The expression of the renin-angiotensin-aldosterone system in the skin and its effects on skin physiology and pathophysiology.

The local renin-angiotensin-aldosterone system (RAAS) is fully expressed in the human skin at the mRNA and protein level. Local RAAS is known to play a regulatory function in epidermal proliferation, wound healing, scarring, cutaneous heating adaptation, and aging. There are also some indications of its role in the regulation of hair growth and sebum secretion. Impaired wound healing, skin diseases associated with diabetes, cancer development, psoriasis, and scleroderma may be related to changes in skin RAAS activity. Extensive research has shown that RAAS-modulating drugs can affect the skin when applied orally or topically, creating new therapeutic approaches against dermatological diseases.

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 microM) lifts the inhibitory effect of 5-LO (0.2 U/ml). Moreover, when treated with an activator of 5-lipoxygenase calcium ionophore A23187 (10 microM) 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.

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.

Inhibitory effect of leukotrienes on luteinizing hormone release.

The aim of this work was to study the effect of high concentrations of leukotrienes on luteinizing hormone (LH) secretion in rat anterior pituitary cells. We also investigated the effect of leukotrienes in parallel with gonadotropin-releasing hormone (GnRH) action. Experiments were on cells gained from trypsinized pituitaries of female rats. Tests were performed by superfusion of the cells attached to cytodex-1 carrier beads. The LH content in samples of perfusate was estimated by radioimmunoassay. This work reports 48% inhibition of basic LH release by action of leukotriene C4 in superfused cells when applied continuously at a concentration of 100 nmol/l. Moreover, we have shown that leukotrienes suppressed GnRH-induced LH secretion in rat pituitary cells when applied in parallel to GnRH (1 nmol/l) as a 4-min pulse at a concentration of 0.1 nmol/l. GnRH-induced LH release was reduced to 66% of its value by leukotriene (LT) B4 (0.1 nmol/l) action; also to 54% by LTC4, 66% by LTD4 and 74% by LTE4 action. In contrast, arachidonic acid (50 pmol/l) and its other 5-lipoxygenase metabolites: 5-hydroperoxyeicosatetraenoic acid (5-HPETE) (50 pmol/l), or 5-hydroxyeicosatetraenoic acid (5-HETE) (50 pmol/l), had no inhibitory effect on GnRH-induced LH release. Arachidonic acid and 5-HETE potentiated GnRH-induced LH release up to 249% and 429%, respectively, when applied in parallel with GnRH (1 nmol/l) as a 4-min pulse at a concentration of 10 pmol/l. In our earlier work we have shown that several leukotrienes are potent stimulants of LH release. The present report documents the finding that the 5-lipoxygenase pathway is also involved in the inhibitory regulation of hormone release in anterior pituitary cells.

Action of vasopressin in superfused human granulosa cells in vitro.

Arginine-vasopressin as well as luteinizing hormone (LH)/follicle-stimulating hormone (FSH) stimulate progesterone release in superfused human granulosa cells. Extracellular administration of inositol triphosphate (10(-6) mol/l) or calcium ions (10(-4) mol/l) mimics the action of both arginine-vasopressin and LH/FSH and evokes progesterone secretion in superfused granulosa cells.

Action of recombinant follicle stimulating hormone in superfused human granulosa cells in vitro.

The aim of this work was to compare the action of recombinant follicle stimulating hormone (rFSH) and urinary FSH (uFSH). Moreover we aimed to compare the secretory efficiency of continuous versus pulsatile stimulation by rFSH in superfused human luteal cells. Progesterone concentration was measured in culture medium by radioimmunoassay. Action of rFSH and uFSH was compared in static cultures of human granulosa cells at doses of 0.001-10 IU/ml. The secretory efficiency of both rFSH and uFSH was found to be similar in a defined range of concentrations (0.001-0.3 IU/ml). At concentrations of 1 and 10 IU/ml, the action of uFSH was significantly more potent than rFSH, up to 139% (P < 0.01) and 133% (P < 0.01) respectively. A concentration of 0.3 IU/ml of rFSH was most potent in static cultures, and evoked progesterone release up to 80 mg/ml. For a stimulation period of up to 4 h, the action of rFSH and uFSH in human granulosa cells was time-dependent and differences between them were not significant. Irregularities were observed at > 4 h stimulation time. In another experiment, in superfused human granulosa cells, we showed that the stimulatory effectiveness of pulsatile rFSH administration (time interval 60 min, application time 10 min) was greater for progesterone release (3973 ng of progesterone/1 IU rFSH) than was continuous administration (848 ng of progesterone/1 IU rFSH). In conclusion, the secretory action of rFSH is similar to that of uFSH for defined times and doses. Moreover, pulsatile rFSH administration is more efficient at stimulating the release of progesterone than continuous administration.

Role of leukotriene C4 in follicle-stimulating hormone (FSH) secretion in female rat pituitary.

Leukotriene C4, at doses of 0.01 and 0.1 nmol/l added to superfused cells in pulse of 4-min duration, evoked follicle-stimulating hormone (FSH) release up to 12- to 26-fold of basal secretion. Higher and lower concentrations of leukotriene C4 were not able to induce FSH secretion. Gonadotropin-releasing hormone (GnRH)-induced FSH release was reduced by 38-57% by the leukotriene receptor antagonist FPL 55712 (10 mumol/l). Moreover, we have shown that FSH release occurs parallel to leukotriene C4 synthesis in rat anterior pituitary cells. Mellitin (100 nmol/l), an activator of phospholipase A2, induced FSH and radioactivity secretion in rat anterior pituitary cells previously preincubated for 24 h with [3H]arachidonic acid (AA).

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%.

Action of leukotriene B4 in human granulosa cells in vitro.

Leukotriene B4 directly enhanced progesterone release from superfused human granulosa cells. This secretory effect was observed in concentrations from 10(-12) to 10(-10) M. Lower and higher concentrations failed to affect progesterone release. When we analysed the high performance liquid chromatography profile of supernatant from human granulosa cell cultures, we detected a leukotriene B4 peak. In conclusion, these data support the hypothesis that leukotriene B4 may participate in the intracellular mechanism of progesterone release in human granulosa cells.

Production of leukotrienes in gonadotropin-releasing hormone-stimulated pituitary cells: potential role in luteinizing hormone release.

Gonadotropin-releasing hormone (GnRH) stimulated the formation of two major metabolites of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4, as well as luteinizing hormone (LH) release in primary cultures of rat anterior pituitary cells. Several lines of evidence suggested the presence of a GnRH-dependent pituitary endocrine system in which LTs act as second messengers for LH release: (i) GnRH-dependent LT formation was observed within 1 min and immediately preceded GnRH-induced LH release, whereas exogenous LTs stimulated LH release at low concentrations; (ii) the dose responses of GnRH-induced LT production and LH release were similar and both effects required the presence of extracellular Ca2+ ions; (iii) GnRH-induced LH release was blocked by up to 45% following the administration of several LT receptor antagonists; (iv) LTE4 action on LH secretion was entirely abolished by LT receptor antagonists; and (v) an activator of protein kinase C acted synergistically with LTE4 to induce LH release. The major source of LT formation in the pituitary cell cultures appeared to be the gonadotrophs, as shown by GnRH receptor desensitization experiments. The results demonstrate the presence of a GnRH-activatable 5-lipoxygenase pathway in anterior pituitary cells and provide strong support for the hypothesis that LTs play a role in LH release in the GnRH signaling pathway.

Action and formation of inositol bisphosphate and inositol trisphosphate in rat anterior pituitary cells.

Inositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, administered exogenously at a concentration of 3 x 10(-5) mol/l increased LH release in superfused rat pituitary cells by 950 +/- 267% and 281 +/- 83%, respectively. This stimulatory effect was reversible and dose-dependent. Other inositol phosphates (inositol 1-monophosphate, inositol 1,4,5,6-tetrakisphosphate, inositol 1,3,4,5,6-pentakisphosphate and inositol 1,2,3,4,5,6-hexakisphosphate), tested in vitro, did not significantly influence LH release. In saponin-permeabilized cells, the rate of basal and stimulated LH release was twice that in non-permeabilized cells. Penetration of inositol bisphosphate and inositol trisphosphate into saponin-treated pituitary cells did not increase the secretory potency of these agents compared with their effect on non-permeabilized cells. The new findings document that inositol trisphosphate formation occurs within 5-45 s after GnRH (10(-7) mol/l) administration and seems to be involved in mediating the rapid, first phase of LH release, whereas inositol bisphosphate formation occurs after 3-15 min and is probably related to later phases of LH secretion. Our results suggest that inositol bisphosphate and inositol trisphosphate are important regulators of the release of luteinizing hormone and can exert their effects not only intracellularly, but also extracellularly.

Differences in luteinizing hormone release stimulated in rat anterior pituitary cells by leukotriene C4 and by gonadotropin-releasing hormone in vitro.

Continuous administration of leukotriene C4 (LTC4, 10(-10) M) to superfused rat anterior pituitary cells increased LH release for 40 min only, whereas in a parallel experiment gonadotropin-releasing hormone (GnRH, 10(-9) M) evoked a continuous increase in hormone secretion. In contrast to GnRH, LTC4 did not desensitize rat anterior pituitary cells. The secretory action resulting from the administration of LTC4 (10(-10) M) was abolished for 40 min after previous stimulation. The results documented the dual action of LTC4 on LH exocytosis.

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.

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.

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.

Arachidonic acid and its lipoxygenase metabolites stimulate prolactin release in superfused pituitary cells.

The direct effect of leukotrienes and other lipoxygenase products on prolactin release has been assessed. Arachidonic acid and its lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) stimulated the release of prolactin in superfused rat pituitary cells in a dose-dependent manner. Leukotrienes (LT) A4, B4, C4 and E4 provoked a very marked biphasic and dose-dependent secretion of prolactin from superfused cells. Maximal effects were achieved with leukotrienes at a concentration of 3 X 10(-11) to 3 X 10(-10) M but LTD4 did not affect peptide release under these conditions. The metabolites were more potent than arachidonic acid in affecting hormone secretion. Pulses of 4 minutes duration of these fatty acids may even elicit a more pronounced response than thyrotrophin-releasing hormone (TRH). Nordihydroguaiaretic acid (NDGA 10(-6) M), a lipoxygenase inhibitor, prevented the effect of arachidonic acid on peptide secretion. Repeated TRH (10(-7) M) administration to pituitary cells led to a reduction in cell response, which may also be observed in cells pre-treated with pulsatile 5-HETE or 15-HETE. These data support previous findings that arachidonic acid and its lipoxygenase metabolites may play a role in the secretory mechanism of prolactin release in pituitary cells.

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.

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.

The role of novel arachidonic acid metabolites in GnRH action on gonadotrophin release in vitro.

Lipoxygenase metabolites of arachidonic acid were shown to stimulate gonadotrophin release dose-dependently in rat pituitary cells. The secretory activity of the arachidonate metabolite leukotriene C4 (LTC4) was biphasic and 10-fold more potent than that of the physiological stimulus gonadotrophin-releasing hormone (GnRH). In pre-labelled, superfused pituitary cells, GnRH dose-dependently enhanced the release of [3H]arachidonic acid, which occurred simultaneously with the secretion of luteinizing hormone (LH). When cells were pre-treated with GnRH for 24 h no response to a further stimulus by GnRH (10(-7) M) could be observed for either [3H]arachidonate nor LH, demonstrating that also in desensitized cells these two mechanisms react similarly. In addition, a GnRH antagonist did not affect the release of arachidonate or LH. These results suggest that arachidonic acid may be involved in the mechanism of GnRH action on gonadotrophins via its lipoxygenase metabolites and LTC4 could act as a very potent intracellular stimulus of LH secretion.