Synergistic enhancement of collagenous protein synthesis by human gingival fibroblasts exposed to nifedipine and interleukin-1-beta in vitro.

Gingival overgrowth commonly occurs coincident to therapy with calcium channel blockers. The biologic mechanism for this condition is unknown; however, many clinicians suggest that poor oral hygiene may contribute to development of the overgrowth. This study tests the hypothesis that collagenous protein synthesis by gingival fibroblasts is synergistically enhanced when they are exposed to both nifedipine (N) and the pro-inflammatory cytokine, interleukin-1-beta, a cytokine expressed in inflamed gingiva. Human gingival fibroblasts were isolated from biopsies of normal gingiva and cells separated into two groups. Group 1 was exposed to media containing 0, 5, 50, or 500 pg/ml IL-1-beta, or 10(-7) M N for 7 days; Group 2 was exposed to those concentrations of IL-1-beta +10(-7) M N. [3H]-proline was added to the medium for the final 24 h. Cells and matrix were harvested and radioactivity determined by liquid scintillation analysis. Means (d.p.m./10(3) cells) were compared by factorial ANOVA and Scheffé comparisons. Collagenous protein synthesis was significantly reduced by 5 pg/ml IL-1-beta +10(-7) M N and enhanced by 500 pg/ml IL-1-beta +10(-7) M N as compared to N or IL-1-beta alone. Thus, patients may be more susceptible to gingival overgrowth coincident to nifedipine therapy as a result of the synergistic enhancement of connective tissue synthesis by these agents.

Site-specific variations in metabolism by human fibroblasts exposed to nifedipine in vitro.

Nifedipine induces overgrowth of gingival tissues in some patients. However, other collagenous tissues in their body do not overgrow. The purpose of this study was to compare effects of serial dilutions of nifedipine on the in vitro metabolism of fibroblasts derived from normal gingiva, nifedipine-induced hyperplastic gingiva, knee capsular ligament, and dermis. The data suggested that nifedipine affects the metabolism of fibroblasts derived not only from gingiva, but also from other collective connective tissues. Thus, nifedipine-responder cells are present in tissues other than gingiva. There was an inverse relationship between in vivo tissue levels of IL-1-beta and in vitro responsiveness to nifedipine of fibroblasts derived from that tissue. Nifedipine-induced overgrowth of connective tissues, other than gingiva, probably does not occur either because of the relatively slow rate of collagenous protein synthesis by resident fibroblasts or because of alterations in collagen deposition/resorption within susceptible tissues produced by nifedipine on collagenase synthesis.

Comparative metabolism of 3H-glucosamine by fibroblast populations exposed to cyclosporine.

Cyclosporine and nifedipine therapy produces gingival overgrowth in many patients. Neither the mechanism underlying this undesirable side effect nor the possibility of synergism between these drugs is known, although many renal transplant patients receive both drugs. This study compared the rates of 3H-glucosamine utilization by three groups of fibroblasts: untreated gingival fibroblasts, fibroblasts from gingival overgrowth tissue of a patient receiving both cyclosporine and nifedipine, and normal gingival fibroblasts exposed to cyclosporine-A in vitro. Significant differences in the rates of deposition of 3H-glucosamine into the extracellular matrix by each group of gingival fibroblasts were demonstrated, suggesting that increased rates of deposition of proteoglycans into the gingival extracellular matrix by fibroblasts should be further investigated as a biologic mechanism for gingival overgrowth.

Serum modulation of insulin action: effects on rat gingival fibroblast metabolism.

Serum is reported to reduce the sensitivity of cells in culture to insulin. The effect of serum concentration in the growth medium on the responsiveness of control (C) and streptozotocin diabetic (D) rat gingival fibroblasts to insulin was measured by monitoring cellular DNA, RNA, total protein and medium hydroxyproline (collagen) levels, as well as the cellular uptake of C14-alpha-NH2-isobutyrate (alpha-AIB) and H3-2-deoxyglucose (2DG). The cells were grown in alpha-MEM at 5, 10, 15 or 20% FCS with 0, 10(-12), 10(-10), 10(-8) and 10(-6) M insulin used at each serum level. Insulin effects in the absence of serum were not assessed. For both the C and D rat cells, the DNA increased proportionately with increasing serum and insulin levels. In contrast, RNA and total cell protein increased with increase in insulin and decrease in serum, the magnitude of the effect being greater in C than in D cells. The insulin stimulation of both 2DG and alpha-AIB uptake and of collagen secretion varied inversely with serum concentrations. The magnitude of the insulin-serum interaction on metabolite uptake was greater for the D rat cells. These data indicate that serum significantly reduced the cell response to insulin stimulated metabolite uptake and collagen secretion, but was without apparent effect on the intracellular insulin responsive parameters. They suggest that serum factor(s) interfere with the availability of insulin to the cell and that the D rat cells are most affected.

Differential effects of megavitamin E on prostacyclin and thromboxane synthesis in streptozotocin-induced diabetic rats.

Diabetic subjects tend to develop microvascular complications believed to be due to platelet hyperaggregability. This increased platelet sensitivity is though to be the result of an imbalance of PGI2 and TXA2 production in diabetes. This study sought to determine whether megavitamin E supplementation could restore PGI2/TXA2 balance in streptozotocin-diabetic rats. Endogenous release of PGI2 by isolated aorta, determined via radioimmunoassay of its stable metabolite, 6-keto-PGF1 alpha, was significantly greater (P less than 0.05) in rats receiving 100x the normal vitamin E requirement than in untreated diabetic rats. PGI2 synthesis was negatively correlated with plasma glucose levels (r = -0.87, P less than 0.05) in non-fasted rats at sacrifice. Vitamin E supplementation, at both the 10x and the 100x level, significantly depressed (P less than 0.05) thrombin-stimulated synthesis of TXA2 in washed platelet. PGI2 and TXA2 production were expressed as a ratio. Megavitamin E therapy appears to increase this ratio over that seen in the diabetic animal. The data suggest that vitamin E, at high levels, exerts an ameliorating influence of the PGI2/TXA2 imbalance of diabetes.

Pituitary response to gonadotropin-releasing hormone in diabetic male rats.

The rise in serum lutenizing hormone concentration after treatment with gonadotropin-releasing hormone was less in diabetic castrated male rats than control castrates. In intact male rats, gonadotropin-releasing hormone treatment resulted in higher serum luteinizing hormone concentrations in diabetic than in control rats.

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen.

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.

Effect of alloxan-diabetes on alpha-amylase and sialic acid levels in the parotid and submandibular glands of rats.

The relationship of diabetes to salivary gland metabolism has been investigated by comparing the levels of alpha-amylase, sialic acid and total protein in the parotid and submandibular glands, plasma and kidneys of control and alloxan-diabetic rats. A significant decrease in alpha-amylase activity was found in both the parotid and submandibular glands of the diabetic rats by use of both a chemical and an electrophoretic assay. Plasma and kidney levels of the enzyme were not altered in the diabetic rats. Sialic acid levels were not affected by the alloxan-induced diabetes. Although the total protein concentration was significantly altered only in the kidneys of the alloxan-diabetic rats, the electrophoretic patterns of both the parotid and submandibular gland soluble proteins were markedly different between the control and alloxan-diabetic rats. The electrophoretic data indicate that protein metabolism in general, and the alpha-amylase concentration specifically, are altered in the salivary glands of alloxan-diabetic rats.

The effect of streptozotocin-induced diabetes and of insulin supplementation on glycogen metabolism in rat liver.

The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.

Some effects of experimentally-induced diabetes on pituitary-testicular relationships in rats.

The effect of experimentally-induced diabetes mellitus on reproductive organ weights, serum and pituitary gonadotropin levels and serum testosterone levels was studied in 3-month old rats. In experiment 1, intact rats were treated with alloxan monohydrate or streptozotocin. In experiments 2 and 3, intact and castrated rats were rendered diabetic with alloxan (experiment 2) or streptozotocin (experiment 3). The duration of each experiment was 3 weeks. In each experiment diabetes resulted in body weight losses or reduced body weight gain, elevated serum glucose concentrations and reduced assessory sex gland weights (intact rats). Serum levels of testosterone were depressed (P less than 0.05 or P less than 0.01) in diabetic rats. Serum levels of LH were significantly (P less than 0.05) lower in intact diabetics than in controls when pooled data from the three experiments were compared. Serum levels of FSH were not affected by diabetes. Pituitary concentrations of FSH were elevated (P less than 0.05) in diabetics in two of the three experiments, while LH concentrations were elevated (P less than 0.05 or P less than 0.01) in diabetics in all experiments. The hypersecretion of gonadotropins in castrated rats was not affected by diabetes.