A. Przylipiak

Stimulation of luteinizing hormone release by melittin and phospholipase A2 in rat pituitary cells

Gonadotropin release in rat pituitary monolayer cultures was stimulated by phospholipase A2, as well as by its activator melittin. A dose-dependent stimulation of luteinizing hormone secretion by melittin was observed in a dose range of 10(-8) to 10(-4) M. A higher dose (1 mM) melittin had a sub-optimal effect. The stimulatory action of melittin was calcium-dependent and blocked by phospholipase A2 inhibitors, chloroquine and quinacrine. Similar to melittin, phospholipase A2 enhanced the effect of LH release in a dose range of 0.1-100 units/ml. The effect of this enzyme was also calcium-dependent with optimal calcium concentrations at 1.5 mM, as obtained also for melittin. In superfusion experiments, the stimulatory action of melittin and phospholipase A2 was reproducible in their effects on LH release in gonadotrophs. In addition, melittin (10(-7) M) stimulated LH and 3H-arachidonic acid efflux in superfused pituicytes following prelabelling with radiolabelled arachidonate. These data suggest that phospholipase A2, which releases arachidonic acid from phospholipids, may participate in controlling gonadotropin secretion in gonadotrophs, since arachidonic acid and its metabolites have previously been found to enhance gonadotropin release.

Stimulation of gonadotropin release by arachidonic acid and its lipoxygenase metabolites in superfused pituitary cells

Luteinizing hormone and follicle stimulating hormone secretion was stimulated by 4 min pulses of arachidonic acid (3 X 10(-5) to 10(-4)M) in superfused rat pituitary cells. The effect of its lipoxygenase metabolites, 5-hydroxy-6,8,11,14-eicosatetranoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) was more potent on hormone release when added in the same dose. Using 3 X 10(-5)M 5-HETE, its releasing activity on gonadotropins was comparable to that of GnRH (10(-9)M). 15-HETE (3 X 10(-5)M) was even more potent on LH and FSH secretion than 5-HETE. The secretory profile induced by 5-HETE and 15-HETE was also similar to that shown for GnRH, resulting in a rapid increase and a more prolonged decline of the hormone release. The addition of these fatty acids to superfused pituitary cells did not alter the response of the cells to their physiological ligand. These findings give further support to the proposal that metabolites of arachidonic acid may be involved in receptor-mediated mechanisms of gonadotropin release in pituitary cells.

INFLUENCE OF ETHANOL ON IN VITRO GROWTH OF HUMAN MAMMARY CARCINOMA CELL LINE MCF-7

We have shown that ethanol in concentrations of 0,0001%-10% significantly enhances 3H-thymidine uptake in cultured human mammary carcinoma cell line MCF-7. Methanol only enhances 3H-thymidine uptake in a concentration of 0,01%.

Leukotrienes stimulate gonadotropin release in vitro

Monolayer cell cultures of female rat anterior pituitaries were used to investigate the effect of leukotrienes (LT) LTA4, LTB4, LTC4, LTD4, LTE4 and other lipoxygenase metabolites of arachidonic acid (5-HETE, 5-HPETE, and 15-HETE) in vitro. 3H-arachidonic acid was rapidly incorporated into pituicytes and its release was enhanced by gonadotropin releasing hormone (GnRH) in superfused pituitary cells. Leukotrienes were found to be very potent stimulators of the release of luteinizing hormone (LH) when added as pulses to superfused pituicytes. At equimolar concentrations, LTA4, LTB4, LTC4 and LTE4 were found to be more potent than the physiological stimulus GnRH. LTD4 did not affect gonadotropin secretion. Other lipoxygenase metabolites of arachidonic acid, such as 5-HETE, 5-HPETE and 15-HETE were less effective on the exocytosis of LH. These results suggest that leukotrienes are potential mediators of GnRH action on gonadotropin secretion and are possible sites of regulation of pituitary function.

Epidermal growth factor stimulates luteinizing hormone and arachidonic acid release in rat pituitary cells.

Epidermal growth factor (EGF) directly enhanced luteinizing hormone (LH) release from dispersed rat pituitary cells in monolayer cultures as well as in superfusion columns. This 2.3-fold stimulatory effect was dose and time dependent and was also reconfirmed in a superfusion system. Retinal, a protein kinase C inhibitor, counteracted the EGF effect only partially. Further experiments were therefore carried out to investigate alternate EGF mechanisms. Nordihydroguaiaretic acid and chloroquine suppressed the stimulatory effect of EGF in a dose-dependent manner. Moreover, EGF (10(-7) M) stimulated [3H]arachidonate release from pre-labelled rat pituitary cells. This indicates that phospholipase A2 and arachidonic acid may be involved in EGF action on LH release from rat pituicytes.

Influence of 5-Lipoxygenase on in vitro Growth of Human Mammary Carcinoma Cell Line MCF-7

The aim of this study was to investigate the direct effect of 5-lipoxygenase (5-LO) on the growth of human mammary cancer cells MCF-7 in vitro. Cell growth was measured according to the level of 3H-thymidine incorporation. 5-LO was shown to inhibit 3H-thymidine incorporation. The inhibitory effect was 19, 42 and 78% when administered at concentrations of 0.1, 0.2 or 0.5 U/ml, respectively. Its effect was time- and dose-dependent and was statistically significant at concentrations of 0.2 and 0.5 U/ml. We have also shown that the specific 5-LO inhibitor MK-886 (1 µM) lifts the inhibitory effect of 5-LO (0.2 U/ml). Moreover, when treated with an activator of 5-lipoxygenase calcium ionophore A23187 (10 µM) MCF-7 cells synthesize LTB4. The results of this study are evidence of the role of 5-lipoxygenase in the regulation of human mammary cancer cells growth in vitro.

Exogenous action of 5-lipoxygenase by its metabolites on luteinizing hormone release in rat pituitary cells

The stimulatory effect of exogenously administered potato 5-lipoxygenase (0.1-0.3 U/2 ml) on luteinizing hormone (LH) release was demonstrated in rat anterior pituitary cells in a superfusion system. Nordihydroguaiaretic acid (NDGA), an inhibitor of 5-lipoxygenase, abolished the effect of the enzyme on LH secretion. The secretory effect on LH after 5-lipoxygenase administration was biphasic and dependent on Ca2+ indicating that 5-lipoxygenase affects LH release through its oxygenation reaction. Another series of experiments demonstrated that activation of 5-lipoxygenase, expressed as production of leukotriene (LT) B4 and C4 (728 +/- 127 pg/10(6) cells and 178 +/- 23 pg/10(6) cells, respectively) occurs in rat pituitary cells after addition of Ca2+ ionophore A23187. However, LTB4 and LTC4 were not formed by pituitary cells that had previously been desensitized by gonadotropin-releasing hormone (GnRH), the physiological ligand of LH release. These results are consistent with a role of 5-lipoxygenase metabolites in the mechanism of GnRH-induced LH secretion.

Influence of leukotrienes on in vitro growth of human mammary carcinoma cell line MCF-7

OBJECTIVES The aim of this work was to study the action of leukotrienes on the growth of human mammary cancer cells MCF-7. STUDY DESIGN The growth of the cells was measured by incorporation of 3H-thymidine. The action of leukotriene (LT)B4, LTD4, LTC4, LTE4 or arachidonate (AA) was tested in human mammary cancer cells MCF-7 in vitro. RESULTS LTB4 or LTD4 but not LTC4 or LTE4 reduced significant incorporation of 3H-thymidine in MCF-7 cells up to 52% or 56% respectively, when administered in concentrations 0.1-1000 pM. Agents in concentrations of 0.01 pM or 10000 pM did not effect 3H-thymidine incorporation. We have shown, that MCF-7 cells synthesise LTB4 when treated with calcium ionophor A23187 (10 microM). Leukotriene-antagonist LY171883 (10 microM) lifts inhibitory effects of LTB4 or LTD4. Arachidonic acid (10 microM) inhibits 3H-thymidine incorporation up to 72%. 5-lipoxygenase inhibitor MK-886 (100 nM) lifts the inhibitory effect of arachidonate. CONCLUSIONS LTB4 or LTD4 inhibits MCF-7 breast cancer cell growth. LT-receptors mediate the growth-inhibitory effect of LTB4 or LTD4.

Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts.

Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9. Conclusion This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of β1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.