Frank Hertelendy

Nω-Nitro-L-arginine, an inhibitor of nitric oxide synthesis, increases blood pressure in rats and reverses the pregnancy-induced refractoriness to vasopressor agents

OBJECTIVE With N omega-nitro-L-arginine, a potent inhibitor of nitric oxide synthesis, we tested the hypothesis that nitric oxide plays a functional role in the blunted pressor responsiveness seen during pregnancy. STUDY DESIGN A group of six pregnant rats were instrumented on the fourteenth day of gestation and studied on days 19 and 20, as well as 7 days post partum. Another group of six virgin rats were similarly prepared and used 5 days after surgery. Blood pressure and heart rate were monitored in conscious freely moving animals before and during the administration of drugs or placebo. Results were analyzed, by one-way repeated-measures analysis of variance, with Dunnetts t test, or by paired t test where applicable. RESULTS Basal mean arterial pressure and heart rate were 90.8 +/- 3.0 mm Hg and 330 +/- 6 beats/min in pregnant animals and 107.1 +/- 3.2 mm Hg and 315 +/- 7 beats/min in nonpregnant animals. Pressor responses to angiotensin II, vasopressin, and norepinephrine were attenuated in gravid animals. Infusion of N omega-nitro-L-arginine significantly and in a dose-dependent manner increased mean arterial pressure and reduced heart rate. These effects could be completely reversed by L-arginine administration. Changes in mean arterial pressure were higher during pregnancy as compared with postpartum values. N omega-nitro-L-arginine infusion potentiated pressor responses to all three vasopressors, resulting in dose-response curves that were significantly shifted to the left, making them virtually identical in pregnant and postpartum rats. CONCLUSION Our data support the emerging view that nitric oxide plays a key role in the regulation of blood pressure during pregnancy.

The nuclear transcription factor NF-κB mediates interleukin-1β–induced expression of cyclooxygenase-2 in human myometrial cells

Abstract Objective: Up-regulation of prostaglandin production by gestational tissues in the setting of intrauterine infection has been implicated as an important contributor to preterm labor and parturition. In this study we investigated the possible role of the nuclear transcription factor NF-κB in interleukin-1 signaling, leading to the expression of cyclooxygenase 2 and prostaglandin production in human myometrial cell cultures. Study Design: Human myometrial smooth muscle cells from an immortalized line were used as a model system between passages 20 and 35. Growth-arrested cell cultures were stimulated with human recombinant interleukin 1, and the activation of NF-κB was assessed by the degradation of the inhibitory protein IκB-α (Western analysis), as well as by nuclear binding of NF-κB by using an electrophoretic mobility shift assay. The abundance of cyclooxygenase-2 messenger ribonucleic acid and protein was measured by Northern and Western analyses, whereas prostaglandin (prostaglandin I 2 and prostaglandin E 2 ) production was determined by specific radioimmunoassays. Results: Within 15 minutes of stimulation with interleukin 1, 90% of IκB-α was degraded. This was temporally associated with nuclear translocation and binding of NF-κB. Within 30 minutes, cyclooxygenase 2 messenger ribonucleic acid appeared, with steady-state levels increasing up to 4 hours. This was followed by an up to 80-fold increase in cyclooxygenase 2 protein and a corresponding time-dependent increase in prostaglandin production. When IκB-α degradation was blocked with calpain I inhibitor, NF-κB translocation, cyclooxygenase 2 messenger ribonucleic acid and protein expression, and prostaglandin synthesis were also inhibited. Conclusion: Stimulation of human myometrial cells with interleukin 1 leads to rapid activation of the transcription factor NF-κB, which is functionally linked to the expression of cyclooxygenase 2 messenger ribonucleic acid, protein, and prostaglandin synthesis. (Am J Obstet Gynecol 1999;181:359-66.)

Interleukin-1 and tumor necrosis factor stimulate arachidonic acid release and phospholipid metabolism in human myometrial cells°.

OBJECTIVE Our aim was to evaluate the effects of the cytokines interleukin-1 and tumor necrosis factor on arachidonic acid release in human myometrial cells. STUDY DESIGN Primary monolayer cultures of human myometrial cells prelabeled with tritiated arachidonic acid were exposed to interleukin-1 or tumor necrosis factor for varying periods and the release of tritiated arachidonic acid and its loss from phospholipids were measured by radiochromatography. To gain some information on the biologic action of interleukin-1 the contractile response to oxytocin was measured in myometrial strips preincubated with this cytokine. Data were statistically evaluated with analysis of variance or Students test. RESULTS Both cytokines caused a dose-dependent increase in tritiated arachidonate release that was suppressed by the protein synthesis inhibitor cycloheximide. Tritiated arachidonic acid release was maximal after 24 hours of stimulation with interleukin-1. Both interleukin-1 and tumor necrosis factor stimulated the release of the isotopically labeled fatty acid from phosphatidylcholine. In addition, interleukin-1 also increased the loss of arachidonic acid from phosphatidic acid and significantly potentiated the oxytocin-evoked myometrial contractility. CONCLUSIONS Both interleukin-1 and tumor necrosis factor enhance arachidonic acid release, probably by inducing the synthesis of phospholipase A2 and possibly other enzymes involved in the metabolism of phospholipids. In turn, arachidonic acid itself may act as a second messenger, synergizing with other uterotonic agents, as well as serving as the precursor for prostaglandins and various other bioactive eicosanoids.

Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells☆☆☆★

OBJECTIVE The objective of our study was to test the hypothesis that oxytocin promotes prostaglandin production by up-regulating cyclooxygenase-2 via activation of mitogen-activated protein kinase cascade in human myometrial cells. STUDY DESIGN Confluent cultures of human myometrial cells obtained from uterine specimens of premenopausal women undergoing hysterectomy were serum starved for 48 hours before oxytocin stimulation. Prostacyclin levels, as 6-keto-prostaglandin F(1) (alpha), were measured by radioimmunoassay, and the cellular cyclooxygenase-2 protein content was determined by Western blot. Mitogen-activated protein kinase activity was assessed by measuring the phosphorylation of myelin basic protein. RESULTS In a time- and dose-dependent manner oxytocin promoted prostacyclin production in human myometrial cells. Maximal responses were observed after 8 hours of stimulation at a dose of 100 nmol/L. This effect was mainly due to the expression of cyclooxygenase-2 protein. Within 5 minutes oxytocin significantly stimulated mitogen-activated protein kinase, as compared with the expression in untreated controls. The maximal increase in enzyme activity (2.5-fold) was obtained at 45 minutes. A selective inhibitor of mitogen-activated protein kinase activation (PD98059), as well as herbimycin, a tyrosine kinase inhibitor, and the transcriptional blocker actinomycin D, suppressed oxytocin-induced cyclooxygenase-2 expression and prostacyclin production. The stimulatory action of oxytocin was also sensitive to inhibition by pertussis toxin but appeared to be independent of protein kinase C activation. CONCLUSION Our data indicate a largely unrecognized signal transduction mechanism for oxytocin, involving G-protein-coupled activation of mitogen-activated protein kinase and cyclooxygenase-2 gene expression, leading to increased prostaglandin production in human myometrial cells. This signaling pathway complements the rapid activation of the phosphoinositide cycle and may be responsible for sustained release of prostaglandins in uterine tissues, promoting labor and parturition.

Prostaglandins and steroid hormones in plasma and ovarian follicles during the ovulation cycle of the domestic hen (Gallus domesticus)

Abstract The concentrations of prostaglandins E and F (PGE, PGF), estradiol (E 2 ), estrone (E 1 ), and progesterone (P) were determined by sensitive and specific RIAs in the plasma and ovarian follicular tissue collected from the same hens which were killed at frequent intervals during the ovulatory cycle. Plasma levels of both PGE and PGF peaked at the time of oviposition/ovulation (OP/OV) with PGE showing a second peak 5 hr before these events. Plasma E 1 and E 2 began to rise 7 hr prior to OP/OV reaching maximum levels just before OP. Plasma P rose to a broad preovulatory peak 2–6 hr before OP falling abruptly at OP. The concentration of PGs in the two largest postovulatory follicles (POF-1, POF-2) were similar, both fluctuating considerably during the cycle (10–220 ng/g). On the other hand, a sharp 20-fold increase in PGF levels of the largest preovulatory (PRF) was observed shortly after OP/OV. PGE concentrations in this follicle were relatively low during most of the ovulatory cycle with the exception of a significant peak 5–7 hr before OP/OV. P levels in PRF, ranging from 13.2 to 165.5 ng/g, were also higher than those in the POFs, particularly between 2 to 6 hr prior to OP/OV. E 1 concentrations in all three follicles peaked between 6 and 9 hr after OP/OV and fell to a nadir 17 hr after these events. E 1 then began to rise in the preovulatory phase of the cycle with E 2 , which reached a significant peak 6 hr before OP/OV. The increase in plasma PGs at the time of OP gives further evidence that these substances are involved in this process. It is suggested that the plasma PGE increase may be related to the preovulatory LH surge and ovarian PGs may play a role in the establishment of the follicular hierarchy.

Prostaglandin-induced premature oviposition in the coturnix quail

Abstract Intrauterine injection of prostaglandin E 1 , E 2 , A 1 , F 2α , F 1β and F 2β all induced premature egglaying in the coturnix, PGE 1 being the most potent. As little as 5 ng of PGE 1 yielded 5/5 successful ovipositions within a few minutes. Arachidonic acid and phospholipase A were also effective, indicating that the avian uterus can synthesize prostaglandins from exogenous or endogenous precursors. Indomethacin (0.1mg) inhibited the action of both arachidonic acid and phospholipase A but not that of PGE 1 . It is concluded that the avian uterus is more responsive to prostaglandins than the mammalian myometrium and thus may serve as a highly sensitive model for further studies.

Role of calcium in luteinizing hormone-induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).

The effects of calcium on steroidogenesis and cyclic AMP production in chicken granulosa cells were examined. For the expression of full steroidogenic effects by LH, forskolin, and 8-bromo cyclic AMP the presence of calcium in the incubation medium was essential, the optimal concentration being 1 mM. Moreover, calcium antagonists (verapamil, TMB-8) significantly suppressed steroidogenesis in response to all three agonists. The metabolism of 25-hydroxycholesterol and the conversion of pregnenolone to progesterone, however, were not affected by the lack of calcium or by coincubation with calcium antagonists. LH-stimulated cyclic AMP production was also suppressed in calcium-deficient medium and in the presence of the putative calcium channel blocker, verapamil. However, TMB-8 did not affect LH-induced cyclic AMP production. Moreover, neither forskolin- nor IBMX-induced cyclic AMP accumulation was significantly affected by lack of calcium or verapamil. These results are interpreted to indicate that the continuous presence of extracellular calcium is essential for hormonal regulation of steroidogenesis and is important for events both proximal and distal to cyclic AMP generation up to pregnenolone synthesis.

Effects of mammalian gonadotropins on progesterone release and cyclic nucleotide production by isolated avian granulosa cells

Abstract Cyclic AMP and, to a lesser extent, cyclic GMP have been implicated as mediators of gonadotropin-induced steroidogenesis in ovarian tissue and cells of mammals The functional role of these cyclic nucleotides in steroidogenesis in avian granulosa cells has not yet been investigated. In the present study the release of progesterone and levels of cyclic nucleotides were measured in granulosa cells isolated by a nonenzymatic procedure from the largest preovulatory follicle of laying hens 22–24 hr prior to ovulation. Bovine LH(NIH-B10) promoted progesterone production in a dose-related manner. Although steroidogenesis was maximally stimulated by 0.1–0.2 μg/ml bLH, cyclic nucleotide levels remained unaffected even at 5 μg/ml bLH at which concentration progesterone release was significantly inhibited when compared to maximal responses. Similarly, oFSH while stimulating steroidogenesis, although less effectively than bLH, failed to significantly increase cyclic AMP production. Theophylline potentiated the steroidogenic effect of gonadotropins and raised basal levels of the cyclic nucleotides. Dibutyryl cyclic AMP (BU 2 cAMP) induced a dose-dependent release of progesterone while dibutyryl cyclic GMP (BU 2 cGMP) had an inhibitory effect. Agonists of adenylate cyclase such as isoproterenol and cholera toxin in combination with theophylline stimulated progesterone production while heparin, an inhibitor of adenylate cyclase, completely blocked the steroidogenic effect of bLH. Moreover, imidazole, a phosphodiesterase activator, suppressed both progesterone and cyclic AMP production. It is suggested therefore that a small fraction of intracellular cyclic AMP, but not cyclic GMP, which cannot be accurately detected by the RIA method employed, plays an important role in LH-promoted steroidogenesis in chicken granulosa cells.

Block of oxytocin-induced parturition and oviposition by prostaglandin inhibitors.

Abstract The possibility that oxytocin-induced labor is mediated, at least in part, by endogenous release of prostaglandins was investigated in two animal models using inhibitors of prostaglandin synthesis. Term pregnant rabbits failed to deliver after an i.v. oxytocin challenge (100 mU) when they were pretreated with an oral dose of indomethacin (10–25 mg) 45–60 minutes prior to oxytocin, while control rabbits began to deliver within a few minutes. Similarly, oxytocin- but not prostaglandin E 1 -induced oviposition in the coturnix quail was inhibited by indomethacin. 5,8,11,14-Eicosatetraynoic acid, another inhibitor of prostaglandin synthesis, was also effective in blocking oxytocin-promoted oviposition. Based on these observations on two quite diverse species, it is suggested that prostaglandins may play a universal role in the expulsion of the uterine content.

Progesterone production in granulosa cells of the domestic fowl: effects of incubation media, pH, cell density and some other factors

Abstract The objective of this study was to determine the optimal conditions for the short term incubation of chicken granulosa cells. Compared to mechanically-dispersed cells, collagenase-treatment yielded granulosa cells of greater viability and responsiveness to ovine LH (oLH) stimulation. The rate of progesterone secretion by enzyme-dispersed chicken granulosa cells was subsequently compared in five different culture media during incubation periods of up to 24 hr. Under room air as the gas phase, Medium 199 (M199) containing Hanks salts and Hams F-12 medium (F-12) were the most effective, whereas Krebs-Ringer bicarbonate-buffered glucose solution (KRBG) and Dulbeccos medium were the poorest. When pH and the ionic strength of KRBG was maintained by continuous gassing with O 2 :CO 2 (95:5), progesterone production was similar to that obtained with Hepes-buffered M199. The pH optimum was found to be 7.4, although within the range of 6.6–8.5 granulosa cells remained responsive to LH stimulation. The optimal cell density was observed to be 1 × 10 4 to 5 × 10 5 /ml. Although time course studies showed that both basal and stimulated progesterone production peaked by about 12 hr of incubation regardless of the media composition, the amounts released were significantly greater in M199 and F-12 during more prolonged (up to 24 hr) incubations. Glucose, a key medium ingredient for hormone-stimulated steroidogenesis, could be replace by pyruvate. On the other hand, lactate was inhibitory. It is concluded that the mature ovarian follicles of the domestic fowl are an excellent source of pure granulosa cells that can be obtained in high yield after a brief treatment with collagenase. These cells remain viable up to 24 hr and continue to produce large amounts of progesterone in response to LH when incubated in an appropriate medium and at optimal cell density.