysis of [14C]arachidonate metabolites, was markedly (95%) reduced in the media of 1-h colon incubates from indomethacin- or aspirin-treated rats, compared with control rats. Moreover, the cyclic (c)AMP content of mucosa of segments from indomethacin- or aspirin-treated rats was significantly lower than that of control rats. Prolonged incubation (4-24 h) of colonic segments from indomethacin-treated rats, in the absence of indomethacin in vitro, led to an eventual return of [3H]dThd incorporation into DNA, iPGE, and mucosal cAMP to control values. Conversely, inclusion of indomethacin (0.25 mM) in the incubations (6 h) of colonic segments from indomethacin-treated rats resulted in persistent suppression of iPGE and mucosal cAMP, as well as persistent enhancement of [3H]dThd incorporation into mucosal DNA. However, incubation of colonic segments from control rats (no in vivo drug exposure) with indomethacin or aspirin in vitro for periods up to 24 h failed to alter DNA synthesis, despite marked reduction in media iPGE and lower mucosal cAMP. The latter observations suggested that additional in vivo factors initiated the enhancement of DNA synthesis in indomethacin- or aspirin-treated rats. Exogenous PGE2, D2, I2, or F2 alpha, each of which increased the endogenous mucosal cAMP content of incubated colonic segments from control, indomethacin- or aspirin-treated rats, all suppressed [3H]dThd incorporation into mucosal DNA in vitro. Dibutyryl cAMP, but not dibutyryl cGMP, had an analogous suppressive effect on in vitro [3H]dThd incorporation into DNA. Thus, the present observations are consistent with an inhibitory action of endogenous colonic PG synthesis on the proliferative activity of colonic epithelium. This action may be mediated through cAMP.     

Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation.

An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring. We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts. Using primary cultures of these cells, we have shown increases in [3H]thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml). Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II. In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in [3H]thymidine incorporation. IL-1beta and platelet-derived growth factor together produced an increase in [3H]thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers. Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4. 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.     

Growth factor-dependent initiation of DNA replication in nuclei isolated from an interleukin 3-dependent murine myeloid cell line.

To study the proliferative response of hematopoietic cells to growth factors at the molecular level, we developed a cell-free system for growth factor-dependent initiation of genomic DNA replication. Nuclei were isolated from the IL-3-dependent cell line NFS/N1-H7 after a 10-h period of IL-3 deprivation. Cytosolic and membrane-containing subcellular fractions were prepared from proliferating NFS/N1-H7 cells. Nuclei from the nonproliferating cells (+/- IL-3) showed essentially no incorporation of [3H]thymidine during a 16-h incubation with a mixture of unlabeled GTP, ATP, UTP, CTP, dGTP, dATP, dCTP, and [3H]dTTP. When the combination of IL-3, a cytosolic fraction, and a membrane-containing fraction from proliferating cells was added to nuclei from nonproliferating cells, a burst of [3H]thymidine incorporation into DNA began after a 12-h lag period, attained a maximal rate at 16 h, and reached a level of 860 pmol thymidine/10(6) nuclei at 24 h (corresponding to replication of approximately 56% total mouse genomic DNA). This DNA synthesis was inhibited approximately 90% by the specific DNA polymerase alpha inhibitor aphidicolin. Deletion of a single cellular component or IL-3 from the system resulted in a marked reduction of DNA replication (-membrane, 80 +/- 4%; -cytosol, 90% +/- 4%; -IL-3, 74 +/- 7% inhibition). This model requires a growth factor (IL-3), a sedimentable cell fraction containing its receptor and possibly additional membrane-associated components, and a cytosolic fraction. It appears to recapitulate the molecular events required for progression from early G1 to S phase of the cell cycle induced by IL-3 binding to its receptor.     

A new type of vasodilator, HA1077, an isoquinoline derivative, inhibits proliferation of bovine vascular smooth muscle cells in culture

The effects of a newly developed vasodilator agent, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride], were investigated on the proliferation of cultured bovine aortic vascular smooth muscle cells (VSMC). HA1077 (10-100 microM) inhibited both fetal calf serum-induced proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC in a dose-dependent manner. When quiescent cells were stimulated with platelet-derived growth factor followed by insulin, HA1077 (1-30 microM), administered together with either stimulation, showed dose-dependent inhibition of [3H]thymidine incorporation. Further reduction of [3H]thymidine incorporation was observed when HA1077 was present at both stimulations, suggesting that HA1077 suppresses DNA synthesis acting in both competence and progression stages. After stimulation with fetal calf serum, quiescent VSMC started and ceased DNA synthesis in 15 to 18 hr and 24 hr, respectively. HA1077 inhibited [3H]thymidine incorporation when it was added either from 12 hr to 15 hr or from 21 hr to 24 hr after serum stimulation. In addition, when percent inhibition of [3H]thymidine incorporation by continuous exposure to HA1077 was examined as a function of the time it was added, reductions of the value were observed at 0 to 3 hr, 12 to 18 hr and 21 to 24 hr. Thus, we concluded that HA1077 suppresses DNA synthesis of bovine VSMC acting at the G0/G1 and the G1/S phase transitions and also in the S phase of the cell cycle. It is suggested that this agent may act as a potent inhibitor of VSMC proliferation as well as a vasodilator.     

Effects of Misoprostol on Bone Resorption and Formation in Organ Culture

Prostaglandins are potent stimulators of bone resorption and formation. Because misoprostol is an analog of prostaglandin E(1) (PGE(1)), we have examined its effects on resorption and formation in organ culture. The results were compared with PGE(2) which can stimulate resorption and both stimulate and inhibit bone formation. Resorption, measured as the release of previously incorporated (45)Ca from 5-day cultures of 19-day fetal-rat long bones, was increased by misoprostol 1.5-fold at 10(minus sign6) M and twofold at 10(minus sign5) M. The effect at 10(minus sign6) M was abolished by addition of indomethacin (10(minus sign6) M). PGE(2) was approximately 100 times more potent and was not affected by indomethacin in this system. In 21-day fetal-rat calvariae, cultured for 24 h in the presence of cortisol (10(minus sign7) M), misoprostol produced a dose-related increase in TdR incorporation between 10(minus sign5) and 10(minus sign7) M. PGE(2) appeared to be only 10-fold more potent in this response. The effects of misoprostol on incorporation of [(3)H]proline into collagenase digestible protein (CDP) and noncollagen protein (NCP) were measured in 72-h cultures, either with continuous treatment or 24-h treatment followed by 48 h in control medium. With continuous treatment at 10(minus sign6) M, misoprostol increased labeling of CDP twofold. A similar effect was observed with 24 h of treatment at 10(minus sign5) M, followed by 48 h in control medium. Again, PGE(2) was approximately 10-fold more potent than misoprostol. When calvariae were treated with insulin-like growth factor I, which increases CDP labeling by 2.5-fold, the effects of misoprostol and PGE(2) were inhibitory. We conclude that misoprostol resembles PGE(2) in its effects on bone but is less potent. Moreover, misoprostol may be relatively less effective in stimulating resorption than in stimulating formation. Therefore, an increase in bone turnover, possibly with a net anabolic effect, might occur in vivo with long-term misoprostol treatment. Misoprostol effects on bone turnover in humans could be evaluated using the sensitive biochemical markers for bone resorption and formation which are currently available.     

Thymidine transport and metabolism in choroid plexus: effect of diazepam and thiopental

Choroid plexus contains an active transport (influx) and a facilitated diffusion (efflux) system for nucleosides. The ability of diazepam and thiopental to inhibit active transport or facilitated diffusion of thymidine in choroid plexus was measured in vitro under various conditions. When isolated rabbit choroid plexuses were incubated in artificial cerebrospinal fluid containing 1 microM [3H] thymidine for 10 min at 37 degrees C under 95% O2-5% CO2, diazepam (10 microM) and thiopental (500 microM) doubled the tissue-to-medium ratios of [3H] thymidine from 8 to 15 to 16. These results were not due to metabolism or intracellular binding but rather to inhibition of [3H] thymidine efflux from choroid plexus. Diazepam, unlike thiopental, inhibited [3H] thymidine efflux in a concentration-dependent manner. When isolated choroid plexuses were incubated in artificial cerebrospinal fluid containing low concentrations of [3H] thymidine (6 nM) to allow intracellular conversion of [3H] thymidine into [3H] thymidine phosphates and [3H] DNA, both diazepam (10 microM) and thiopental (500 microM) altered [3H] thymidine accumulation and metabolism consistent with inhibition of facilitated diffusion but not active transport of thymidine. These studies provide evidence that, at toxic but not therapeutic concentrations, diazepam and thiopental alter facilitated nucleoside transport in the choroid plexus.